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BURGER'S MEDICINAL

CHEMISTRY AND
DRUG DISCOVERY 4

Sixth Edition
Volume 3: Cardiovascular Agents
and Endocrines

Edited by

Donald J. Abraham
Department of Medicinal Chemistry
School of Pharmacy
Virginia Commonwealth University
Richmond, Virginia

WILEY-
INTERSCIENCE
A john Wiley and Sons, Inc., Publication
PREFACE ,

Editors, Editorial Board Members, and sixth edition, we devote an entire subsection
Wiley and Sons have worked for three of Volume 4 to cancer research; we have also
a half years to update the fifth edition of reviewed the major published Medicinal
ger's Medicinal Chemistry and Drug Dis- Chemistry and Pharmacology texts to ensure
wvery. The sixth edition has several new and that we did not omit any major therapeutic
unique features. For the first time, there will classes of drugs. An editorial board was consti-
an online version of this major reference tuted for the first time to also review and sug-
rk. The online version will permit updating gest topics for inclusion. Their help was
and easy access. For the first time, all volumes greatly appreciated. The newest innovation in
are structured entirely according to content this series will be the publication of an aca-
and published simultaneously. Our intention demic, "textbook-like" version titled, "Bur-
was to provide a spectrum of fields that would ger's Fundamentals of Medicinal Chemistry."
new or experienced medicinal chem- The academic text is to be published about a
biologists, pharmacologists and molecu- year after this reference work appears. It will
iologists entry to their subjects of interest also appear with soft cover. Appropriate and
as well as provide a current and global per- key information will be extracted from the ma-
spective of drug design, and drug develop- jor reference.
There are numerous colleagues, friends,
Our hope was to make this edition of and associates to thank for their assistance.
Burger the most comprehensive and useful First and foremost is Assistant Editor Dr.
published to date. To accomplish this goal, we John Andrako, Professor emeritus, Virginia
expanded the content from 69 chapters (5 vol- Commonwealth University, School of Phar-
es) by approximately 50% (to over 100 macy. John and I met almost every Tuesday
s in 6 volumes). We are greatly in debt for over three years to map out and execute
thors and editorial board members the game plan for the sixth edition. His contri-
icipating in this revision of the major ref- bution to the sixth edition cannot be under-
work in our field. Several new subject stated. Ms. Susanne Steitz, Editorial Program
ave emerged since the fifth edition ap- Coordinator at Wiley, tirelessly and meticu-
Proteomics, genomics, bioinformatics, lously kept us on schedule. Her contribution
mbinatorial chemistry, high-throughput was also key in helping encourage authors to
screening, blood substitutes, allosteric effec- return manuscripts and revisions so we could
tors as potential drugs, COX inhibitors, the publish the entire set at once. I would also like
etatins, and high-throughput pharmacology to especially thank colleagues who attended
are only a few. In addition to the new areas, we the QSAR Gordon Conference in 1999 for very
have filled in gaps in the fifth edition by in- helpful suggestions, especially Roy Vaz, John
cluding topics that were not covered. In the Mason, Yvonne Martin, John Block, and Hugo
Preface

Kubinyi. The editors are greatly indebted to Dukat, Martin Safo, Jason Rife, Kevin k e p -
Professor Peter Ruenitz for preparing a tem- olds, and John Andrako in our Department
plate chapter as a guide for all authors. My of Medicinal Chemistry, School of Pharmacy,
secretary, Michelle Craighead, deserves spe- Virginia Commonwealth University for sug-
cial thanks for helping contact authors and gestions and special assistance in reviewing
reading the several thousand e-mails gener- manuscripts and text. Graduate student
ated during the project. I also thank the com- Derek Cashman took able charge of our web
puter center at Virginia Commonwealth Uni- site, http://www.burgersmedchem.com, an-
versity for suspending rules on storage and other first for this reference work. I would es-
e-mail so that we might safely store all the pecially like to thank my dean, Victor
versions of the author's manuscripts where Yanchick, and,Virginia Commonwealth Uni-
they could be backed up daily. Last and not versity for their support and encouragement,
least, I want to thank each and every author, Finally, I thank my wife Nancy who under-
some of whom tackled two chapters. Their stood the magnitude of this project and pro-
contributions have provided our field with a vided insight on how to set up our home office
sound foundation of information to build for as well as provide John Andrako and me
the future. We thank the many " reviewers of lunchtime menus where we often dreamed of
manuscripts whose critiques have greatly en- getting chapters completed in all areas we se-
hanced the presentation and content for the lected. To everyone involved, many, many
sixth edition. Special thanks to Professors thanks.
Richard Glennon, William Soine, Richard
Westkaemper, Umesh Desai, Glen Kel- DONALD J . ABRAHAM
logg, Brad Windle, Lemont Kier, Malgorzata Midlothian, Virginia
Dr. Alfred Burger

rhotograph or Professor Burger followed by his comments to the American Chemical Society 26th Medicinal
Chemistry Symposium on June 14, 1998. This was his last public appearance at a meeting of medicinal
chemists. As general chair of the 1998 ACS Medicinal Chemistry Symposium, the editor invited Professor
Burger to open the meeting. He was concerned that the young chemists would not know who he was and he
might have an attack due to his battle with Parkinson's disease. These fears never were realized and his
comments to the more than five hundred attendees drew a sustained standing ovation. The Professor was 93,
and it was Mrs. Burger's 91st birthday.
Opening Remarks

ACS 26th Medicinal Chemistry Symposium


June 14, I998
Alfred Burger
University of Virginia

It has been 46 years since the third Medicinal Chemistry Symposium met at the University of
Virginia in Charlottesville in 1952. Today, the Virginia Commonwealth University welcomes
you and joins all of you in looking forward to an exciting program.

So many aspects of medicinal chemistry have changed in that half century that most of the
new data to be presented this week would have been unexpected and unbelievable had they
been mentioned in 1952. The upsurge in biochemical understandings of drug transport and
drug action has made rational drug design a reality in many therapeutic areas and has made
medicinal chemistry an independent science. We have our own journal, the best in the world,
whose articles comprise all the innovations of medicinal researches. And if you look at the
announcements of job opportunities in the pharmaceutical industry as they appear in
Chemical & Engineering News, you will find in every issue more openings in medicinal
&emistry than in other fields of chemistry. Thus, we can feel the excitement of being part of
this medicinal tidal wave, which has also been fed by the expansion of the needed research
training provided by increasing numbers of universities.

The ultimate beneficiary of scientific advances in discovering new and better therapeutic
agents and understanding their modes of action is the patient. Physicians now can safely look
forward to new methods of treatment of hitherto untreatable conditions. To the medicinal
scientist all this has increased the pride of belonging to a profession which can offer predictable
intellectual rewards. Our symposium will be an integral part of these developments.

xii
CONTENTS

1 CARDIAC DRUGS: 3 MYOCARDIAL INFARCTION


ANTIANGINAL, VASODILATORS, AGENTS, 155
ANTIARRHYTHMIC, 1 George E. Billman
Gajanan S. Joshi Ruth A. Altschuld
Allos Therapeutics, Inc. The Ohio State University
Westminster, Colorado Columbus, Ohio
James C. Burnett
Virginia Commonwealth University 4 ENDOGENOUS VASOACTIVE
Richmond, Virginia PEPTIDES, 193
Donald J. Abraham James L. Stanton
Institute for Structural Biology and Randy L. Webb
Drug Discovery Metabolic and Cardiovascular
School of Pharmacy and Diseases
Department of Medicinal Chemistry Novartis Institute for Biomedical
Virginia Commonwealth University Research
Richmond, Virginia Summit, New Jersey

2 DIURETIC AND URICOSURIC 5 HEMATOPOIETIC AGENTS, 251


AGENTS, 55 Maureen Harrington
Cynthia A. Fink Indiana University
Jeffrey M. McKenna Walther Oncology Center
Lincoln H. Werner Indianapolis, Indiana
Novartis Biomedical Research
Institute 6 ANTICOAGULANTS,
Metabolic and Cardiovascular ANTITHROMBOTICS, AND
Diseases Research HEMOSTATICS, 283
Summit, New Jersey
Gregory S. Bisacchi
Bristol-Myers Squibb
Princeton, New Jersey
xiii
xiv . Contents

7 ANTIHYPERLIPIDEMIC 10 IRON CHELATORS AND


AGENTS, 339 THERAPEUTIC USES, 479
Michael L. Sierra Raymond J. Bergeron
Centre de Recherches James S. McManis
Laboratoire GlaxoSmithKline William R. Weimar
Les Ulis, France Jan Wiegand
Eileen Eiler-McManis
8 OXYGEN DELIVERY BY College of Pharmacy
ALLOSTERIC EFFECTORS OF University of Florida
HEMOGLOBIN, BLOOD Gainesville, Florida
SUBSTITUTES, AND PLASMA
EXPANDERS, 385 11 THYROID HORMONES AND
THYROMIMETICS, 563
Barbara Campanini
Stefano Bruno Denis E. Ryono
Samanta Raboni Discovery Chemistry
Andrea Mozzarelli
Gary J. Grover
Department of Biochemistry and
Metabolic Diseases Biology
Molecular Biology
Bristol-Myers Squibb
National Institute for the Physics of
Princeton, New Jersey
Matter
University of Parma Karin Mellstrom
Parma, Italy Cell Biology
Karo Bio AB
Huddinge, Sweden
9 INHIBITION OF SICKLE
HEMOGLOBIN
POLYMERIZATION AS A BASIS 12 FUNDAMENTALS OF STEROID
FOR THERAPEUTIC APPROACH CHEMISTRY AND
TO SICKLE-CELL ANEMIA, 443 BIOCHEMISTRY, 593
Constance Tom Noguchi Robert W. Brueggemeier
Alan N. Schechter Pui-kai Li
National Institute of Diabetes, Division of Medicinal Chemistry
Digestive and Kidney Diseases, and Pharmacognosy
National Institutes of Health College of Pharmacy
Laboratory of Chemical Biology The Ohio State University
Bethesda, Maryland Columbus, Ohio
John D. Haley
OSI Pharmaceuticals Inc. 13 FEMALE SEX HORMONES,
Uniondale, New York CONTRACEPTIWS, AND
FERTILITY DRUGS, 629
Donald J. Abraham
Virginia Commonwealth University Peter C. Ruenitz
Department of Medicinal Chemistry College of Pharmacy
Richmond, Virginia University of Georgia
Athens, Georgia
Contents

14 MALE SEX HORMONES, 15 ANTI-INFLAMMATORY


ANALOGS, AND ANTAGONISTS, STEROIDS, 747
679 Mitchell A. Avery
John R. Woolfrey
Robert W. Brueggemeier University of Mississippi-University
Division of Medicinal Chemistry Department of Medicinal Chemistry
and Pharmacognosy School of Pharmacy
The Ohio State University, College University, Mississippi
of Pharmacy
Columbus, Ohio INDEX, 881
BURGER'S
MEDICINAL CHEMISTRY
AND
D R U G DISCOVERY
CHAPTER ONE

Cardiac Drugs: Antianginal,


- -

Vasodilators, and
Antiarrhythmics
JOSHI
GAJANANS.
Allos Therapeutics, Inc.
Westminster, Colorado

JAMES C. BURNETT
Virginia Commonwealth University
Richmond, Virginia

DONALD J. ABRAHAM
1"
Institute for Structural Biology and Drug Discovery
, School of Pharmacy and Department of Medicinal Chemistry
.. Virginia Commonwealth University
Richmond, Virginia

Contents
1 Introduction, 2
2 Cardiac Physiology, 2
2.1 Heart Anatomy, 3
2.2 Electrophysiology, 3
2.3 Excitation and Contraction Coupling, 4
3 Ion Channels, 6
3.1 Channel Gates, 6
3.2 Sodium Channels, 7
3.3 Potassium Channels, 7
3.4 Calcium Channels, 7
4 Antianginal Agents and Vasodilators, 8
4.1 Factors Affecting Myocardial Oxygen Supply,
8
4.2 Factors That Govern Myocardial
Oxygen Demand, 9
4.3 Types of Angina, 9
4.4 Etiology and Causes of Angina, 10
4.5 Treatment, 11
4.5.1 Treatment of Angina, 11
4.5.2 Prevention, 11
Burger's Medicinal Chemistry and Drug Discovery 4.6 Vasodilators, 11
Sixth Edition, Volume 3: Cardiovascular Agents and 4.6.1 Mechanism of Action, 11
Endocrines 4.6.2 Vasodilating Agents, 13
Edited by Donald J. Abraham 4.6.3 Pharmacokinetics and Tolerance of
ISBN 0-471-37029-0 O 2003 John Wiley & Sons, Inc. Organic Nitrates, 15
Cardiac Drugs: Antianginal, Vasodilators, and Antiarrhythmics

4.6.4 Side Effects, 15 5.1.3 Heart Block, 29


4.7 Calcium Channel Blockers, 15 5.1.4 Reentry Phenomenon, 30
4.7.1 Applications, 15 5.2 Types of Cardiac Arrhythmias, 31
4.7.2 Arylalkylamines and Benzothiazepines, 5.3 Classification of Antiarrhythmic Drugs,31
16 5.4 Perspective: Treatment of Arrhythmias, 33
4.7.3 1-4 Dihydropyridine Derivatives, 20 5.5 Class I: Membrane-Depressant Agents, 34
4.7.4 Other Therapeutics, 28 5.5.1 Class IA Antiarrhythmics, 34
4.7.5 Cardiac Glycosides, 28 5.5.2 Class IB Antiarrhythmics, 36
4.7.6 Angiotension-ConvertingEnzyme (ACE) 5.5.3 Class IC Antiarrhythmics, 37
Inhibitors and P blockers, 28 5.6 Class 11: P-Adrenergic Blocking Agents, 38
4.7.7 Glycoprotein IIbIIIIa Receptor 5.7 Class 111: Repolarization Prolongators, 40
Antagonists, 29
5.8 Class TV: Calcium Channel Blockers, 43
4.7.8 Anti-Clotting Agents, 29
5.9 Miscellaneous Antiarrhythmic Agents, 44
5 Antiarrhythmic Agents, 29
6 Future Trends and Directions, 45
5.1 Mechanisms of Cardiac Arrhythmias, 29
5.1.1 Disorders in the Generation of 6.1 Antiarrhythmics: Current
Electrical Signals, 29 and Future Trends, 45
5.1.2 Disorders in the Conduction of the 6.2 Antianginal Agents and Vasodilators: Future
Electrical Signal, 29 Directions, 46

1 INTRODUCTION The development of unique, novel, and tis-


sue-specific cardiac drugs to replace or supple-
It is an exciting time for drug discovery, be- ment existing therapies for various cardiac
cause we are in the midst of a rapid evolution disorders continues to generate significant
towards one of medicine's ultimate goals- and growing attention, and has evolved hand-
moving from treating the symptoms of dis- in-hand with research that has facilitated a
eases to the absolute prevention of diseases. better understanding of the underlying causes
One of the major milestones that will aid in of cardiac disease states.
realizing this goal is the first draft of the hu- The subject of cardiovascular disorders and
their treatment is vast and diverse. This chap-
man genome map, which was recently com-
ter focuses on areas relevant to the antiangi-
pleted. The announcement of this milestone
nal, vasodilating, and antiarrhythmic drugs.
marked what will be seen in the future as a
The cardiac physiology, pathophysiology, and
turning point in the search for new medicines causes of these common diseases are reviewed
that will address the cause, versus the symp- before considering the drugs used in their
toms, of many human ailments. treatment. For additional information, the
Over the last several decades, tremendous reader is referred to other chapters in this se-
advances in basic and clinical research on car- ries that cover advances and updates on ther-
diovascular disease have greatly improved the apeutics and treatments of other cardiovascu-
prevention and treatment of this, the nation's lar ailments such as myocardial infarction,
number one killer of men and women of all antithromobotics, antihyperlipidemic agents,
races. It is estimated that approximately 40% oxygen delivery, nitric oxide, angiogenesis,
of Americans (approximately 60 million be- and adrenergics and adrenergic blocking
tween the ages of 40-70 years) suffer from agents. This chapter makes no attempt to pro-
some degree of this disease (1-3). During the vide comprehensive reviews of literature re-
second half of the 20th century, the problem of lated to these fields.
treating heart disease has been at the fore-
front of the international medical communi-
ties' consciousness. This is reflected in the 2 CARDIAC PHYSIOLOGY
World Health Organizations 1967 classifica-
tion of cardiovascular disease as the world's The human heart and physiological processes
most serious epidemic. that are altered during cardiovascular disease
2 Cardiac Physiology

are reviewed as background to the mecha- (CAD). This leads to myocardial ischemia,
nisms of action of therapeutics used to treat which is the cause of myocardial infarction
angina and arrhythmia. However, for in-depth (heart attack) and angina pectoris.
details about heart anatomy and physiology,
2.2 Electrophysiology
the reader is referred to textbooks and reviews
(4). With the exception of differences in calcium
ion uptake and release, the mechanisms of
2.1 Heart Anatomy
contraction of human skeletal and cardiac
The human heart consists of four chambers: muscle are generally the same. However, un-
the right and left atria and the right and left like skeletal muscle, which requires neuronal
ventricles. Blood returning from the body col- stimulation, heart muscle contracts automat-
lects in the right atrium, passes into the right ically. A heartbeat is composed of a rhythmic
ventricle, and is pumped to the lungs. Blood contraction and relaxation of the heart muscle
returning from the lungs enters the left mass, and is associated with an action poten-
atrium, passes into the left ventricle, and is tial in each cell. The constant pumping action
pumped into the aorta. Valves in the heart of the heart depends on the precise integration
prevent the backflow of blood from the aorta of electrical impulse generation, transmission,
to the ventricle, the atrium, and the veins. and myocardial tissue response.
Heart muscle (the myocardium) is com- A heartbeat involves three principle electri-
posed of three types of fibers or cells. The first cal events. First, an electrical signal to con-
type of muscle cells, found in the sinus and tract is initiated. This is followed by the prop-
atrioventricular node, are weakly contractile, agation of the impulse signal from its point of
autorhythmic, and exhibit slow intercellular origin over the rest of the heart. Finally, the
conduction. The second type, located in the signal abates, or dies away. Cardiac arrhyth-
ventricles, are the largest myocardial cells, mias develop when any of these three events
and are specialized for fast impulse conduc- are disrupted or impaired.
tion. These cells constitute the system for Figure 1.1 displays the principle compo-
propagating excitation over the heart. The re- nents of the heart involved in cardiac impulse
maining myocardial cells (the third type) are generation and conduction. In a normal
strongly contractile and make up the bulk of healthy heart, the electrical impulse signal to
the heart. contract is initiated in the sinoatrial (SA)
Muscle cells in the heart abut very tightly node, which is located at the top of the right
from end to end and form fused junctions atrium (Fig. 1.1). Following depolarization of
known as intercalated discs. This serves two the SA node, the impulse spreads out into the
functions. First, when one muscle cell con- atria through membrane junctions in an or-
tracts, it pulls on cells attached to its ends. ' derly fashion from cell to cell. The atria con-
Second, when cardiac cells depolarize, the tract first. Following, as the impulse for con-
wave of depolarization travels along the cell traction spreads over this part of the heart
membrane until it reaches the intercalated toward the ventricles, it is focused through
disc, where it moves on to the next cell. Thus, specialized automatic fibers in the atria
heart muscle contracts in a unified and coor- known as the atrioventricular (AV) node (Fig.
dinated fashion. Large channels, referred to as 1.1). At this node, the impulse is slowed so that
gap junctions, pass through the intercalated the atria finish contracting before the impulse
discs and connect adjacent cells. These con- is propagated to myocardial tissue of the ven-
nections play an important role in transmit- tricles. This allows for the rhythmic pumping
ting the action potential from one cell to an- action that allows blood to pass from the atria
other. to the ventricles.
Myocardial cells receive nutrients from cor- After the electrical impulse emerges from
onary arteries that branch from the base of the AV node, it is propagated by tissue known
the aorta and spread over the surface of the as the bundle of His, which passes the signal
organ. Blockage of sections of these coronary on to fast-conducting myocytes known as Pur-
arteries occurs during coronary artery disease kinje fibers. These fibers conduct the impulse
4 Cardiac Drugs: Antianginal, Vasodilators, and Antiarrhythmics

Superior vena cava

Sinoatrial node
lnternodal pathways

Atrioventricular node

Right bundle branch

0.2 0.4 0.6


Left posterior fascicle Time (s)
Figure 1.1. Action potentials and the conducting system of the heart. Shown are typical transmem-
brane action potentials of the SA and AV nodes, specialized conducting myocardial cells, and non-
specialized myocardial cells. Also shown is the ECG plotted on the same time scale. Courtesy of

to surrounding, nonspecialized myocardial based on the electrical potential differences


cells. The transmission of the impulse results that exist across cell membranes. These po-
in a characteristic electrocardiographic pat- tentials arise because of several factors: (1)
tern that can be equated to predictable myo- intracellular fluid is rich in potassium (K+)
cardial cell membrane potentials and Na+ and and poor in sodium (Nat) (the reverse is true
Kt fluxes in and out of cells. of extracellular fluid); (2)the cell membrane is
Following contraction, heart muscle fibers more permeable to K+ than it is to Nat; (3)
enter a refractory period during which they anions in the intracellular fluid are mostly or-
will not contract, nor will they accept a signal ganic and fixed, and do not diffuse out through
to contract. Without this resting period, the the membrane; and (4) cells use active trans-
initial contraction impulse that originated in port to maintain gradients of Naf and K t . In
the SA node would not abate, but would con- most cardiac cells the transmembrane poten-
tinue to propagate over the heart, leading to tial difference is approximately -90 mV.
disorganized contraction (known as fibrilla- Stimulation, either electrical or chemical,
tion). can depolarize the cell membranes by causing
conformational changes that open selective
2.3 Excitation and Contraction Coupling
membrane ion channels. This allows Na' to
Myocardial pacemaker cells, usually in the SA flow into the cell and reduce the negative in-
node, initiate an action potential that travels tracellular charge. The transmembrane po-
from cell to cell through the intercalated discs. tential is reduced to a threshold value, which
This opens calcium channels and leads to a produces an action potential that is transmit-
small influx of extracellular calcium ions, ted in an all-or-none fashion along the cellular
which triggers events leading to muscle con- membrane. As the action potential travels
traction. along the cell membrane, it induces a rise in
The biophysical property that connects ex- the levels of free, or activator, calcium (Ca2+)
citation impulse and muscle contraction is within the cell. This, in turn, initiates the in-
2 Cardiac Physiology

+25 11
primarily by the opening of an outward-recti-
fying K+ channel and the closure of the cal-
cium channels. The repolarization that occurs
during this phase involves the interplay of sev-
eral different types of potassium channels.
Following phase 3, the transmembrane poten-
tiaI is restored to its resting value (phase 4;
Fig. 1.2).
Cells of the nodal tissue and specialized
conducting myocytes, such as Purkinje fibers,
can spontaneously depolarize and generate ac-
tion potentials that propagate over myocardial
tissue. This is referred to as automaticity, and
all of these cells have pacemaker potential. In
automatic cells, the outward leak of Kt slows
after repolarization, whereas Naf continues
Cell membrane (NKAY to leach into the cell. This results in a steady-
state increase in intracellular cations and
leads to depolarization. The action potential
phase 4 of such cells is not flat, as observed in
Out Na Fig. 1.2, but becomes less negative until it
Figure 1.2. Diagrammatic representation of an ac- reaches a threshold that triggers the opening
tion potential of a nonautomatic ventricular cell, of an L-type calcium channel in nodal tissue,
showing the principle ion fluxes involved in mem- or the sodium channel in conducting tissue.
brane depolarization and repolarization. The mem- Thus, phase 0 in nodal tissue is caused by the
brane potential in millivolts is given on the vertical influx of Ca2+ and not Na+. Figure 1.1 dis-
axis. This denotes the electrical potential of the in- plays the action potentials for a selection of
ner face of the membrane relative to the outer face. cardiac cells having spontaneous and non-
Phases of the potential are numbered 0, 1,2,3,and spontaneous depolarizability. The electrical
4 and are described in detail in the text.
activity of myocardial cells produce an electri-
cal current that can be measured and recorded
teraction between actin and myosin, which as an electrocardiogram (ECG).
leads to muscle contraction. The time taken by an automatic myocyte to
The action potential of a non-automatic depolarize spontaneously is dependent on the
ventricular myocyte is shown in Fig. 1.2. It is maximum negative value of the resting mem-
divided into five phases (0-4).The rapid mem- brane potential and the slope of phase 4. Un-
brane depolarization, phase 0 (also referred to der normal circumstances, cells of the SA node
as the upstroke), results from the opening of depolarize before other potential pacemaker
fast sodium channels, and is augmented by . cells, because the maximum value of the trans-
Ca2+entering through calcium channels. Fol- membrane potential is approximately -60 mV
lowing depolarization there is a brief initial and the upward slope of phase 4 is steep. Thus,
repolarization (phase 1; termed early repolar- the SA node is normally the pacemaker for the
ization), caused by the closing of the sodium rest of the heart. However, if the impulse from
channels, and a brief outward movement of the SA node is slowed or blocked, or if the
K+ ions. This is followed by a plateau period process of depolarization is accelerated in
(phase2), during which the slow influx of Ca2+ other automatic cells, non-SA cells may initi-
through an L-type calcium channel occurs ate a wave of depolarization that either re-
(Fig.1.2). This phase is most notable because places the SA node impulse or interferes with
it creates a prolonged refractory period during it. Heartbeats that originate from non-SA
which the muscle cannot be re-excited. Phase pacemaker activity are referred to as ectopic
3 is the repolarization period and is caused beats.
6 Cardiac Drugs: Antianginal, Vasodilators, and Antiarrhythmics

The spontaneous impulse rate of automatic


cells depends on the slope of action potential (resting)
phase 4, the magnitude of the maximum dia- h gate open
stolic potential, and the threshold potential.
Changes in any of these values can occur dur- R e c o v y
ing disease states, or from the effects of small (slow)
"drug" molecules. pl-Adrenergic receptor
agonists increase heart rate by increasing
phase 4 of the pacemaker cell action potential. (inactive) h gate open
h gate shut m gate open
Cholinergic drugs that are agonists of musca- Inactivation
rinic receptors slow the heart by decreasing (slow)
the phase 4 slope. Thus, compounds that block
muscarinic receptors (atropine-like) increase Figure 1.3. Simplified represenltation of the gating
mechanism in voltage-activated sodium, potassium,
heart rate, while compounds that block beta and calcium channels. The model hypothesizes
receptors slow the heart. three states, closed resting, open active, and closed
inactive, and two gates, h and m. The figure depicts
what are generally considered to be the essential
3 ION CHANNELS features of gating, which include a closed "resting"
state that is capable of rapidly opening in response
The ion channel transmembrane protein con- to changes in membrane potential followed by a re-
sists of subunits designated as a, P, y, and 6. fractory period in which the channel slowly returns
The a subunit is the major component of Na+, to the resting state.
K+, and Ca2+ channels that spans the mem-
ing the time that the channel is in the inacti-
brane and is tetrameric in nature. Each unit of
vated state, before returning to the resting
this tetramer is designated as a domain, and
state. Potassium channel blockers increase
each domain is made up of six segments desig-
the duration of the action potential, because
nated as S1, S2, S3, S4, S5, and S6. The S5 and
potassium currents are responsible for repo-
S6 segments are linked to each other in a spe-
larizing the membrane during the action po-
cific arrangement so as to form the lining of
tential. Calcium channel blockers slow im-
the ion channels. The S4 segment of each do-
pulse conduction through the SA and AV
main contains many lysine and arginine resi-
dues that act in response to changes in the nodes.
membrane potential, and are thus involved in
3.1 Channel Gates
the opening (voltage gating) of the channel. It
is believed that the S4 segment constitutes the The term gating refers to the process during
"m" gate (5-ll), whereas a polypeptide chain which external stimuli cause conformational
that links the S6 segment of domain I11 to the changes in membrane proteins, leading to the
S1 segment of domain IV constitutes the "h" opening and closing of ion channels. It has
gate (12,131. Other transmembrane subunits been theorized that ion channels have at least
such as p, y, and 6 are believed to play a regu- two gates, referred to as m and h, and that
latory role and mainly contribute to the posi- both gates must be open for ions to pass
tioning and conformation of the a subunit in through the channel (14). According to this
the membrane. model, channel gates cycle through three
Many of the drugs used to treat angina and states: (1) closed resting (R);(2) open active
cardiac arrhythmias exert their therapeutic (A); and (3) closed inactive (I).
effects by blocking Na+, K f , and Ca2+ ion The gating model shown in Fig. 1.3 is a
channels. In the case of sodium channel block- basic outline of the channel gating mechanism
ers, this results in a decrease in the slope of and is useful for describing drug action (15,
phase 0 of the action potential, and thereby 16). In the closed resting state, the h gate is
decreases the V ,, or rate of conduction of open and the m gate is shut. During depolar-
the impulse. Sodium channel blockade can ization the m gate switches to the open posi-
also prolong the refractory period by increas- tion and the channel is activated, allowing the
3 Ion Channels

fast passage of ions through the channel. De- channels can be constructed. Recently, it has
polarization also initiates the channel inacti- been reported that some of the K+ channels
vation so that the channel begins to move from contain only two transmembrane segments (9,
the open to the closed inactive state. In the 10). Many K' channels are classified as recti-
closed inactive state both the m and h gates fying, which means that they are unidirec-
are shut, and the channel does not respond to tional (or transport ions in one direction only),
further repolarization until it has moved back and that their ability to pass current varies
to the closed resting state, during which the h with membrane potential.
gate is again open and the m gate is shut. The inward-rectifying K+ current (usually
designated I,) allows Kt to move out of the
3.2 Sodium Channels cell during phase 4 of the action potential, but
is closed by depolarization; the outward-recti-
There is strong evidence indicating that the fying K+ current (usually designated I,) is
amino acid sequence of sodium channels has
opened by depolarization. Hence, the I ,
been conserved over a long period. As indi-
makes a substantial contribution to the value
cated earlier, the inward voltage dependent
of the resting (phase 4) membrane potential;
Na+ channel consists of four protein subunits
the I, is the major outward current contribut-
designated as a, P, y, and 6. The a subunit, the
ing to repolarization (19). ATP-sensitive Kt
major component of Nat channel made up of
channels are activated if the ATP level in the
200 amino acids, is subdivided into four co-
heart decreases as observed in myocardial
valently bound domains and contains binding
ischemia (20). This leads to the inward flow of
sites for a number of antiarrhythmic com-
Ca2+ ions, thereby reducing myocardial con-
pounds and other drugs. Nat channels are
tractility and conserving energy for basic cell
found in neurons, vertebrate skeletal muscle,
survival processes.
and cardiac muscle. Electrophysiological stud-
The Kf channels are highly selective and
ies, indicating that Na+ channels favor the
are 100-fold more permeable to K+ than to
passage of Naf over K+, point to the fact that
Naf (21). Certain types of molecules bind ex-
Na' channels must be narrow, and ion con- tracellularly and block the voltage-gated K+
ductance depends on the ionic size (ionic ra-
channels (16). These include several peptide
dius of Nat is 0.95 A compared with that of K+ toxins, as well as small charged organic mole-
being 1.33 A) and possibly steric factors (7). cules such as tetraethylammonium, 4-amino-
There is also some evidence indicating that
pyridine, and quinine. Other molecules have
voltage-dependent cardiac Naf channels exist
been found to be K+ channel openers. These
in two isoforms: fast and slow (17). Activation
compounds act on the ATP-sensitive Kf cur-
of the fast Na+ channels in cardiac cells pro- rent and provide cardioprotection during isch-
duces the rapid influx of Na+ and depolarizes
emia. K' channel opener molecules also relax
the membrane in all cardiac myocytes, except
smooth muscle cells and may increase the cor-
nodal tissue, where Naf channels are either
onary blood flow during angina (22-28).
absent or relatively few in number (18). Na+
channels are almost all voltage-gated, and 3.4 Calcium Channels
gates open in response to changes in mem-
Calcium ions are essential for the chain of
brane potential.
events that lead to myocardial contraction.
The role of calcium in the cardiac cycle has
3.3 Potassium Channels
been studied extensively for years. Four types
Potassium channels are outward voltage-de- of voltage-dependent calcium channels with
pendent channels. Similar to Nat channels, specific function and location have been iden-
the major Kf channel subunit consists of four tified. These include (1) the L-type (found
domains, but unlike the a subunit of Na' mainly in skeletal, cardiac, and smooth muscle
channels, the domains of the K+ channels are cells); (2) the T-type (located in pacemaker
not covalently linked. At least 10 genes code cells); (3) the N-type (found in neuronal cells);
for the Kt channel domains, which means and (4) the P-type (located at neuromuscular
that hundreds of combinations of four-domain junctions) (29-31). L-type Ca2+ channels are
Cardiac Drugs: Antianginal, Vasodilators, and Antiarrhythmics

Suggested binding site sparse, L- and T-type Ca2+ channels are re-
for dihydropyridines sponsible for depolarization.

4 ANTIANGINAL AGENTS
A N D VASODILATORS

Angina pectoris is the principle symptom of


ischemic heart disease and is caused by an im-
balance between myocardial oxygen demand
and oxygen supply by coronary vessels. Such
Suggested binding site
'phenylalkylamines an imbalance can result from increased myo-
cardial oxygen demand caused by exercise, de-
Figure 1.4. Suggested structure of an L-type cal- creased myocardial oxygen delivery, or both.
cium channel from skeletal muscle, showing the five Angina pectoris is always associated with sud-
protein subunits that comprise the channel. Phos- den, severe chest pain and discomfort, al-
phorylation sites are indicated by P. Binding sites though some individuals do not experience
for phenylalkylamine and dihydiopyridine calcium pain with ischemia. Thus, angina occurs be-
channel blockers are also shown. Courtesv
" of Trend. cause the blood supply to the myocardium
Pharmacol. Sci.
through coronary vessels is insufficient to
meet the metabolic needs of the heart muscle
for oxygen (32).
formed by a complex arrangement of five pro-
tein subunits designated as the d, a2, p, y, 4.1 Factors Affecting Myocardial
and S subunits, which are comprised of Oxygen Supply
polypeptide chains oPdifferent lengths. The Blood Oxygenation and Oxygen Extraction
arrangement of these subunits is shown in Involving Tissue Ischemia. A normal and
-
Fia. 1.4. The tetrameric a1 subunit is the most healthy- heart extracts about 75%of blood ox-
important functional component forming the ygen at rest; however, increased coronary
Ca2' channel and is responsible for producing blood flow and extraction results in an in-
the pharmacological effects of calcium chan- crease in oxygen supply. Ischemic heart dis-
nel blockers. Ca2+ channels play an important ease develops when there is a deficiency in the
role in cellular excitability by allowing the supply of blood and oxygen to the heart and is
rapid influx of Ca2+, which depolarizes the typically caused by a narrowing of the coro-
cell. The resulting increase in intracellular nary arteries, a condition known as coronary
Ca2+ is essential for the regulation of Ca2+- artery disease (CAD)or coronary heart disease
dependent processes including excitation- (CHD). CAD is a consequence of the compli-
contraction coupling, excitation-secretion
-
cated pathological process involving the devel-
coupling, and gene regulation. It has been sug- opment of atherosclerotic lesions in the
gested that dihydropyridine calcium channel coronary arteries, in which cholesterol, tri-
blockers exert their effects by binding at the glycerides, and other substances in the blood
top of the channel near the cytoplasmic en- deposit in the walls of arteries, narrowing
trance, whereas arylalkylarnines bind at the them. The narrowing limits the extraction and
bottom of the channel between domains I11 flow of oxygen rich blood to the heart.
and IV of the a1 subunit. The other hydropho- Pulmonary Conditions. Sometimes acute
bic a2, p, y, and 6 subunits may play a role in and chronic bronchopulmonary disorders
positioning the a1 subunit in the membrane. such as pneumonia, bronchitis, emphysema,
L-type channels are activated slowly by partial tracheobronchitis, chronic asthmatic bronchi-
depolarization of the cell membrane and inac-
-
tis, tuberculosis, and primary amyloidosis of
tivated by full depolarization and by increas- the lung affect oxygen extraction and its sup-
ing Ca2+ concentration. In nodal tissues, ply to the heart, causing severe ischemia. Also,
where fast sodium channels are absent or if the heart does not work as efficiently as it
4 Antianginal Agents and Vasodilators

should, it reduces the cardiac output. This atherosclerosis may inhibit the flow of oxygen-
causes the congestion of fluid in the tissues rich blood, resulting in ischemia.
and leads to swelling (edema). Occasionally, Cardiac Contractility (Inotropic State). Re-
the fluid collects in the lungs and interferes duction in the cardiac output causes a reflex
with breathing, causing shortness of breath at activation of the sympathetic nervous system
rest or during exertion. Edema is also exacer- to stimulate heart rate and contractility, fur-
bated by a reduced ability of the kidneys to ther leading to greater oxygen demand. If the
dispose of sodium and water. The retained wa- coronary arteries are occluded and incapable
ter further increases the edema (swelling). of delivering the needed oxygen, an ischemia
Coronary Vascular Conditions. Various con- will occur.
ditions such as coronary collateral blood flow, Preload-Venous Pressure and Its Impact on
coronary arterial resistance affected by the Diastolic Ventricular Wall Tension and Ventric-
nervous system, accumulation of local metab- ular Volumes. It has been suggested that an
olites, tissue death, endothelial function, dia- important strategy in the treatment of cardiac
stolic blood pressure, and endocardial-epicar- function is reduction of the work load of the
dial blood flow contribute significantly to the heart, by reducing the number of heart beats
pathogenesis of angina. per minute and the work required per heart
beat defined by preload and afterload. Preload
4.2 Factors That Govern Myocardial is defined as the volume of blood that fills the
Oxygen Demand heart before contraction. Contraction of the
Heart rate. A significant change in the reg- great veins increases preload, whereas dila-
ular beat (fast or slow) or rhythm of the heart tion of veins reduces preload.
(arrhythmias) may affect the myocardial oxy- Afterload-Systolic Pressure Required to Pump
gen demand. Excessive slowing of heartbeat is Blood Out. Afterload is defined as the force
called bradycardia and is sometimes associ- that the heart must generate to eject blood
ated with fatigue, dizziness, and lightheaded- from the ventricles. It largely depends on the
ness or fainting. The various symptoms of bra- resistance of arterial vessels. Contraction of
dycardia have been categorized as sinus these vessels increases afterload, whereas di-
bradycardia, junctional rhythm, and heart lation reduces afterload.
block. These symptoms can easily be corrected 4.3 Types of Angina
with an electrical pacemaker, which is im-
planted under the skin and takes over the Stable Angina. Stable angina is also called
functioning of the natural pacemaker. Con- chronic angina, exertional angina, typical or
versely, a rapid heartbeat is referred to as classic angina, angina of effort, or atheroscle-
tachycardia. Tachycardias are classified into rotic angina. The main underlying pathophys-
two types: supraventricular and ventricular. iology of this, the most common type of an-
Different types of abnormal rapid heart beats gina, is usually atherosclerosis, i.e., plaques
have been categorized as sinus tachycardia that occlude the vessels or coronary thrombi
(normal response to exercise), atrial tachycar- that block the arteries. This type of angina
dia, atrial fibrillation, atrial flutter, AV nodal usually develops by "exertion", exercise, emo-
re-entry, AV reciprocating tachycardia, pre- tional stress, discomfort, or cold exposure and
mature atrial contractions, ventricular tachy- can be diagnosed using EKG. Therapeutic ap-
cardia, and premature ventricular contrac- proaches to treat this type of angina include
tions. Electrocardiographic monitoring is increasing the myocardial blood flow and de-
needed for the correct diagnosis of arrhyth- creasing the cardiac preload and afterload.
mias. Vasospastic Angina. It is also called variant
Because exercise stimulates the heart to angina or Prinzmetal's angina. It is usually
beat faster and more forcefully, more blood, caused by a transient vasospasm of coronary
and hence more oxygen, is needed by the myo- blood vessels or atheromas at the site of
cardium to meet this increased workload. Nor- plaque. This can easily be seen by EKG
mally this is accomplished by dilation of coro- changes in ST elevation that tend to occur at
nary blood vessels; however, sometimes rest. Sometimes chest pain develops even at
10 Cardiac Drugs: Antianginal, Vasodilators, and Antiarrhythrnics

rest. A therapeutic approach to treat this type precursors of the LDLs. The LDLs are charac-
of angina is to decrease vasospasm of coronary terized by high levels of cholesterol, mainly in
arteries, normally provoked by a-adrenergic the form of highly insoluble cholesteryl esters.
activation in coronary vasculature. However, However, there is a strong relationship be-
a-adrenergic activation is not the only cause of tween high LDL levels and coronary heart dis-
vasospasm. ease and a negative correlation between HDL
Unstable Angina. It is also called preinfarc- and heart disease. Total blood cholesterol is
tion angina, crescendo angina, or angina at the most common measurement of blood cho-
rest. It is usually characterized by recurrent lesterol, and various total blood cholesterol
episodes of prolonged attacks at rest and re- levels and risk factors accepted by most physi-
sults from small platelet clots (platelet aggre- cians and the American Heart Association are
gation) at an atherosclerotic plaque site that discussed next. In general, for people who
may also induce local vasospasm. This type of have total cholesterol levels lower than 200
angina requires immediate medical interven- mg/dL, heart attack risk is relatively low (un-
tion such as cardiac bypass surgery or angio- less a person has other risk factors). If the
plasty, because it could ultimately lead to myo- total cholesterol level is 240 mg/dL, the person
cardial infarction (MI). Treatment regimens has twice the risk of heart attack as someone
include inhibition of platelet aggregation and who has a cholesterol level of 200 mg/dL. Cho-
thrombus formation, vasodilation of coronary lesterol levels of 240 mg/dL are considered
arteries (angioplasty), or decrease in cardiac high, and the risk of heart attack and, indi-
load. rectly, of stroke is greater. About 20% of the
U.S.population has high blood cholesterol lev-
4.4 Etiology and Causes of Angina
els. The LDL cholesterol level also greatly af-
The risk factors for the development of CHD fects the risk of heart attack, and indirectly, of
and angina pectoris are genetic predisposition, stroke. Lower LDL cholesterol levels correlate
age, male sex, and a series of reversible risk with a lower risk. Sometimes the ratio of total
factors. The most important factors include cholesterol to HDL cholesterol is used as an-
high-fat and cholesterol-rich diets (32,33,34), other measure. In this case, the goal is to keep
lack of exercise, inability to retain normal car- the ratio below 51;the optimum ratio is 3.5:l.
diac function under increased exercise toler- It is assumed that people with high triglycer-
ance (35, 361, tobacco and smoking (because ides (more than 200 mg/dL) have underlying
nicotine is a vasoconstrictor) (371, excessive diseases or genetic disorders. In such cases,
alcohol drinking, carbohydrate and fat meta- the main treatment is to change the lifestyle
bolic disorders, diabetes, hypertension (38, by controlling weight and limiting carbohy-
39), obesity (40,411, and the use of drugs that drate intake, because carbohydrates raise tri-
produce vasoconstriction or enhanced oxygen glyceride levels and lower HDL cholesterol
demand. The increased cholesterol levels levels.
caused by the consumption of a diet rich in During the last few years, there has been
saturated fat stimulates the liver to produce reliable evidence that coronary artery disease
cholesterol, a lipid needed by all cells for the (CAD) is a complex genetic disease. In fact, a
synthesis of cell membranes and in some cells number of genes associated with lipoprotein
for the synthesis of other steroids, is the prin- abnormalities and genes influencing hyper-
cipal reversible determinant of risk of heart tension, diabetes, obesity, immune, and clot-
disease. Low density lipoproteins (LDLs, also ting systems play important roles in athero-
referred to as "bad" cholesterol) transport sclerotic cardiac disorders. Researchers have
cholesterol from liver to other tissues, identified genes regulating LDL cholesterol,
whereas high density lipoproteins (HDLs, also HDL cholesterol, and triglyceride levels based
referred as "good" cholesterol) transport cho- on common apo E genetic variation (42-46).
lesterol from tissues back to the liver to be Many genes linked to CAD are involved in de-
metabolized. Triglycerides are transported termining how the body removes low density
from the liver to the tissues mainly as very low lipoprotein (LDL) cholesterol from the blood-
density lipoproteins (VLDLs). VLDLs are the stream. If LDL is not properly removed, it ac-
4 Antianginal Agents and Vasodilators

cumulates in the arteries and can lead to CAD. the total amount of fat in the diet, being phys-
The protein that removes LDL from the blood- ically active (because exercise can help to in-
stream is called the LDL receptor (LDLR). In crease HDL), avoiding cigarette smoking and
1985, Michael Brown and Joseph Goldstein exposure to secondhand smoke, and also by
were awarded a Nobel prize for determining reducing sodium intake (49). In individuals
that a mutation in this gene was responsible whose cholesterol level does not r e s ~ o n dto di-
for familial hypercholesterolemia (FH). Peo- etary intervention and in those having genetic
ple with FH have abnormally high blood levels predisposition to high cholesterol levels, drug
of LDL (47). therapy may be necessary. There are now sev-
As with LDLR, mutations in the apo E gene eral very
- effective medications available which
affect blood levels of LDL. Although, more have been proven to be effective for treating
than 30 mutant forms of apo E have been iden- elevated cholesterol and preventing heart at-
tified, people carrying the E4 version of the tacks and death. These include statins such as
gene tend to have higher cholesterol levels atorvastatin, cerevastatin. fluvastatin, lova-
than the general population, whereas choles- statin, pravastatin, and simvastatin (which
terol levels in people with the E2 version are lower LDL cholesterol by 30-50% and in-
significantly lower. The apo E gene has also crease HDL) and fibrates such as bezafibrate,
been implicated in Alzheimer's disease (48). fenofibrate, and gemfibrozil (which lower ele-
vated levels of blood triglycerides and increase
4.5 Treatment
HDL).
In general, the action of various therapeutic Several bile acid sequestrant antilipemic
drugs occurs through (1)alteration of myocar-. agents such as questran, colestid, and welch01
dial contractility or heart rate, (2) modifica- are also used as an adjunct therapy to decrease
tion of conduction of the cardiac action poten- elevated serum and LDL cholesterol levels in
tial, or (3) vasodilatation of coronary and the management of type IIa and IIb hyperlipo-
peripheral vessels. Therefore, the contents of proteinemia. These drugs are known to reduce
this section focus on therapeutics that apply to the risks of coronary heart disease (CHD) and
the treatment of angina and/or act as vasodi- myocardial infarction. The bile acid seques-
lators. trant antilipemic drugs are known to have re-
duced or no GI absorption and are normally
4.5.1 Treatment of Angina. The various regarded as safe in pregnant patients.
treatment modalities of different kinds of an-
4.6 Vasodilators
gina include the following: (1) prevention of
precipitating factors; (2) use of nitrates as va- A number of the simple organic nitrates and
sodilators to treat acute symptoms; (3)use of nitrites find application in both the short- and
prophylactic treatment using a choice of drugs long-term prophylactic treatment of angina
among antianginal agents, calcium channel pectoris, myocardial infarction, and hyperten-
blockers, and P-blockers; (4)surgeries such as sion. Most of these nitrates and nitrites are
angioplasty, coronary stenting, and coronary formulated by mixing with suitable inert ex-
artery bypass surgery; and (5)anticoagulants cipients such as lactose, dextrose, mannitol,
and the use of antithromobolytic agents. alcohol, and propylene glycol for safe han-
dling, because some of these compounds are
4.5.2 Prevention. Even though cardiovas- heat sensitive, very flammable, and powerful
cular disease remains the leading cause of explosives. The onset, duration of action, and
death in the United States, most risk reduc- potency of organic nitrates could be attributed
tion strategies have traditionally focused on to structural differences. However, there is no
detection and treatment of the disease. How- relationship between the number of nitrate
ever, some of the risk factors of cardiac dis- groups and the activity.
eases are reversible and changes in lifestyle
could significantly contribute towards de- 4.6.1 Mechanism of Action. The nitrates
creasing mortality from CHD. One can reduce and nitrites are simple organic compounds
the risk of hypercholesterolemia by reducing that metabolize to a free radical nitric oxide
12 Cardiac Drugs: Antianginal, Vasodilators, and Antiarrhythmics

:a
: Nitrovasodilators -
Enzyme

Endothelium

Figure 1.5. Suggested mechanism of action of nitrate and nitrites used as vasodilators to generate
NO, the most potent (endogenous)vasodilator that induces a cascade of reactions resulting in smooth
muscle relaxation and vasodilation.

(NO) at or near the plasma membrane of vas-


cular smooth muscle cells. In 1980, Furchgott
and Zawadzski first discovered that NO is the
- of the sympathetic nervous system, which in-
creases the heart rate and the myocardial con-
tractility. Reduction in the ventricular wall
most potent endogenous vasodilator (50). NO tension decreases the myocardial oxygen con-
is a highly reactive species with a very short sumption. At the same time, nitrates improve
half-life of few seconds. It is an endothelium myocardial oxygen supply by increasing the
derived relaxing factor (EDRF) that influ- coronary blood flow to the endocardium. Thus,
ences vascular tone. Nitric oxide induces vaso- nitrates alter the imbalance of myocardial ox-
dilation by stimulating soluble guanylate cy- ygen consumption and supply, which is the
clase to produce cyclic GMP (cGMP) as shown basis of angina pectoris. The main pharmaco-
in Fig. 1.5. The latter eventually leads to de- logical effect of organic nitrates is relaxation
phosphorylation of the light chains of myosin of vascular smooth muscle, which results in
(51). The resulting hemodynamic effect pro-
vasodilation. Organic nitrates provide an ex-
duces dilation of epicardial coronary arteries,
ogenous source of NO that augments the ac-
systemic resistance vessels, and veins (52,531.
It is this dilation that causes a reduction in the tions of EDRF, which is impaired during cor-
coronary vascular resistance and is responsi- onary artery diseases (55). It has been
ble for the efficacy of these compounds (54- suggested that nitrates may be useful as anti-
56). Thus, the main action of the nitrates and platelet and antithrombic agents in the man-
nitrites involves peripheral vasodilation,- ei- agement of intracoronary thrombi (56). Al-
ther venous (low doses), or both venous and though the exact mechanism of action of
arterial (higher doses). A result of pooling of nitrates on antiplatelet aggregation is un-
blood in the veins is a reduction in the venous known, it is postulated that activation of
return and the ventricular volume (preload). cGMP inhibits the calcium influx, resulting in
This reduction in reduction in the volume de- fibrinogen binding to glycoprotein IIb/IIIa
creases oxygen demand on the heart and the receptors.
pain of angina is relieved quickly. Further- All vasodilators can be divided into three
more, it has been found that nitrates exert types depending on their pharmacological site
their effect only on the large coronary vessels. of action. These include cerebral, coronary,
This is because minor vessels lack the ability and peripheral vasodilators. In this chapter,
to convert nitrate to NO. (Fig. 1.5). only coronary and peripheral vasodilators,
The use of nitrates leads to reflex activation represented in Fig. 1.6, are reviewed.
4 Antianginal Agents and Vasodilators

0N02 ON02 0 N 0 2

Me O~NO&ONO~ 02N0
9 , 0 N O 2
Amyl Nitrite (1) Glyceryltrinitrate or GTN (2)
Pentaerythritol tetranitrate (3)

lsosorbide dinitrate (4) lsosorbide mononitrate (5) lsoxsuprine Hydrochloride (6)

0
Nicorandil (7)
Figure 1.6. Chemical structures of various currently used vasodilators for the treatment of angina.

4.6.2 Vasodilating Agents. Various com- artery disease by nasal inhalation for acute
pounds that are currently used as vasodilators relief of angina pectoris. It has also been used
are described below. Structures of these com- to treat heart murmurs resulting from steno-
pounds are depicted in Fig. 1.6, and some of sis and aortic or mitral valve irregularities.
their properties such as bioavailability, half- Amyl nitrate acts within 30 s after administra-
life, and some possible side effects are illus- tion, and its duration of action is about 3-5
trated in Table 1.1. min. However, this drug has a number of ad-
Amyl Nitrite (1) (Fig. 1.6, Table 1.1): This verse side effects such as tachycardia and
drug is an aliphatic compound with an un- headache.
pleasant odor. It is a volatile and inflammable Glyceryl trinitrate or GTN (2) (Fig. 1.6, Ta-
liquid and is immiscible in water. Amyl nitrate ble 1.1): Glyceryl trinitrate is a short-acting
can be administered to patients with coronary trinitrate ester of glycerol, with a duration of

Table 1.1 Currently Used Vasodilators


Name Uses Side Effects
Amy1 nitrite (1) Angina pectoris, cyanide poisoning, heart Tachycardia, CNSa
murmurs
Glyceryl trinitrate (2) Angina pecto&, hypertension, acute MI, CNSa
refractory heart failure
Pentaerythritol tetranitrate (3) Prophylactic anginal attacks CNSa
Isosorbide dinitrate (4) Angina pectoris, congestive heart failure, Reflex tachycardia, CNSa
dysphasia
Isosorbide mononitrate (5) Angina pectoris, congestive heart failure CNS," GI intolerance
Isoxsuprine HCl(6) Peripheral vascular diseases (Burger's CNS," Tachycardia
disease, Raynaud's disease),
arteriosclerosis obliterans
Nicorandil(7) Antianginal (not available in USA) Hypotension
-- -

"CNSadverse effects include headache, dizziness, nausea, vomiting, diarrhea, flushing, weakness, rash, and syncope.
14 Cardiac Drugs: Antianginal, Vasodilators, and Antiarrhythmics

action of approximately 30 min. GTN is easily drug is routinely used for the relief of acute
absorbed through the skin and has a strong angina pectoris, as well as in the short-and
vasodilating effect. In fact, glyceryl trinitrate long-term prophylactic management of an-
is the only vasodilator known to enhance cor- gina. It can also be used in combination with
onary collateral circulation and is capable of cardiac glycosides or diuretics for the possible
preventing myocardial infarction induced by treatment of congestive heart failure (57-59).
coronary occlusion. As a result, it is widely Isosorbide mononitrate (5)(Fig. 1.6, Table
used in preventing attacks of angina versus 1.1): Isosorbide mononitrate is the major ac-
stopping these attacks once started. GTN can tive metabolite of isosorbide dinitrate and oc-
be administered by sublingual, transdermal, or curs as a white, crystalline, odorless powder.
intravenous route. However, about 40-80% of Similar to dinitrate, mononitrate is freely sol-
the dose is normally lost during IV administra- uble in water and alcohol. The mononitrate is
tion because of the absorption by plastic ma- available commercially as conventional tab-
terial used to administer the dose. lets, as extended release formulation capsules,
Pentaerythritol Tetanitrate or PETN (3) or as controlled release coated pellets. The ex-
(Fig. 1.6, Table 1.1): PETN is a nitric acid es- tended release formulations and tablets
ter of the tetrahydric alcohol pentaerythritol. should be stored in tight, light resistance con-
Because PETN is a powerful explosive, it is tainers at room temperature.
normally mixed and diluted with other inert Isosorbide mononitrate is readily absorbed
materials for safe handling purposes and to from the GI tract and is principally metabo-
prevent accidental explosions. PETN is lized in the liver. But unlike isosorbide dini-
mainly used in the prophylactic management trate, it does not undergo first pass hepatic
of angina to reduce the severity and frequency metabolism, and therefore the bioavailability
of attacks. It has a similar mechanism of ac- of isosorbide mononitrate in conventional or
tion as GTN and effects vascular smooth mus- extended release tablets is very high (100%
cle cells to induce vasodilation as other ni- and 80%, respectively). About 50% of a dose of
trates. PETN's duration of action can be isosorbide mononitrate undergoes denitration
prolonged by using a sustained release to form isosorbide, followed by partial dehy-
formulation. dration to form sorbitol. Mononitrate also un-
Isosorbide Dinitrate (4) (Fig. 1.6, Table dergoes glucuronidation to form 5-mononi-
1.1): This compound forms white crystalline trate glucuronide. None of the indicated
rosettes that are soluble in water. Isosorbide metabolites show pharmacological activity.
dinitrate can be administrated by oral, sublin- Similar to isosorbide dinitrate, the mononi-
gual, or intrabuccal routes, and the approxi- trate is used for the acute relief of angina pec-
mate onset and duration of action depends on toris, for prophylactic management in situa-
the administration route and various dosage tions likely to provoke angina attacks, and also
forms. The approximate onset and duration of for the long-term management of angina pec-
action of various dosage forms of isosorbide toris (60-61).
dinitrate are as follows: Isoxsuprine Hydrochloride (6) (Fig. 1.6,
Table 1.1): This vasodilator is structurally re-
Duration lated to nylidrin and occurs as a white crystal-
Dosage Forms Onset of Action line powder that is sparingly soluble in water
(62). Isoxsuprine causes vasodilation by direct
Oral l h 5-6 h relaxation of vascular smooth muscle cells,
Extended release 30 min 6-8h which decreases the peripheral resistance. It
Chewable within 3 min 0.5-2 h
Sublingual within 3 min 2h
also stimulates p-adrenergic receptors, and at
high doses can reduce blood pressure. This
drug is used as an adjunct therapy in the man-
Isosorbide dinitrate is metabolized to the agement of peripheral vascular diseases such
corresponding mononitrates (2 and 5 mononi- as Burger's disease, Raynaud's disease, arte-
trate) within several minutes to hours, de- riosclerosis obliterans, and for the relief of ce-
pending on the route of administration. This rebrovascular insufficiency (63-66).
4 Antianginal Agents and Vasodilators

Nicorandil (7) (Fig. 1.6, Table 1.1): Nic- are not as widely distributed. At plasma con-
orandil is a nicotinamide analog possessing a centrations of 50-500 ng/mL, approximately
nitrate moiety. It exhibits a dual mechanism 30-60% are bound to plasma proteins.
of action, acting as both a nitrovasodilator and
a potassium channel activator (67, 68). Nic- 4.6.4 Side Effects. The principle side ef-
orandil offers cardioprotection and has been fects of nitrates include dilation of cranial ves-
shown to improve the myocardial blood flow. sels. This causes headaches, and it can be a
This results in decreased systemic vascular re- limiting factor in the doseage used. More seri-
sistance and blood pressure, pulmonary capil- ous side effects are tachycardia and hypoten-
lary wedge, and left ventricular end-diastolic sion, which result in a corresponding increase
pressure (69,70).It is relatively well tolerated in myocardial oxygen demand and decreased
when used orally or intravenously in patients coronary perfusion-both of which have an
with stable angina. adverse effect on the myocardial oxygen bal-
However, Falase et al. reported that the use ance. Another well-documented problem is
of nicorandil in patients undergoing cardio- the development of tolerance to nitrates.
pulmonary bypass surgery needs further eval- Blood vessels become hypo- or non-reactive to
uation as severe vasodilation and hypotension the drugs, particularly if large doses, frequent
requiring significant vasoconstrictor support dosing regimens, or long-acting formulations
has been observed (71). are used. To avoid this, nitrates are best used
intermittently, allowing a few hours without
4.6.3 Pharmacokinetics and Tolerance of treatment during a 24-h period.
Organic Nitrates. All organic nitrates exhibit
4.7 Calcium Channel Blockers
similar pharmacological effects. The foremost
factor contributing to the pharmacokinetics of Verapamil was the first calcium-channel
glycerol trinitrates (GTN), and other longer blocker (CCB). It was first used in Europe
adingorganic nitrates, is the existence of high (1962) and then in Japan for its antiarrhyth-
capacity hepatic nitrate reductase in the liver. mic and coronary vasodilator effects. The
This enzyme eliminates the nitrate groups in a CCBs have become prominent cardiovascular
stepwise process. But in serum, nitrates are drugs during the last 40 years. Many experi-
metabolized independent of glutathione (54, mental and clinical studies have defined their
72, 73). In general, the organic nitrates are mechanism of action, the effects of new drugs
well absorbed from the oral mucosa following in this therapeutic class, and their indications
administration lingually, sublingually, intra- and interactions with other drugs.
buccally, or as chewable tablets. The organic Calcium plays a significant role in the exci-
nitrates are also well absorbed from the GI tation-contraction coupling processes of the
tract and then undergo first pass metabolism heart and vascular smooth muscle cells, as
in the liver. Nitroglycerin is well absorbed well as in the conduction of the heart cells. The
through the skin if applied topically as an oint- membranes of these cells contain a network of
ment or transdermal system. Orally adminis- numerous inward channels that are selective
tered nitrates and topical nitroglycerin are for calcium. The activation of these channels
relatively long acting. However, the rapid de- leads to the plateau phase of the action poten-
velopment of tolerance to the hemodynamic tial of cardiac muscle cells. Please refer to Sec-
and antianginal effects of various dosage tion 3.4 for a detailed discussion on calcium
forms is known to occur with continuous ther- channels, their mechanism of action, and their
apy. Therefore, an approximately 8 hlday ni- role in cardiovascular diseases.
trate-free period is needed to prevent toler-
ance. Slow release transdermal patches of 4.7.1 Applications. Calcium channel block-
GTN are the most favored dosage form for ing agents are the first drugs of choice for the
achieving prolonged nitrate levels. Highly li- management of Prinzmetal angina. Because
pophilic nitrates, following IV infusion, are of fewer adverse side effects on glucose ho-
widely distributed in to vascular and periph- meostasis, lipid, and renal function, it has also
eral tissues, whereas less lipophilic nitrates been suggested that extended release or inter-
16 Cardiac Drugs: Antianginal, Vasodilators, and Antiarrhythmics

mediate-long acting calcium channel blocking dent processes by hindering the release of cal-
agents may be useful in the management of cium and sodium influx across fast sodium
hypertension in patients with diabetes melli- channels. Thus, bepridil exhibits both calcium
tus. However, data from limited clinical stud- and sodium channel blocking activity and also
ies indicate that patients with impaired glu- possesses electrophysiological properties sim-
cose metabolism receiving calcium channel ilar to those of class I antiarrhythmic agents,
blockers are at higher risks of nonfatal MI and which prolong QT and QTc intervals (77). Al-
other adverse cardiovascular events than though the precise mechanism of action re-
those receiving ACE inhibitor or P-adrenergic mains to be fully determined, this drug re-
agents (74). duces (in a dose-dependent manner) heart
A number of recent reviews describing the rate and arterial pressure by dilating periph-
use of Ca2+channel blockers in the treatment eral arterioles and reducing total peripheral
of hypertension are available (75). New Ca2+ resistance. This leads to a modest decrease
channel blockers have greater selectivity and (less than 5 mm Hg) in systolic and diastolic
can be used to treat hypertension in the pres- blood pressure. When administered IV, it also
ence of concomitant diseases, such as angina reduces left ventricular contractility and in-
pectoris, hyperlipidemia, diabetes mellitus, or creases filling pressure.
congestive heart failure. Reflex tachycardia Although bepridil hydrochloride is usually
and vasodilator-induced headache are the ma- administered orally for the treatment of
jor side effects that limit the use of these chronic stable angina, it is not the first drug
agents as antihypertensives (75). of choice because of its arrhythmogenic poten-
Based on their pharmacophore and chemi- tial and associated agranulocytosis. Conse-
cal structure, the calcium channel blockers quently, it is administered only in patients
can be divided into three different classes that have failed to respond to other antiangi-
of compounds. These include (1) arylalkyl- nal agents (78, 79).
amines, (2) benzothiazepines, and (3)1-4 di- When used alone or in combination with
hydropyridines. These drugs have broad appli- other antianginal agents, it is as effective as
cations in cardiovascular therapy because of P-adrenergic blocking agents or other dihy-
their effects such as (1) arterial vasodilation dropyridine calcium channel blockers. How-
resulting in reduced afterload, (2) slowing of ever, bepridil can aggravate existing arrhyth-
impulse generation and conductance in nodal mias or induce new arrhythmias to the extent
tissue, and (3) reduction in cardiac work and of potentially severe and fatal ventricular
sometimes myocardial contractility, i.e., nega- tachyarrhythmias, related to an increase in
tive inotropic effect to improve myocardial ox- QT and QTc interval (80). Bepridil is rapidly
ygen balance. Each of the above classes of com- and completely absorbed after oral adminis-
pounds and their pharmacological action will tration and is 99% bound to plasma proteins.
be discussed in detail. Diltiazem Hydrochloride (9) (Fig. 1.7, Ta-
ble 1.2): Like bepridil, diltiazem is also a non-
4.7.2 Arylalkylamines and Benzothiazepines. dihydropyridine calcium channel blocker, but
These drugs vary in their relative cardiovascu- it belongs to a benzothiazepine family of com-
lar effects and clinical doses but have the most pounds (81, 82) Diltiazem is a light sensitive
pronounced direct cardiac effects (e.g., vera- crystalline powder that is soluble in water and
pamil, Fig. 1.7). formulated as either a hydrochloride or
Bepridil Hydrochloride (8) (Fig. 1.7, Table malate salt. Diltiazem has a pharrnacologic
1.2): Bepridil is a nondihydropyridine calcium profile that is similar to other calcium channel
channel blocking agent with antianginal and blockers, i.e., it acts by inhibiting the trans-
antiarrhythmic properties. This compound in- membrane influx of extracellular calcium ions
hibits calcium ion influx across L-type (slow, across the myocardial cell membrane and vas-
low voltage) calcium channels (76). However, cular smooth muscle cells (83, 84). However,
unlike other agents, it also inhibits calcium unlike dihydropyridine calcium channel
ion influx across receptor operated channels blockers, diltiazem exhibits inhibitory effects
and inhibits intracellular calmodulin-depen- on the cardiac conduction system-mainly at
4 Antianginal Agents and Vasodilators

OMe

Me0 OMe

Verapamil(l1)

Diltiazem(9) Clentiazem (10)

OMe

Gallopamil (12)

Fendilii (14) Prenylamine (15) Terodilime (16)

h g s 10,12,14,15 and 16 are not available in USA and drug # 13 has been discontinued in USA in 1998.

Figure 1.7. Chemical structures of various currently used arylalkylamines and benzothiazepines
used as antianginal agents and vasodilators.
the atrioventricular (AV) node and minor si- Clentiazem (10) (Fig. 1.7, Table 1.2): Clen-
nus (SA) node. The frequency-dependent ef- tiazem is a chlorinated derivative of diltiazem
fed of diltiazem on AV nodal conduction selec- and is currently undergoing clinical evalua-
tively decreases the heart's ventricular rate tion for the treatment of angina pectoris and
during tachyarrhythmias involving the AV hypertension in Europe. The primary mecha-
node. However, in patients with SA node dys- nism of clentiazem responsible for the antihy-
function, it decreases the heart rate and pro- pertensive effects seems to be reduction in the
longs sinus cycle length, resulting in sinus ar- peripheral arterial resistance caused by cal-
rest. Diltiazem has little to no effect on the QT cium channel blockade (86,87).
interval. Verapamil Hydrochloride (11) (Fig. 1.7,
Diltiazem is administered orally as hydro- Table 1.2): Like diltiazem, verapamil is also a
chloride salt tablets or extended release caD- non-dihydropyridine calcium channel blocker.
sules for the treatment of printzmetal angina, It is available as a racemic mixture and occurs
chronic stable angina, and hypertension. IV as a crystalline powder that is soluble in water.
infusion is the preferred formulation for the The L-isomer of verapamil, which is 2-3 times
treatment of supraventricular tachyarrhyth- more active than the corresponding D-isomer
mias. A controlled study also indicated that for its pharmacodynamic response on AV con-
the simultaneous use of diltiazem and a Pad- duction, has been shown to inhibit the ATP
renergic blocking agent in patients with dependent calcium transport mechanism of
chronic stable angina reduced the frequency the sarcolemma (88).This drug has a pharma-
of attacks and increased exercise tolerance cological mechanism of action that is similar
(85). to other calcium channel blocking agents-it
Table 1.2 Properties of Arylalkylamines and Benzothiazepines
Oral
Name Bioavailibility (%) Half-Life (h) Uses Side Effects
Bepridil HCl (8) Rapid and good 26-64 Chronic stable angina CNS, Ventricular arrhythmias
Dilitazem HCl(9) 80 2-11 Prinzmetal angina, MI, hypertension Hypotension, GI, CNS,"
Supraventricular arrhythmias bradycardia
Clentiazem (10) 'ND ND Hypertension (not available in USA) ND
Verapamil HCl(11) 90 2-8 Supraventricular tachyarrhythmias Bradycardia, AV block, edema,
Angina, MI, hypertension, hypertrophic CNS," hepatic
A
SQ cardiomyopathy
Gallopamil (12) ND ND Angina pectoris, hypertension, ND
supraventricular tachycardia, ischemia
(not available in USA)
Mibefradil(13) ND ND Discontinued use in USA in 1998 for safety reasons (drug-drug interaction)
Fendiline (14) ND ND Angina pectoris (not available in USA) ND
Prenylamine (15) ND ND Prophylactic angina pectoris (not in USA) Hypotension
Terodiline (16) ND ND Suspended caused by cholinergic activity in addition to Ca2+channel antagonist
activity (not available in USA)
"CNS adverse effects include headache, dizziness, nausea, vomiting, diarrhea, flushing, weakness, rash, and syncope.
bND:No data obtained due to limited literature information.
4 Antianginal Agents and Vasodilators

reduces afterload and myocardial contractil- tween mibefradil and P-blockers, digoxin, ve-
ity. However, verapamil also exerts negative rapamil, and diltiazem, especially in elderly
dromotropic effects on the AV nodal conduc- patients, resulting in one death and three
tion and is also classified as a class IV antiar- cases of cardiogenic shock with intensive sup-
rhythmic agent (89). The effects of verapamil port of heart rate and blood pressure. There-
on nodal impulse generation and conduction fore, the manufacturer voluntarily withdrew
are useful in treating certain types of arrhyth- mibefradil from U.S.market in 1998 (100).
mias. However, its effects on myocardial con- Fendiline (14) (Fig. 1.7, Table 1.2): Fendi-
tractility may cause complications in patients line is used in the long-term treatment of cor-
with heart failure. Therefore, verapamil is onary heart disease. This agent is a coronary
used in the treatment and prevention of su- vasodilator and clinical studies have estab-
praventricular tachyarrhythmia and in hy- lished that it is as therapeutically effective as
pentensive patients not affected by cardiode- both isosorbide and diltiazem in the treatment
pressent effects (90). of angina pectoris (101-105). Recently, the ac-
Verapamil is also administered orally in the tion of fendiline on cardiac electrical activity
treatment of prinzmetal angina and chronic has also been investigated in guinea pig papil-
stable angina and is as effective as any other lary muscle. Results from these studies sug-
p-adrenergic blocking agent or calcium chan- gest that a frequency- and concentration-de-
nel blocker. IV verapamil is the drug of choice pendent block of Na' and L-type Ca2+
for the management of supraventricular channels occurs in the presence of fendiline,
tachyarrhythmias including rapid conversion leading to inhibition of fast and slow conduc-
to sinus rhythm of paroxysmal supraventricu- tion and inactivation of Ca2+channels (106).
lar tachycardias (PSVT) (those associated Further studies have shown that fendiline
with Wolff-Parkinson-White or Lown-Ganong- also induces an increase in Ca2+ concentra-
Levine syndrome) and temporary relief of tion in Chang liver cells by releasing stored
atrial fibrillation. It is also used as a mono- Ca2+ in an inositol 1,4,5-triphosphate inde-
therapy or in combination with other anti- pendent manner and by causing extracellular
hypertensive agents for the treatment of Ca2+influx (107).
hypertension. Prenylamine (15)(Fig. 1.7, Table 1.2): Pre-
Gallopamil (12) (Fig. 1.7, Table 1.2): Gallo- nylamine is a homolog of fendiline and is used
pamil is a more potent methoxy analog of ve- in the treatment of chronic coronary insuffi-
rapamil and has demonstrated efficacy in both ciency and prophylaxis of anginal paroxysms.
effort and rest angina, hypertension, and su- The latter is recognized by a disturbance in
praventricular tachycardia (91-94). F'urther- brain blood circulation and sometimes hyper-
more, intracoronary administration of gallo- tension, but prenylamine is not sufficiently ef-
pamil may be useful in treating myocardial fective in very acute anginal paroxysms (108).
ischemia during percutaneous transluminal Because it is a coronary vasodilator, it acts as a
coronary angioplasty (95). calcium antagonist, but without any substan-
Intrarenal gallopamil has shortened the tial effect on the contractility of the myocar-
course of acute renal failure. It has been sug- dium. However, it improves the vascular blood
gested that the role of inhaled gallopamil in circulation and thereby oxygen supply of the
asthma remains to be defined, and well-con- myocardium.
- It also decreases the amount of
trolled potential comparisons with verapamil norepinephrine and serotonin in the myocar-
are needed to define the place in therapy of dium and brain and therefore possesses a
gallopamil for all indications. slight blocking effect on p-adrenergic recep-
Mibefradil(13) (Fig. 1.7, Table 1.2): Mibe- tors. Because this agent enhances the antihy-
fradil is a T- and L-type calcium channel pertensive effect of p blockers, its dosage must
blocker (CCB) that was FDA approved in for be closely monitored. If given in high doses
the management of hypertension and chronic during tachycardia, it can lead to deceleration
stable angina (96-99). However, postmarket- of cardiac activity (109).
ing surveillance discovered potential severe Terodiline (16) (Fig. 1.7, Table 1.2):
life-threatening drug-drug interactions be- Terodiline is an alkyl analog of fendiline and is
20 Cardiac Drugs: Antianginal, Vasodilators, and Antiarrhythmics

used as a calcium channel antagonist. This idly; they are not soluble in water; and because
agent also possesses anticholinergic and vaso- of their depressive effects on myocardium,
dilator activity (110). When administered they have negative inotropic effects. CCBs ac-
twice daily, terodiline is effective in the treat- count for almost $4 billion in sales, and dihy-
ment of urinary urge incontinence (110). dropyridines like lercanidipine are the fastest
Comparative studies with other agents used in growing class of CCB. There are 13 derivatives
urge incontinence are required to determine if of DHP calcium channel blockers currently li-
the dual mechanism of action and superior ab- censed for the treatment of hypertension. Ex-
sorption of terodiline offer clinical advantages. amples of the most prescribed drugs include
Safety concerns about ventricular arrhyth- amlodipine, felodipine, isradipine, lacidipine,
mias have suspended general clinical investi-
lercanidipine, nicardipine, nifedipine, and ni-
gations.
soldipine. Currently, thiazide diuretics or p
4.7.3 1-4 Dihydropyridine Derivatives. blockers are recommended as first-line thera-
This is an important class of drugs that are peutics for hypertension. Calcium channel
broadly used as vasodilators (Figs. 1.8 and blockers, ACE inhibitors, or a blockers may be
1.9). In general, 1,Cdihydropyridines demon- considered when first-line therapy is not toler-
strate slight selectivity towards vascular ver- ated, contraindicated, or ineffective.
sus myocardial cells and therefore have Amlodipine Besylate (17) (Fig. 1.8, Table
greater vasodilatory effects than other cal- 1.3): Amlodipine belongs to a 1,Cdihydropyr-
cium channel blockers. 1,Cdyhydropyrdines idine family of compounds possessing struc-
are also known to possess insignificant elec- tural resemblance to nifedipine, felodipine, ni-
trophysiological and negative inotropic effects modipine, and others. This drug is a calcium
compared with verapamil or diltiazem. The di- channel blocking agent with a long duration of
hydropyridines have no significant direct ef- action. It is mainly used orally either alone or
fects on the heart, although they may cause in combination with other antihypertensive
reflex tachycardia. Some of the properties of agents to treat hypertension and prinzmetal
first and second generation dihydropyridines and chronic stable angina (112, 113).
are given in Table 1.3. Representative drugs Aranidipine (18) (Fig. 1.8, Table 1.3):
from this class are shown in Figs. 1.8 and 1.9. Aranidipine is a 1,4-dihydropyridine calcium
Most of the newer drugs have longer elimina- channel blocker with vasodilating and antihy-
tion half-lives, but also show higher rates of pertensive activity, and therefore is used for
hepatic clearance and hence low bioavailabili- the treatment of hypertension (114-116).
ties. The only exception is amlodipine, which This compound is used either alone or in com-
has a much higher bioavailability (60%)and a bination with a diuretic or p blocker, for the
long elimination half-life. Several metabolic once-daily treatment of mild-to-moderate es-
pathways of DHP-type calcium channel block- sential hypertension. Aranidipine is under in-
ers have been identified in humans. The most vestigation for the treatment of angina pecto-
important metabolic pathway seems to be the ris, but available data are limited to preclinical
oxidation of the 1,Cdihydropyridine ring into animal studies. It decreases T-type and L-type
pyridine catalyzed by the cytochrome P450 calcium currents in a concentration-depen-
(CYP) 3A4 isoform and the oxidative cleavage dent manner. The duration of aranidipine's
of carboxylic acid (111). Calcium antagonists antihypertensive effect is longer than that of
are known to block calcium influx through nifedipine and nicardipine. Aranidipine does
the voltage-operated calcium channels into not significantly affect heart rate, cardiac out-
smooth muscle cells. Several of the com- put, or stroke volume index at rest or after
pounds in the 1,CDHPcategory such as nifed- exercising in patients with mild-to-moderate
ipine, nisoldipine, or isradipine have been hypertension. However, it significantly in-
shown to be useful in the management of cor- creases left ventricular fractional shortening
onary artery diseases. However, these calcium (FS)and left ventricular ejection fraction (EF)
antagonists have some major disadvantages: at rest. It does not adversely affect the hemo-
they are photosensitive and decompose rap- dynamics of lipoprotein or carbohydrate me-
MeOOC MeOOC COOCH2COMe

Me =,""TiH2
H
H
Amlodipine (17) Aranidipine (18) Barnidipine (19)

Benidipine (20) Cilnidipine (21) Efonidipine (22)

..
Me Me Me
Me
\
MeOOCg O C H 2 M e MeOOC
F

CI

Elgodipine (23) Felodipine (24) lsradipine (25)

Drugs 18, 19,20,21,22 and 23 are not available in USA.

Figure 1.8. Chemical structures of various 1,4-dihydropyridines,currently used as calcium channel


blockers that are used as antianginal and antihypertensive agents, which cause vasodilation.
Table 1.3 Properties of Commonly Used 1,4-Dihydropyridine (Calcium Channel Blockers)
Oral
Name Bioavailability Half-Life (h) Uses Possible Side Effects
Amlodipine besylate (17) 64 .934-58 Hypertension, prinzmetal angina Hernodynamic, renal
Aranidipine (18) ND" NDa Hypertension, angina pectoris LVFS,b LVEF" at rest
Barnidipine (19) NDa ND" Angina pectoris, hypertension CNSd
Benidipine (20) ND" NDa Hypertension, angina pectoris CNSd
Cilnidipine (21) ND" ND" Hypertension CNSd
Efonidipine (22) ND" Long Angina pedoris CNSd
Elgodipine (23) NDa ND" Angina pedoris, vasodilator CNSd
during PTCAe
Felodipine (24) 16 10-18 Angina, mild hypertension Reflex tachycardia, angina
Isradipine (25) 19 8 Stable angina Antiatherogenic effects
Lacidipine (26) ND" ND" Hypertension CNSd
N
w Lercanidipine (27) NDa Short Hypertension CNS,d peripheral edema
Manidipine (28) NDa NDa Hypertension CNSd
Nicardipine (29) 15-40 11-12
Nifedipine (30) 45 Prinzmetal and chronic angina, Slight (- )inotropic effect
hypertension
Nilvadipine (31) NDa NDa Hypertension, angina pectoris
Nimodipine (32)f 12 1 Subarachnoid hemorrhage
Nisoldipine (33) 4 15-16 Hypertension, angina pectoris
Nitredipine (34) 16 8-10 Hypertension
"ND: No data obtained because of limited literature information. Compounds 18,19,20,21,22, and 23 are not available in USA.
bLVFS:Left Ventricular Fractional Shortening.
"LVEF: Left Ventricular Ejection Fraction.
dCNSadverse effects include headache, dizziness, nausea, vomiting, diarrhea, flushing, weakness, rash, and syncope.
"PTCA: Percutaneous Transluminal Coronary Angioplasty.
fNimodipine's selectivity for cerebral arterioles makes it useful to treat subarachnoid hemorrhage and not hypertension as with other dihydropyridines. It is also used to treat
migraines.
24 Cardiac Drugs: Antianginal, Vasodilators, and Antiarrhythmics

tabolism, and the pharmacokinetics of arani- dihydropyridine derivatives were evaluated


dipine are not altered in the elderly or in for their state dependent inhibition of L-type
patients with renal failure (114-116). Ca2+channels, and revealed that structurally
Barnidipine (19) (Fig. 1.8, Table 1.3): related DHPs act in distinct ways to inhibit
Barnidipine is a long-acting calcium antago- the L-type channel in the resting, open, and
nist that was launched in Japan in September inactivated states. Cilnidipine and related
1992 under the brand name Hypoca. Barnidip- DHPs seem to exert their blocking action on
ine is used in Europe under the brand name the open channel by binding to a receptor dis-
Vasexten. As a long-acting calcium antago- tinct from the known DHP-binding site (130).
nist, this drug requires administration only Furthermore, the effect of cilnidipine on left
once a day for the treatment of angina and ventricular (LV) diastolic function in hyper-
hypertension, and it is available as a modified tensive patients, as assessed by pulsed doppler
release formulation with a gradual and long echocardiography and pulsed tissue doppler
duration of action (117-119). It is a selective imaging, has been examined. These studies
calcium channel antagonist that reduces pe- suggest that changes in LV diastolic perfor-
ripheral vascular resistance secondary to its mance in patients with essential hyperten-
vasodilatory action (120). Recently it was sug- sion, following cilnidipine treatment, were bi-
gested that Barnidipine administration for a phasic, displaying an initial increase in early
week decreased the blood pressure and made diastolic transmitral flow velocity and a later
the sodium balance negative by increasing uri- increase in early diastolic LV wall motion ve-
nary sodium excretion in patients with essen- locity. The initial and later changes can be re-
tial hypertension. The natriuretic effect of this lated to an acute change in afterload and im-
drug could contribute at least in part to its provement in LV relaxation (131).
antihypertensive effect (121). Also, the possi- Efonidipine (22) (Fig. 1.8, Table 1.3): Efo-
ble use of benidipine for protection against ce- nidipine is a new, long-acting dihydropyridine
rebrovascular lesions in salt-loaded stroke- calcium channel blocker derivative used in the
prone spontaneously hypertensive rats was treatment of hypertension (132-135). When
evaluated by magnetic resonance imaging the effect of efonidipine on endothelin-1
(MRI) (122). (ET-1) in open-chest anesthetized dogs was
Benidipine (20) (Fig. 1.8, Table 1.3): Beni- studied, it was concluded that efonidipine at-
dipine is a new dihydropyridine with potent, tenuates ET-1-induced coronary vasoconstric-
long-lasting calcium antagonism (123). The tion, and therefore would be useful for some
administration of benidipine once daily effec- patients with variant angina, in which ET-1 is
tively decreases blood pressure and attenuates involved in the genesis of coronary vasocon-
blood pressure response to mental stress. Re- striction (136).
flex tachycardia, deterioration of diurnal Recently, to gain insight into the renopro-
blood pressure change, and excessive lowering tective mechanism of efonidipine hydrochlo-
of nighttime blood pressure have not been ride, the acute effects of efonidipine on pro-
observed after benidipine administration. teinuria, glomerular hemodynamics, and the
Therefore, it has been suggested that benidip- tubuloglomerular feedback (TGF) mechanism
ine may be useful for the treatment of elderly in anesthetized 24- to 25-week-old spontane-
hypertensive patients with cardiovascular dis- ously hypertensive rats (SHR) with glomeru-
ease and as an antianginal medication. How- lar injury were evaluated. The results indicate
ever no clinical data is currently available that efonidipine attenuates the TGF response
(124). in SHR by dilating the afferent arteriole, thus
Cilnidipine (21) (Fig. 1.8, Table 1.3): Cilni- maintaining the level of renal plasma flow
dipine is a unique calcium antagonist that has (RPF) and glomerular filtration rate (GFR)
both L-type and N-type voltage-dependent cal- despite reduced renal perfusion pressure
cium channel blocking activity (125-129). (137).
Cilnidipine is under investigation for the Elgodipine (23) (Fig. 1.8, Table 1.3): This
treatment of hypertension in Europe. compound is a novel type of DHP that is very
Recently, cilnidipine, its analogs, and other selective and a potent coronary vasodilator
4 Antianginal Agents and Vasodilators

calcium channel blocker (138). Elgodipine is mulation, the plasma level of felodipine also
very stable to light (2% degradation after 1 declines polyexponentially, with a mean ter-
year of exposure to room light and tempera- minal half-life of 11-16 h.
ture) compared with other currently available The bioavailability of felodipine is influ-
compounds, which are water soluble and de- enced by the presence of high fat or carbohy-
compose within 24 h. It is very selective for drates and increases approximately twofold
vascular smooth muscle, in particular, coro- when taken with grapefruit juice. A similar
nary vessels. Because elgodipine is more than finding has been seen with other dihydropyri-
100-fold more selective for coronarv " vessels
dine calcium antagonists, but to a lesser ex-
versus cardiac fibers, it has few negative ino- tent than that seen with felodipine (145-147).
tropic effects. Elgodipine seems to be poten-
Felodipine produces dose-related decreases
tially useful as a coronary vasodilator during
in systolic and diastolic blood pressure, which
PTCA (percutaneous transluminal coronary
angioplasty).Its stability and solubility allows correlates with the plasma concentration of
for intracoronary administration in patients felodipine. Felodipine can lead to increased ex-
with stable angina. Furthermore, because of a cretion of potassium, magnesium, and calcium
lack of negative inotropic effects, it is also ad- (148). It has been recommended that to pre-
ministered in with moderate heart vent side effects, individuals who are taking
failure (139-141). felodipine should avoid grapefruit and its juice
Some of the preliminary electrophysiologi- (149). This is because grapefruit (juice) is an
cal data in volunteers have shown that elgo- inhibitor of cytochrome P450 isoforms 3A4
dipine differs from other calcium channel and 1A2, which are needed for the normal me-
blockers in its effects on atria-ventricular tabolism of felodipine.
conduction. Isradipine (25) (Fig. 1.8, Table 1.3): Israd-
The chemical stability of elgodipine allows ipine is a calcium antagonist that is available
for its incorporation into suitable polymeric for oral administration and is used in the man-
matrixes for transdermal administration. Pre- agement of hypertension, either alone or con-
liminary" data in vitro and in vivo in volunteers currently with thiazide-type diuretics (150-
have shown that elgodipine penetrates into 153). Isradipine binds to calcium channels
the skin. Studies are in progress to determine with high affinity and specificity and inhibits
the daily effective dose, and therefore, the fea- calcium flux into cardiac and smooth muscle.
sibility of transdermal patches (142). In patients with normal ventricular function,
Felodipine (24) (Fig. 1.8, Table 1.3): Felo- isradipine's afterload reducing properties lead
dipine is a member of the DHP calcium chan- to some increase in cardiac output. Effects in
nel blocker family. This compound is insoluble patients with impaired ventricular function
in water but is freely- soluble in dichlorometh- have not been fully studied.
ane and ethanol. Felodipine exists as a racemic In humans, peripheral vasodilation pro-
mixture and is used to treat high blood pres- duced by isradipine results from decreased
sure, Raynaud's syndrome, and congestive systemic vascular resistance and increased
heart failure (143,144).It reversibly competes cardiac output. In general, no detrimental ef-
with nitrendipine and/or other calcium chan- fects on the cardiac conduction system were
nel blockers for dihydropyridine binding sites seen with the use of isradipine.
and blocks voltage-dependent Ca2+ currents Isradipine is 90-95% absorbed and is sub-
in vascular smooth muscle. ject to extensive first-pass metabolism, result-
Following oral administration, felodipine is ing in a bioavailability of about 15-24%. Isra-
almost completely absorbed and undergoes ex- dipine is completely metabolized before
tensive first-pass metabolism. However, fol- excretion, and no unchanged drug is detected
lowing intravenous administration, the in the urine. Six metabolites have been char-
plasma concentration of felodipine declines acterized in blood and urine, with the mono
triexponentially, with mean disposition half- acids of the pyridine derivative and a cyclic
lives of 4.8 min, 1.5 h, and 9.1 h. Following oral lactone product accounting for >75% of the
administration of the immediate-release for- material identified. The reaction mechanism
26 Cardiac Drugs: Antianginal, Vasodilators, and Antiarrhythmics

ultimately leading to metabolite transforma- pertrophy, and changed the expression of


tion to the cyclic lactone is complex. genes for ANP and interstitial components of
Lacidipine (26) (Fig. 1.9, Table 1.3): Laci- extracellular matrix induced by isoproterenol
dipine also belongs to the DHP class of calcium (163).
channel blockers (154). Recently it was shown Nicardipine (29) (Fig. 1.9, Table 1.3): Ni-
that lacidipine can slow the progression of ath- cardipine belongs to the 1,4-dihydropyridine
erosclerosis more effectively than atenolol, ac- calcium channel blocking family of com-
cording to the results of the European lacidip- pounds. It is usually administered either
ine study on atherosclerosis (155). The orally or by slow continuous IV infusion (when
improvement of focal cerebral ischemia by oral administration is not viable), for the
lacidipine may be partly caused by long-last- treatment of chronic stable angina and the
short-term management of hypertension. It is
ing improvement of collateral blood supply to
used as a monotherapy or in combination with
the ischemic area (156).
other antianginal or antihypertensive drugs.
When comparative effects of both lacidip- Nifedipine (30) (Fig. 1.9, Table 1.3): The
ine and nifedipine were measured, both drugs principle physiological action of nifedipine is
reduced blood pressure significantly during a similar to other 1,4-dihydropyridine deriva-
24-h period with one dosage daily; only lacidip- tives. This drug functions by inhibiting the
ine reduced left ventricular mass significantly transmembrane influx of extracellular cal-
after 12 weeks of treatment (157). cium ions across the myocardial membrane
Lercanidipine (27) (Fig. 1.9, Table 1.3): and vascular smooth muscle cells, without af-
Lercanidipine is a member of the dihydropyr- fecting plasma calcium concentrations. Al-
idine calcium channel blocker class of drugs. though- the exact mechanism of action of nifed-
Recently, a New Drug Application with the ipine is unknown, it is believed to deform the
U.S. FDA to market lercanidipine for the slow calcium channel and hinder the ion-con-
treatment of hypertension has been submit- trol gating mechanism of the calcium channel
ted. This drug has been available in European by interfering with the release of calcium ions
countries for more than 4 years, with an estab- from the sarcoplasmic reticulum. The inhibi-
lished record of anti-hypertensive effect and tion of calcium influx dilates the main coro-
safety in millions of patients (158, 159). In nary and systemic arteries because of the im-
fact, lercanidipine has grown to be the third ~edimentof the contractile actions of cardiac
<

most prescribed CCB in Italy. Lercanidipine and smooth muscle. This reduced myocardial
prevents calcium from entering the muscle contractility results in increased myocardial
cells of the heart and blood vessels, which en- oxygen delivery, while decreasing the total pe-
ables the blood vessels to relax, thereby lower- ripheral resistance associated by a modest
ing blood pressure. It has a short plasma lowering of systemic blood pressure, small in-
half-life, but its high lipophilicity allows accu- crease in heart rate, and reduction in the af-
mulation in cell membranes, resulting in long terload, ultimately leading to reduced myocar-
duration of action. It has been suggested that dial oxygen consumption.
lercanidipine causes fewer vasodilatory ad- Unlike verapamil and diltiazem, nifedipine
verse side effects than other CCBs and is does not exert any effect on SA or AV nodal
therefore being promoted for the treatment of conduction at therapeutic dosage levels. Ni-
isolated systolic hypertension (ISH) in elderly fedipine is administered orally through ex-
patients (160). tended release tablets in various dosage forms.
Manidipine (28) (Fig. 1.9, Table 1.3): Mani- It is mainly used in the treatment of Prinz-
dipine is effective in the treatment of essential metal angina and chronic stable angina. In the
hypertension (161, 162). When the effect of latter case, it is as effective as p-adrenergic
manidipine hydrochloride on isoproterenol- agents or oral nitrates, but is used only when
induced LV hypertrophy and the expression of the patient has low tolerability for adequate
the atrial natriuretic peptide (ANP) trans- doses of these drugs.
forming growth factor was evaluated, it was Nilvadipine (31) (Fig. 1.9, Table 1.3): Nil-
found that manidipine prevented cardiac hy- vadipine is marketed as a racemic mixture for
4 Antianginal Agents and Vasodilators

the treatment of hypertension and angina muscle than on cardiac muscle. The effect of
(164-166). Nilvadipine also provides protec- nisoldipine on blood pressure is principally a
tion against cerebral ischemia in rats having consequence of a dose-related decrease of pe-
chronic hypertension. These effects are depen- ripheral vascular resistance. Whereas nisol-
dent on the duration of treatment (167). Re- dipine, like other dihydropyridines, exhibits a
sults from a clinical study in the United mild diuretic effect, most of the antihyperten-
States, during which a combination of imida- sive activity is attributed to its effect on pe-
pril and a diuretic, P-adrenoceptor antagonist, ripheral vascular resistance.
or a calcium channel blocker (such as nilvad- Nisoldipine is metabolized into five major
ipine) were administered, indicated a reason- metabolites that are excreted in the urine. The
able and safe treatment option when striving major biotransformation pathway seems to be
for additive pharmacodynamic effects not ac- the hydroxylation of the isobutyl ester. A hy-
companied by relevant pharmacokinetic inter- droxylated derivative of the side-chain,
actions (168). present in plasma at concentrations approxi-
Nimodipine (32) (Fig. 1.9, Table 1.3): Ni- mately equal to the parent compound, seems
modipine is a structural analog of nifedipine, to be the only active metabolite and has about
and the S-(-)-enantiomeris primarily respon- 10% of the activity of the parent compound.
sible for the calcium channel blocking activity. Cytochrome P,,, enzymes play a key role in
Substitution of a nitro substituent on the aryl the metabolism of nisoldipine. The particular
ring and planarity of the 1,4-dihydropyridine isoenzyme system responsible for its metabo-
moiety contribute greatly to the pharmacolog- lism has not been identified, but other dihy-
ical effect of nimodipine. Nimodipine is a light dropyridines are metabolized by cytochrome
sensitive yellowish crystalline powder. The P,,, 3A4. Nisoldipine should not be adminis-
mechanism of action of nimodipine is similar tered with grapefruit juice because it inter-
to other calcium channel blockers; however, feres with nisoldivine metabolism. Because
the preferential binding affinity of nimodipine there is very little information available about
towards the cerebral tissue is yet to be fully this drug's use in patients with severe conges-
understood. Nimodipine functions by binding tive heart failure, it should be administered
to the stereoselective high affmity receptor with caution to these vatients.
A

sites on the cell membrane, in or near the cal- Recently, antianginal and anti-ischemic ef-
cium channel, and inhibits the influx of cal- fects of nislodipine and ramipril in patients
cium ions. The vasodilatory effect of nimodip- with Syndrome X (typical angina pectoris, pos-
ine also seems to arise partly from the -
itive treadmill exercise test but negative intra-
inhibition of the activities of sodium-potas- venous ergonovine test and angiographically
sium activated ATPase, an enzyme required normal coronary arteries) suggested that they
for the active transport of sodium across the have similar anti-ischemic and antianginal ef-
myocardial cell membranes. fects in patients with Syndrome X (172).
Nisoldipine (33)(Fig. 1.9, Table 1.3): Nisol- Nitrendipine (34) (Fig. 1.9, Table 1.3): This
dipine is a dihydropyridine, similar to nifedi- drug is used to treat mild to moderate hyper-
pine, but is 5-10 times more potent as a vdo- tension (173, 174).
dilator and has little effect on myocardial In summary, calcium antagonists inhibit
contractility. Nisoldipine is available as a long- the influx of extracellular calcium ions into
acting extended release preparation and cells. This results in decreased vascular
seems effective in treating mild-to-moderate smooth muscle tone and vasodilation leads to
hypertension and angina with once-daily oral a reduction in blood pressure. The 1,Cdihy-
administration (169-171). Nisoldipine selec- dropyridine derivatives (aranidipine, cilnidip-
tively relaxes the muscles of small arteries ine, amlodipine, nisoldipine, nifedipine, felo-
causing them to dilate, but has little or no ef- dipine, nitrendipine, and nimodipine) differ
fect on muscles or the veins of the heart. from the benzothiazepine (e.g., diltiazem) and
In vitro studies show that the effects of ni- phenylalkylamine (e.g., verapamil) classes of
soldipine on contractile processes are selec- calcium antagonists with regard to potency,
tive, with greater potency on vascular smooth tissue selectivity, and antiarrhythmic effects.
Cardiac Drugs: Antianginal, Vasodilz tors, and Antiarrhythrnics

In general, dihydropyridine agents are the also find applications to treat and prevent si-
most potent arteriolar vasodilators, producing nus and supraventricular tachycardia and
the least negative inotropic and electrophysi- symptoms of angina pectoris and myocardial
ological effects; in contrast, verapamil and dil- infarction, but only in combination with p-ad-
tiazem slow AV conduction and exhibit nega- renergic blocking agents and in patients with
tive inotropic activity while also maintaining congestive heart failure.
some degree of arteriolar vasodilatation. The exact mechanism of pharmacological
Calcium channel blockers are commonly action of glycosides has not been fully eluci-
used to treat high blood pressure, angina, and dated. However, glycosides exhibit a positive
some forms of arrhythmia. In the treatment of inotropic effect accompanied by reduction in
hypertension and chronic heart failure, a com-
peripheral resistance and enhancement of
bination therapy enhances therapeutic effi-
myocardial contractility resultingin increased
cacy. Pharmacodynamically, combinations of
myocardial oxygen consumption. They also in-
ACE inhibitor plus a diuretic, 0-adrenorecep-
tor antagonist, or calcium channel blocker are hibit the activities of sodium-potassium acti-
the most promising. vated ATPase, an enzyme required for the ac-
tive transport of sodium across the myocardial
4.7.4 Other Therapeutics. For the past two cell membranes.
decades, the cardiovascular drug market has Glycosides are normally administered ei-
lead drug discovery efforts and sales in the ther orally or by N injection and possess a
pharmaceutical industry. A constant flow of half-life of 36 h to 5-7 days in normal patients,
new and effective drugs has kept this sector in depending on the choice of drugs.
its number one position and will continue to
do so in the future. In addition to the above 4.7.6 Angiotension-ConvertingEnzyme (ACE)
indicated classes of drugs, a number of highly
Inhibitors and P blockers. ACE inhibitors pre-
effective drugs have been introduced and are
vent the conversion of angiotension I to angio-
routinely used either alone or in combination
tension 11,a potent vasoconstrictor. This con-
therapy. Some of these categories are dis-
sequentially reduces plasma concentrations of
.
cussed below:
angiotension 11 and hence vasodilation, and
4.7.5 Cardiac Glycosides. Glycosides are a results in attenuation of blood pressure. ACE
distinct class of compounds that are either inhibitors also affect the release of renin from
found in nature or can be synthetically pre- the kidneys and increase plasma renin activity
pared. The natural glycosides are isolated (PRA). It has been suggested that the hypo-
from various plant species namely digitalis tensive effect of ACE inhibitors may decrease
purpurea Linne, digitalis lanata Ehrhart, vascular tone because of angiotension-induced
strophanthus gratus, or acokanthea schim- vasoconstriction and increased sympathetic
peri. Therefore, these compounds are also activity. The reduced production of angioten-
named as digitalis, digoxin, and digitoxin. sion I1 lowers the plasma aldosterone concen-
Currently, digoxin is the only cardiac glyco- tration (caused by less secretion of aldosterone
side commercially available in United States. from the adrenal cortex). Aldosterone is
Glycosides have a characteristic steroid (agly- known to decrease the sodium extraction con-
cone) structure complexed with a sugar moi- centration and water retention, resulting in a
ety at C-3 position of the steroid through the desired hypotensive effect. p Blockers reduce
p-hydroxyl group. the oxygen demand of the heart by slowing the
Glycosides are mainly used in the prophy- heart rate and lowering arterial pressure.
lactic management and treatment of conges- Such drugs include propranolol (Inderal), a and
tive heart failure and atrial fibrillation. They P blockers labetalol (Normdyne, Trandate),
are known to relieve the symptoms of systemic acebutolol (Sectral), atenolol (Tenormin), meto-
venous congestion (right-sided heart failure or pro101 (Toprol), and bisoprolol (Zebeta). These
peripheral edema) and pulmonary congestion drugs are equally effective as calcium channel
(left-sided heart failure). However, glycosides blockers and have fewer adverse effects.
5 Antiarrhythmic Agents

4.7.7 Glycoprotein Ilb/llla Receptor An- pacemaker cells, initiate a cardiac impulse.
tagonists. These compounds are also called The spontaneous electrical depolarization of
blood thinners because they block platelet ac- the SA pacemaker cells is independent of the
tivity. These drugs are very beneficial for nervous system; however, these cells are in-
many patients with angina and do not seem to nervated by both sympathetic and parasympa-
pose an increased risk for stroke, including thetic fibers, which can cause increases or de-
strokes caused by bleeding. Some of the most creases in heart rate as a result of nervous
widely used drugs include Abciximab (ReoPro, system stimulation. Other special cellsjn the
Centocor),eptifibatide (Integrelin),lamifiban, heart also possess the ability to generate an
and tirofiban (Aggrastat). Glycoprotein IIbI impulse, and may influence cardiac rhythm,
IIIa receptor antagonists are used to reduce but are normally surpassed by the dominant
the risk for heart attack or death in many pa- signal generation of SA pacemaker cells.
tients with unstable angina and non-Q-wave When normal pacemaker function is sup-
myocardial infarctions when used in combina- pressed-caused by pathological changes oc-
tion with heparin or aspirin. Patients with un- curring from infarction, digitalis toxicity, or
stable angina showing elevated levels of tropo- excessive vagal tone-or when excessive re-
nin T factor are good candidates for these lease of catecholamines from sympathomi-
drugs. metic nerve fibers occurs, these other auto-
matic cells (including special atrial cells,
4.7.8 Anti-Clotting Agents. Anti-clotting certain AV node cells, the bundle of His, and
agents, either anticoagulants or anti-platelet Purkinje fibers) have the potential to become
drugs, are being used to treat unstable angina, ectopic pacemakers, which can dominant car-
to protect against heart attacks, and to pre- diac rhythm and consequently lead to arrhyth-
vent blood clots during heart surgeries. They mias.
can be used alone or in combinations, depend-
ing on the severity of the condition. Clopi- 5.1.2 Disorders in the Conduction of the
dogrel (Plavix), a platelet inhibitor, has been Electrical Signal. Disorders in the transmis-
shown to be 20% more effective than aspirin sion of the electrical impulse can lead to con-
for reducing the incidence of a heart attack. duction block and reentry phenomenon. Con-
Other promising anti-clotting drugs comprise duction block may be complete (no impulses
argatroban (Novastan), danaparoid (Orga- pass through the block), partial (some im-
ran), and forms of hirudin (bivalirudin lepi- pulses pass through the block), and bi-direc-
drudin or desirudi), a substance derived from tional or unidirectional. During bi-directional
the saliva of leeches. One study suggested that block, an impulse is blocked regardless of the
the hirudin agents may be superior to heparin direction of entry; a unidirectional block oc-
in preventing angina and heart attack, al- curs when an impulse from one direction is
though bleeding is a greater risk with hirudin. completely blocked, while impulses from the
opposite direction are propagated (although
usually at a slower than normal rate).
5 ANTIARRHYTHMIC AGENTS
5.1.3 Heart Block Heart block occurs when
5.1 Mechanisms of Cardiac Arrhythmias
the impulse signal from the SA node is not
The pumping action of the heart involves transmitted through either the AV node or
three principle electrical events: the genera- lower electrical pathways properly. Heart
tion of a signal; the conduction or propagation block is classified by degree of severity: (1)first
ofthe signal; and the fading away of the signal. degree heart block, all impulses moving
When one or more of these events is disrupted, through the AV node are conducted, but at a
cardiac arrhythmias may arise. slower than normal rate; (2) second degree
heart block, some impulses fully transit the
5.1.1 Disorders in the Generation of Electri- AV node, whereas others are blocked (as a re-
cal Signals. In normal heart, cells located in sult, the ventricles fail to beat at the proper
the right atrium, referred to as the SA node or moment); (3) third degree heart block, no im-
30 Cardiac Drugs: Antianginal, Vasodilators, and Antiarrhythmics

pulses reach the ventricles (automatic cells in


the ventricles initiate impulses, but at a \bl
slower rate, and as a result the atria and ven- A (Normal)
tricles beat at somewhat independent rates).

5.1.4 Reentry Phenomenon. The most im-


portant cause of life-threatening cardiac ar-
JY X :a
rhythmias results from a condition known as
Sb' b'
reentry, which occurs when an impulse wave 4-7-
circles back, reenters previously excited tis-
sue, and reactivates these cells. Under normal
conditions, reentry does not occur, as cells be- B (Impeded) I a"
come refractory (unable to accept a signal) for 8

a period of time that is sufficient for the orig-


inal signal to die away. Hence the cells will not
Figure 1.10. Model for reentrant activity. A depo-
contract again until a new impulse emerges larization impulse approaches an obstacle (noncon-
from the SA node. However, there are certain ducting region of the myocardium) and splits into
conditions during which this does not happen, two pathways (A and B) to circumvent the obstacle.
and the impulse continues to circulate. The If pathway B has impeded ability to conduct the
essential condition for reentry to occur in- action potential the following may occur. (1) If im-
volves the development of a cellular refractory pulse B is slowed and arrives at cross junction X
period that is shorter than the conduction ve- after the absolute refractory period of cells depolar-
locity. Consequently, any circumstance that ized by A, the impulse may continue around the
shortens the refractory period or lengthens obstacle as shown by path b andlor follow A along
path b'. In both cases, the impulse is said to be re-
the conduction time can lead to reentry.
flected. (2)If pathway B shows unidirectional block,
Nearly all tachycardias, including fibrilla- impulse A may continue around the obstacle as
tion, are caused by reentry. The length of the shown by path a. If the obstacle is large enough, so
refractory period depends mainly on the rate that cells in cross-region Y are repolarized before
of activation of the potassium current; the the return of a orb, then a circus movement may be
rate of conduction depends on the rate of acti- established. Both (1)and (2) may propagate daugh-
vation of the calcium current in nodal tissue, ter impulses (a"and b") to other parts of the myocar-
and the sodium current in other myocytes. dium. These effects can give rise to coupled beats
The channels controlling these currents are and fibrillation.
the targets for suppressing reentry.
While many conditions can lead to reentry, ized, for example, within the AV node or the
the most common is shown in Fig. 1.10. The end branches of part of the Purkinje system.
conditions needed for this type of reentry are Alternatively, they can be more extensive and
as follows. First, the existence of an obstacle, may give rise to daughter impulses capable of
around which the impulse wave front can spreading to the rest of the myocardium.
propagate, is needed. The obstacle may be in- Unidirectional block occurs in tissue that
fracted or scarred tissue that cannot conduct has been impaired, such that its ability to con-
the impulse. The second condition needed is duct an impulse is completely blocked in one
the existence of a pathway that allows conduc- direction but only slowed in the other. As a
tion at the normal rate around one side of the result of unidirectional block, the impulse can-
obstacle, whereas the other side of the path- not proceed forward along path B (Fig. 1.10),
way is impaired. The impairment may be such and the cells on this path remain in a polarized
that it allows conduction in only one direction state. However, when the impulse traveling
(unidirectional block) or it may allow conduc- along path A reaches a suitable cross-junction
tion to proceed at a greatly reduced rate, such (point X in Fig. 1.10), the impulse proceeds
that when the impulse emerges from the im- back along path a, although at a slower rate
paired tissue, the normal tissue is no longer than normal (indicated by the dotted path
refractory. These pathways are usually local- line, Fig. 1.10). When this impulse reaches an-
5 Antiarrhythrnic Agents

other cross junction 0,it is picked up by path caused by ectopic signals from the ventricles);
A and conducted around the circle. If the ob- and (3) ventricular fibrillation (electrical im-
stacle is large enough, the cells in path A will pulses in the ventricles are fired in a fast and
have repolarized and the path will again be uncontrolled manner, causing the heart to
followed, giving rise to a continuous circular quiver).
movement. When reentry occurs randomly in
5.3 Classification of Antiarrhythmic Drugs
the myocardium, it results in random im-
pulses that lead to cardiac fibrillation. The classification of antiarrhythmic agents is
important for clinical application; however,
5.2 Types of Cardiac Arrhythmias
there is no single classification system that
Arrhythmias can be divided into two catego- has gained universal endorsement. At this
ries-ventricular and supraventricular ar- time, the method proposed by Singh and
rhythmias. Within these two categories, ar- Vaughan Williams (175) continues to be the
rhythmias are further defined by the speed of most enduring classification scheme. Since its
the heartbeats. Bradycardia indicates a very initial conception, this classification method
slow heart rate of less than 60 beatslmin; has undergone several modifications-cal-
tachycardia refers to a very fast heart rate of cium channel blockers have been added as a
more than 100 beatslmin. Fibrillation refers fourth class of compounds (176) and class I
to fast, uncoordinated heartbeats. agents have been subdivided into three groups
Listed below are common forms of arrhyth- to account for their sodium channel blocking
mias grouped according to their origin in the kinetics (177).
heart. Supraventricular arrhythmias include In general, the Singh and Vaughan Wil-
the following: (1) sinus arrhythmia (cyclic liams classification system is based on results
changes in heart rate during breathing); (2) obtained from microelectrode studies con-
sinus tachycardia (the SA node emits impulses ducted on individual heart cells in uitro. Un-
faster than normal); (3) sick sinus syndrome der this system, class I antiarrhythmic agents
(the SA node fires improperly, resulting in ei- include drugs that block sodium channels
ther slowed or increased heart rate); (4) pre- (these compounds have local anesthetic prop-
mature supraventricular contractions (a pre- erties) (178). Compounds in this class are fur-
mature impulse initiation -in the atria causes ther subdivided into three groups: IA, IB, and
the heart to beat prior to the time of the IC (179-181). Class IA drugs are moderately
next normal heartbeat); (5) supraventricular potent sodium channel blockers and usually
tachycardia (early impulse generation in the prolong repolarization; class IB drugs have
atria speed up the heart rate); (6)atrial flutter the lowest potency as sodium channel block-
(rapid firing of signals in the atria cause atrial ers, produce little to no change in action po-
myocardial cells to contract quickly, leading to tential duration, and usually shorten repolar-
afast and steady heartbeat); (7) atrial fibrilla- ization; and class IC drugs, which are the most
tion (electrical impulses in the atria are fired potent of the sodium channel blockers, have
in a fast and uncontrolled manner, and arrive little to no effect on repolarization (182).
in the ventricles in an irregular fashion); and Class I1 drugs act indirectly on the electro-
(8)Wolff-Parkinson-White Syndrome (abnor- physiology of the heart by blocking p-adrener-
mal conduction paths between the atria and gic receptors. Class I11 compounds are agents
ventricles causes electrical signals to arrive in that prolong the duration of the action poten-
the ventricles too early, and subsequently re- tial (increase refractoriness). The mechanism
the atria). of action of these drugs often involves inhibi-
Arrhythmias originating in the ventricles tion of both sodium and potassium channels.
include the following: (1)premature ventricu- Class IV antiarrhythmic agents are calcium
lar complexes (electrical signals from the ven- channel blockers.
tricles cause an early heartbeat, after which The Singh and Vaughan Williams classifi-
the heart seems to pause before the next nor- cation scheme has received broad application
mal contraction of the ventricles occurs); (2) and is used in most textbooks (183-187). In
ventricular tachycardia (increased heart rate fact, based on a Medline search with the key
32 Cardiac Drugs: Antianginal, Vasodilators, and Antiarrhythmics

Table 1.4 Singh


- and Vaughan Williams Classification of Antiarrhythmic Drugs

Class Drum Mechanism of Action


IA quinidine, procainamide, disopyramide Sodium channel blockade, lengthen
refractory period
IB lidocaine, phenytoin, tocainide, mexiletine Sodium channel blockade, shorten
duration of action potential
IC encainide, flecainide, lorcainide, Sodium channel blockade, conduction
moricizine, propafenone slowed
I1 propranolol, esmolol, acebutolol, 1-sotalol Blockade of p-adrenergic receptors, AV
conduction time slowed,
automaticity suppressed
I11 amiodarone, bretylium, sotalol ( d , l ) , Potassium channel blockade,
dofetilide, ibutilide prolonged refractoriness
IV verapamil, diltiazem, bepridil Blockade of slow inward Ca2+
Miscellaneous adenosine, digitoxin, digoxin Miscellaneous

words "antiarrhythmic drug," it was found 4. The metabolites of many of the antiar-
that of 50 consecutive articles (published in rhythmic drugs also contribute to their po-
1998) 56% used the Singh and Vaughan Wil- tency (for example, lorcainide and its me-
liams classification in the title and/or the ab- tabolite, norlorcainide, are both active
stract (188). Table 1.4 lists examples of drugs antiarrhythmic agents).
in each of these classes and summarizes the 5. The classification is based on studies of
mechanisms of action of each class. Note that normal myocardial cells and may not rep-
miscellaneous drugs (189) have been added to resent cell behavior during disease states.
Table 1.4 to account for compounds with
mechanisms of action that do not fit within the 6. The classification may lead to inappropri-
four standard classes. ate administration, because it implies that
The Singh and Vaughan Williams method drugs in the same class have similar favor-
of classification has received strong criticism able and unfavorable effects.
from the Task Force of the Working Group on
Arrhythmias of the European Society of Car- In response to these limitations, a new clas-
diology. In reports published simultaneously sification system, known as the Sicilian Gam-
in Circulation (190) and the European bit, was devised by the Task Force of the
Heart Journal (1911, criticisms were listed in Working Group on Arrhythmias (190, 191).
detail. Key points of contention with regard to This system draws its name from an opening
the Singh and Vaughn Williams classification chess move termed the "Queen's Gambit,"
are listed below: which was designed to provide several aggres-
sive options to the player using it, and as the
1. The classification is incomplete; there is no Task Force was meeting in Sicily at the time,
class for miscellaneous drugs such as this point of origin was also included in the
digoxin and adenosine, which are both clin- title of the classification system. In general,
ically used to treat arrhythmia. the Sicilian Gambit is more flexible than the
2. The effects of a drug in a particular class Singh and Vaughan Williams method and
can result from more than one mechanism takes into account that individual antiar-
(see Table 1.5), and it is difficult to deter- rhythmic agents may have more than one
mine which of the multiple actions are re- mechanism of action. In Table 1.5, antiar-
sponsible for the antiarrhythmic activity. rhythmic agents have been grouped according
3. Antiarrhythmic drugs that are channel to the traditional Singh and Vaughan Wil-
blockers are listed, but channel activators liams method of classification, but also dis-
are not covered. played are the multiple mechanisms of action
5 Antiarrhythmic Agents 33

Table 1.5 Multiple Inhibitory Mechanisms of Antiarrhythmic Drugs


Channels Receptors Pumps

NaK
Fast Med Slow Ca K If a P Mz P ATPase

Procainamide

1111
Propranolol
"'8'i"e;r,).$;a;3;p
:j~;~;;g~~~j~18jzj8j, =

AgonistlAntagonist:

that would clearly lead to substantially differ- describing these agents, but with the caveat
ent clinical effects for compounds within each that this system has limitations.
class, as pointed out by ~ r o ~ o n e nofthe
ts Si-
5.4 perspctive: Treatment of Arrhythmias
cilian Gambit (190).
Criticisms and rebuttals with regard to de- In recent years there have been many changes
velopingan optimal method for classifying an- in the way that arrhythmia is treated; new
tiarrhythmic agents are ongoing (192, 193) technologies, including radiofrequency abla-
and will continue as novel research discoveries tion and implantable devices for atrial and
shed new light on the causes of cardiac ar- ventricular arrhythmias have proven to be re-
rhythmias and the mechanisms of action of markably successful mechanical treatments.
antiarrhythmic drugs. For the scope of this In addition, Cardiac Suppression Trials (CAST)
chapter, the Singh and Vaughan Williams and numerous other studies have provided ev-
method of categorizing antiarrhythmic drugs idence indicating that drugs which act mainly
will serve as a benchmark for organizing and by blocking sodium ion channels--class I
34 Cardiac Drugs: Antianginal, Vasodilators, and Antiarrhythmics

agents under the Singh and Vaughn Williams this class have been observed to have modest
system of classification-may have the poten- effects on repolarization. Drugs in class LA-
tial to increase mortality in patients with quinidine, procainamide, and disopyramide-
structural heart disease (194, 195). Since the have sodium ion channel dissociation rates
CAST results were released, the use of class I that are intermediate between class IB and IC
drugs has decreased, and attention has shifted compounds.
to developing new class I11 agents, which pro- The affinity of the class I antiarrhythmic
long the action potential and refractoriness by agents for sodium channels vary with the state
acting on potassium channels. Of the class I11 of the channel or with the membrane potential
antiarrhythmic agents, amiodarone has been (197). As indicated in Section 3.1, sodium
studied extensively and has proven to be a channels exist in at least three states: R =
highly effective drug for treating life-threat- closed resting, or closed near the resting po-
ening arrhythmias. tential, but able to be opened by stimulation
In addition, new studies have indicated and depolarization; A = open activated, allow-
that combination therapies, for example ad- ing Na+ ions to pass selectively through the
ministration of amiodarone and class I1 membrane; and I = closed inactivated, and un-
p-blockers, or concomitant treatment with im- able to be opened (196). Under normal resting
plantable mechanical devices and drug thera- conditions, the sodium channels are predomi-
pies are effective avenues for treating arrhyth- nantly in the resting or R state. When the
mias. membrane is depolarized, the sodium chan-
These topics are discussed in greater detail nels are active and conduct sodium ions. Fol-
in Section 6. The remainder of Section 5 cov- lowing, the inward sodium current rapidly de-
ers individual antiarrhythmic agents as they cays as the channels move to the inactivated
are categorized under the Singh and Vaughn (I)state. The return of the I state to the R state
Williams classification. is referred to as channel reactivation and is
voltage-and-time-dependent. Class I antiar-
5.5 Class I: Membrane-Depressant Agents
rhythmic drugs have a low affinity for R chan-
Antiarrhythmic agents in this class bind to so- nels, and a relatively high aMinity for both the
dium channels and inhibit or block sodium A and I channels (198).
conductance. This inhibition interferes with An overview of the uses and side effects of
charge transfer across the cell membrane. In- class I antiarrhythmics are displayed in Table
vestigations into the effects of class I antiar- 1.6. In addition to blocking sodium channels,
rhythmics on sodium channel activity have re- the class I compounds have also been observed
sulted in the division of this class into three to effect other ion channels and receptors (Ta-
separate subgroups--referred to as IA, IB, and ble 1.5).
IC (196).
The basis for dividing the class I drugs into 5.5.1 Class IA Antiarrhythmics. Quinidine
subclasses resulted from measured differ- (35) (Fig. 1.11, Table 1.6): Quinidine is the
ences in the quantitative rates of drug binding prototype of the class IA antiarrhythmic
to, and dissociation from, sodium ion channels agents. It is obtained from species of the genus
(196). Class IB drugs, which include lidocaine, Cinchona,and is the D-isomer of quinine. This
tocainide, and mexiletine, rapidly dissociate molecule contains two basic nitrogens: one in
from sodium channels and consequently have the quinoline ring and one in the quinuclidine
the lowest potencies of the class I drugs- moiety. The nitrogen in the quinuclidine moi-
these molecules produce little to no change in ety is more basic. Three salt formulations are
the action potential duration and shorten re- available: quinidine gluconate, quinidine po-
polarization. Class IC drugs, which include en- lygalacturonate (1991, and quinidine sulfate
cainide and lorcainide, are the most potent of (200). Of the three, the gluconate formulation
the class I antiarrhythmics; drugs in this class is the most soluble in water.
display a characteristically slow dissociation Quinidine binds to open sodium ion chan-
rate from sodium ion channels, causing a re- nels, decreasing the entry of sodium into myo-
duction in impulse conduction time. Agents in cardial cells. This depresses phase 4 diastolic
5 Antiarrhythmic Agents

Table 1.6 Class I Antiarrhythmic Agents: Uses and Side Effects


Drug Name Use Side Effects
Class IA
Quinidine (35) Atrial and ventricular tachycardia Hematological, GI, liver
Procainamide (36) Ventricular arrhythmias Hematological, GI, CNS," sensitivity
Disopyramide (37) Ventricular arrhythmias Anticholinergic, hematological
Class IB
Lidocaine (38) Ventricular arrhythmias CNS
Tocainide (39) Ventricular arrhythmias CNS, GI, Hypotension
Mexiletine (40) Ventricular arrhythmias CNS, GI
Phenytoin (41) Improved atrioventricular conduction CNS, gingival hyperpsia, blood
dyscrasias
Class IC
Encainide (42) Ventricular arrhythmias CNS, Ocular
Flecainide (43) Ventricular and supraventricular CNS, Ocular
arrhythmias
Lorcainide (44) Ventricular arrhythmia and CNS, GI
tachycardia, Wolff-Parkinson-white
Syndrome
Propafenone (45) Supraventricular arrhythmias CNS, GI
Moricizine (46) Ventricular arrhythmias CNS, GI, Ocular
"CNS adverse effects include headache, dizziness, nausea, vomiting, diarrhea, flushing, weakness, rash, and syncope.

depolarization (shifting the intracellular primarily metabolized by the liver; a hydroxy-


threshold potential toward zero), decreases lated metabolite, 2-hydroxyquinidine, is equal
transmembrane permeability to the passive in potency to the parent compound (202).
influx of sodium (slowing the process of phase Procainamide (36)(Fig. 1.11, Table 1.6):
0 depolarization, which decreases impulse ve- Procainamide is an amide derivative of pro-
locity), and increases action potential dura- caine. Replacement of the ether oxygen in pro-
tion (201).Physiologically, this results in a re- caine with an amide nitrogen (in procain-
duction in SA node impulse initiation and amide) decreases CNS side effects, rapid
depression of the automaticity of ectopic cells. hydrolysis, and instability in aqueous solution
Qumdine is also thought to ad, at least in part, that results from the ester moiety in procaine.
by binding to potassium channels (Table 1.5). Procainamide is formulated as a hydrochlo-
Quinidineis used to treat supraventricular ride salt of its tertiary amine. The metabolite
and ventricular arrhythmias including atrial of procainamide is N-acetylprocainamide
flutter and fibrillation, and atrial and ventric- (NAPA), which possesses 25% of the parent
ular premature beats and tachycardias. It is drugs activity (203,204).

Quinidine (35) Procainamide (36) Disopyramide (37)


Figure 1.11. Chemical structures of class IA antiarrhythmic agents: sodium channel blockers.
36 Cardiac Drugs: Antianginal, Vasodilators, and Antiarrhythmics

sodium ion channels, decreasing diastolic de-


polarization and prolonging the resting period
(208, 209).
Lidocaine is administered intravenously
for suppression of ventricular cardiac arrhyth-
Lidocaine (38) Tocainide (39) mias and is the prototype compound for class
IB. Its first-pass metabolite, monoethylglycin-
exylidide, results from deethylation of the ter-
tiary amine and is an active antiarrhythmic
agent (210,211).
Tocainide (39) (Fig. 1.12, Table 1.6): To-
cainide is an analogue of lidocaine, but struc-
turally differs in that it possesses a primary,
versus a tertiary, terminal side-chain amine.
Mexiletine (40) Phenytoin (41) In addition, a methyl substituent on the car-
Figure 1.12. Chemical structures of class IB anti- bon atom that is adjacent to the side-chain
arrhythmic agents: sodium channel blockers. amide carbonyl may partially protect this moi-
ety against hydrolysis.
Tocainide has a similar mechanism of ac-
tion to that of lidocaine (212,213). It is orally
Mechanistically, procainamide has the active, and the presence of a primary m i n e
same cardiac electrophysiological effects as allows for formulation as a hydrochloride salt.
quinidine. It decreases automaticity and im- Therapeutically it is used to prevent or treat
pulse conduction velocity, and increases the ventricular tachycardias.
duration of the action potential (205). This Mexiletine (40) (Fig. 1.12, Table 1.6):
compound may be used to treat all of the ar- Structurally, mexiletine resembles lidocaine
rhythmias indicated for treatment with quin- and tocainide in that it contains axylyl moiety.
idine, including atrial flutter and fibrillation, However, it differs in that it possesses a side-
and atrial and ventricular premature beats chain ether versus an amide moiety (as found
and tachycardias. in lidocaine and Cocainide). As a result, me-
Disopyramide (37) (Fig. 1.11, Table 1.6): xiletine is not vulnerable to hydrolysis and has
The electrophysiological effects of this drug a longer half-life than lidocaine (214).
are similar to those of quinidine and procain- Mexiletine possesses a primary m i n e and
amide-decreased phase 4 depolarization and is formulated as a hydrochloride salt that is
decreased conduction velocity (206).This mol- orally active. Its effects on cardiac electro-
ecule contains the ionizable tertiary m i n e physiology are similar to that of lidocaine
that is characteristic of compounds in this (215).It is used in the treatment of ventricular
class, is formulated as a phosphate salt, and is arrhythmias, including ventricular tachycar-
administered both orally and intravenously dias that are life-threatening. However, be-
(207). cause of the proarrhythmic effects of this com-
Because of its structural similarity to anti- pound, it is generally not used with lesser
cholinergic drugs, disopyramide produces side arrhythmias (216).
effects that are characteristic of these com- Phenytoin (41) (Fig. 1.12, Table 1.6): Phe-
pounds, including dry mouth, urinary hesi- nytoin is a hydantoin derivative of the anti-
tancy, and constipation. Clinically it is used to convulsants that does not possess the sedative
treat life-threatening ventricular tachyar- properties of the central nervous system de-
rhythmias. pressants. It is structurally dissimilar to class
I antiarrhythmic compounds and is the only
5.5.2 Class IB Antiarrhythmics. Lidocaine non-basic member of this family of com-
(38)(Fig. 1.12, Table 1.6):Lidocaine is formu- pounds. However, its effects on cardiac cells
lated as a hydrochloride salt that is soluble in are similar to those of lidocaine. Mechanisti-
both water and alcohol. It binds to inactive cally, it depresses ventricular automaticity
5 Antiarrhythmic Agents

Encainide (42) Flecainide (43)

Lorcainide (44) Propafenone (45)

Moricizine (46)
Figure 1.13. Chemical structures of class IC antiarrhythmic agents: sodium channel blockers.

and prolongs the effective refractory period encainide contains a terminal tertiary amine
relative to the action potential duration. It de- and is formulated as a chloride salt. It is
creases the force of contraction, depresses used to treat life-threatening ventricular
pacemaker action, and improves atrioventric- arrhythmias.
ular conduction, especially when adminis- Metabolites of encainide also display activ-
tered in conjunction with digitalis (217). ity. The metabolite ODE, resulting from de-
methylation of the methoxy moiety, is more
5.5.3 Class IC Antiarrhythmics. Encainide potent than encainide (220). A second metab-
(42) (Fig. 1.13, Table 1.6): This compound is a olite, NDE, results from N-demethylation.
benzanilide derivative containing a piperidine Flecainide (43) (Fig. 1.13, Table 1.6): Fle-
ring. Like other class I compounds, it blocks cainide is a benzamidelpiperidine derivative.
sodium channels, depressing the upstroke ve- However. it is structurallv
" dissimilar from en-

locity of phase 0 of the action potential and cainide in that it contains one less benzyl
increasing the recovery period after repolar- group, possesses two lipophilic trifluoroethoxy
ization (218). Encainide has also been shown substituents at the 1 and 4 positions on the
to block the delayed potassium rectifier cur- benzamide ring (versus a single methoxy sub-
rent (219). Like other class I antiarrhythmics, stituent at the 4 position of the benzamide in
Cardiac Drugs: Antianginal, Vasodilators, and Antiarrhythmics

encainide), and lacks a methyl substituent on propafenone decreases the fast inward cur-
the piperidine nitrogen. It is formulated as an rent carried by sodium ions, prolongs the re-
acetate salt, and like encainide, its metabolites fractory period, reduces spontaneous automa-
are active. It possesses cardiac electrophysio- ticity, and depresses triggered activity (227,
logical effects that are similar to those of en- 228).
cainide, i.e., it slows cardiac impulse conduc- Propafenone is indicated in the treatment
tion, and it is used to treat life-threatening of paroxysmal atrial fibrillationhlutter and
ventricular arrhythmias and supraventricular paroxysmal supraventricular tachycardia. It
tachyarrhythmias (221,222). is also used to treat ventricular arrhythmias,
Lorcainide (44) (Fig. 1.13, Table 1.6): Lor- such as sustained ventricular tachvcardias
"
cainide is another benzamidelpiperidine de- that are life-threatening.
rivative in this class. Its mechanism of action Moricizine (46) (Fig. 1.13, Table 1.6): Mori-
is similar to that of encainide-it slows con- cizine is a phenothiazine derivative and is a
duction in myocardial tissue and reduces the structurally unique member of the class IC
speed of depolarization of myocardial fibers, antiarrhythmic agents. Like other agents in
suppressing impulse conduction in the heart this subclass, it decreases the speed of cardiac
(223,224). conduction by lengthening the refractory pe-
Lorcainide is formulated as a hydrochloride riod and shortening the length of the action
salt and is orally active. Metabolism of this period of cardiac tissue (229). Moricizine is
drug results in N-dealkylation of the piperidyl formulated as a hydrochloride salt, and is used
nitrogen to yield norlorcainide (225). This me- to treat life-threatening ventricular arrhyth-
tabolite is as potent as its parent compound, mias.
but its half-life is approximately three times
5.6 Class 11: p-Adrenergic Blocking Agents
longer. Lorcainide is used to treat ventricular
arrhythmia, ventricular tachycardia, and The competitive inhibitors in this class are all
Wolff-Parkinson-White Syndrome. p-adrenergic antagonists that have been
Propafenone (45) (Fig. 1.13, Table 1.6): found to produce membrane-stabilizing or de-
Structurally, propafenone is unlike other com- pressant effects on myocardial tissue at con-
pounds in this class-encainide, flecainide, centrations above normal therapeutic doses.
and lorcainide. Instead, it is an ortho substi- It is hypothesized that their antiarrhythmic
tuted aryloxy propanolamine similar in struc- properties are mainly caused by inhibition of
ture to the major class of p blockers. The race- adrenergic stimulation of the heart (230,231)
mic mixture possesses good Na+ channel by the endogenous catecholamines epineph-
blocking action, whereas the S-(-)-isomer is rine and norepinephrine, which increase the
the potent p blocker. Mechanistically, this slow, inward movement of Ca2+during phase
compound has a direct stabilizing effect on 2 of the action potential. The principle electro-
myocardial membranes, which manifests in a physiological effects of the blocking agents
reduction in upstroke velocity (phase 0) of the manifest as a reduction in the phase 4 slope
action potential (226). In Purkinje fibers, potential of sinus or ectopic pacemaker cells,
and to a lesser extent myocardial fibers, which decreases heart rate and slows ectopic

Table 1.7 Class I1 Antiarrhythmic Agents: Uses and Side Effects


Drug Name Use Side Effects
Propranolol(47) Cardiac arrhythmias Cardiovascular, CNSa
Nadolol(48) Atrial fibrillation Cardiovascular, CNS
1-Sotalol(49) Ventricular arrhythmias Arrhythmogenic, cardiovascular, CNS
Atenolol (50) Atrial fibrillation CNS, GI
Acebutolol(51) Ventricular arrhythmias CNS, GI
Esmolol(52) Supraventricular tachyarrhythmias CNS, GI
Metoprolol(53) Atrial tachyarrhythmias Cardiovascular, CNS
"CNSadverse effects include headache, dizziness, nausea, vomiting, diarrhea, flushing, weakness, rash, and syncope.
5 Antiarrhythrnic Agents

Propranolol (47) Nadolol (48)

Sotalol (49) Atenolol (50)

0 A.CH3
Acetobutolol (51)

CH3

Esmolol (52) Metoprolol (53)


Figure 1.14. Chemical structures of class 11 anti.arrhythmicagents: P-adrenergicblocking agents.

tachycardias. A list of compounds in the class, receptors. Sotalol differs from other members
along with uses and side effects are shown in of this class in that it lacks the -OCH,-
group. This results in a shortening of the char-
With the exception of sotalol (2321, the acteristic ethylamino side-chain (Fig. 1.14).
compounds in this class are all structurally Propranolol(47) is the prototype agent for
similar agents known as aryloxypropano- this class of compounds. Because of the substi-
lamines (Fig. 1.14). This name originates from tution pattern on its aromatic ring, it is not a
the presence of an --OCH,- group located selective P-adrenergic blocking agent (Fig.
between a substituted benzene ring and an 1.14). During propranolol-mediated P-recep-
ethylamino side-chain. The aromatic ring and tor block, the chronotropic, ionotropic, and va-
substituents are the primary determinants sodilator responses to P-adrenergic stimula-
. Substitution of the tion are decreased. Propranolol exerts its
para position of the benzene ring, in tandem antiarrhythmic effects in concentrations asso-
with the absence of meta position substitu- ciated with P-adrenergic blockade, and this
tion, seems to confer selectivity for p l cardiac seems to be its principal antiarrhythmic
40 Cardiac Drugs: Antianginal, Vasodilators, and Antiarrhythmics

Table 1.8 Class I11 AntiarrhHhmic Agents: Uses and Side Effects
Drug Name Use Side Effects
Sotalol (d, 1) (49) Ventricular arrhythmias Arrhythmogenic, cardiovascular,
CNSa
Amiodarone (54) Ventricular arrhythmias Pulmonary toxicity
Bretylium (56) Ventricular arrhythmias and ventricular Cardiovascular, arrhythmogenic,
fibrillation CNS, GI
Ibutilide (57) Atrial fibrillation, ventricular tachycardia Arrhythmogenic, slowed heart
rate, heart failure
Dofetilide (59) Ventricular arrhythmias, atrial Arrhythmogenic, tores de pointes,
fibrillation and flutter CNS, GI
Azimilide (60) Supraventricular tachycardia, atrial
flutter
-

"CNS adverse effects include headache, dizziness, nausea, vomiting, diarrhea, flushing, weakness, rash, and syncope.

mechanism of action (233). It has also been uses and some side effects are shown in Table
shown to possess membrane-stabilizing activ- 1.8.
ity that is similar to quinidine and other anes- Racemic sotalol, dofetilide, and ibutilide
thetic-like drugs. However, the significance of are potassium channel blockers. Potassium
this membrane action in the treatment of ar- channels, particularly the channel giving rise
rhythmias is uncertain, as the concentrations to the "delayed rectifier current," are acti-
required to produce this effect are greater vated during repolarization phase 3 of the ac-
than required for the observance of its tion potential. Sotalol also possesses p-adren-
P-blocking effects. ergic blocking properties (see above), whereas
Nadolol(48) (234) and L-Sotalol (49) (235) ibutilide is also a sodium channel blocker. The
are both nonspecific /.3 blockers (Fig. 1.14), mechanisms of action of amiodarone and bre-
whereas para substitutions on the aromatic tylium, which also prolong the action poten-
rings of atenolol (50) (2361, acetobutolol (51) tial, remain unclear. However, both have so-
(237), esmolol(52) (238), and metoprolo1 (53) dium channel-blocking properties.
(239) all confer P, antagonist selectivity (Fig. Of the compounds listed in this class, so-
1.14). Each of these agents exerts electrophys- talol, dofetilide, and ibutilide are structurally
iological effects that result in slowed heart similar (Fig. 1.15). All three drugs contain a
rate, decreased AV nodal conduction, and in- central aromatic ring with a sulfonamide moi-
creased AV nodal refractoriness. ety and a para-substituted alkylamine side-
chain. Dofetilide, unlike sotalol and ibutilide,
5.7 Class Ill: Repolarization Prolongators
is nearly symmetrical, with two methanesul-
The drugs in this class-amiodarone, brety- fonamides at either end of the molecule.
lium, dofetilide, ibutilide, and (D,L) or racemic Amiodarone (54) (Fig. 1.15, Table 1.8):
sotalol-all produce electrophysical changes Amiodarone is structurally unique in this
in myocardial tissue by blocking ion channels; class, and because it has received a great deal
however, some are selective, whereas others of attention over the past several years for its
are multi-channel blockers (this is not surpris- ability to treat arrhythmias, it is considered to
ing, because there is a high degree of sequence be the prototype compound for this class. Ami-
homology between the different ion channels). odarone is currently the most used drug for
Importantly, all class I11 drugs have one com- patients with life-threatening arrhythmias-
mon e f f e c t t h a t of prolonging the action approximately one-half of the patients cur-
potential, which increases the effective refrac- rently receiving antiarrhythmic drug therapy
tory period without altering the depolariza- are treated with amiodarone (241).
tion or the resting membrane potential (240). It is a benzofuranyl derivative with a cen-
A list of compounds in this class, along with tral diiodobenzoyl substituent and an alkyl
42 Cardiac Drugs: Antianginal, Vasodilators, and Antiarrhythmics

amine side-chain. Mechanistically, this mole- ternary ammonium salt. It is formulated as a


cule prolongs the duration of the action poten- tosylate salt and is soluble in water and
tial and effective refractory period, with min- alcohol. It is administered by intravenous or
imal effect on resting membrane potential intramuscular injection, and is used to treat
(242-244). Amiodarone exhibits mechanisms ventricular fibrillation and ventricular ar-
of activity from each of the four Singh and rhythmias that are resistant to other therapy.
Vaughan Williams classes. In addition, it also The mechanism of antiarrhythmic action
displays non-competitive a-and P-adrenergic of this drug has not been determined. It does
inhibitory properties. It is effective in the not suppress phase 4 depolarization, but it
treatment of life-threatening recurrent ven- does prolong the effective refractory period
tricular arrhythmias and atrial fibrillation
(254). This compound also selectively accumu-
(245) and is orally available as a chloride salt.
lates in neurons and inhibits norepinephrine
Amiodarone contains two iodine substitu-
release, and it has been suggested that its ad-
ents, and consequently does effect thyroid hor-
mones (246). However, its most serious side renergic neuronal-blocking properties are re-
effects involve both the exacerbation of ar- sponsible for its antiarrhythmic activity (255).
rhythmias and pulmonary toxicity. Ibutilide (57) (Fig. 1.15, Table 1.8): Ibutil-
A new, experimental noniodinated benzo- ide is formulated as a fumarate salt and is ad-
furanyl derivative of amiodarone, dronedarone ministered by intravenous injection (256).
(55) (247, 2481, has emerged as a potential This agent prolongs repolarization of cardiac
new member of the class I11 antiarrhythmics. tissue by increasing the duration of the action
It has been found to have similar electrophys- potential and the effective refractory period in
iological effects as amiodarone, but with fewer cardiac cells. It blocks both sodium and potas-
side effects (249). sium channels (257-2591, but unlike sotalol,
(D,L)-Sotalol (49) (Fig. 1.15, Table 1.8): So- does not possess P-adrenergic blocking activ-
talol has been classified as both a class I1 and a ity. Ibutilide is used in the treatment of su-
class I11antiarrhythmic agent. The L isomer is praventricular tachyarrhythmias, such as
classified as a P blocker and is 50 times more atrial flutter and atrial fibrillation (260, 261).
active than the D isomer in this capacity; the However, it may cause ventricular arrhyth-
racemic mixture of this drug is considered to mias and is not recommended for the treat-
be a class I11agent, because it inhibits the rap- ment of this condition.
idly activating component of the potassium Trecetilide (58),a congener of ibutilide, is
channel involved in the rectifier potassium currently being investigated for intravenous
current. and oral treatment of atrial flutter and atrial
Sotalol is used to treat and prevent life- fibrillation. In addition to blocking potassium
threatening ventricular arrhythmias (250). channel receptors, this compound also seems
Additionally, because of its class I1 and I11 ac- to prolong repolarization through other mech-
tivity, it is also effective against supraventric- anisms as well.
ular arrhythmias (251). The mode of action, Dofetilide (59) (Fig. 1.15, Table 1.8):
pharmacokinetics, and therapeutic uses of so- Dofetilide is one of the newest members of the
talol have been reviewed extensively, and in class I11 antiarrhythmic agents and has re-
1993, a report on this drug was published cently received U.S. FDA approval. It prolongs
(252). repolarization and refractoriness without af-
Sotalol is formulated as a hydrochloride fecting cardiac conduction velocity, and is a
salt and can be administered orally. In terms relatively selective blocking agent of the de-
of efficacy, clinical trials have indicated that layed rectifier potassium current (262, 263).
this compound is at least as effective or more Unlike ibutilide and sotalol, this agent does
effective in the management of life-threaten- not inhibit sodium channels or p-adrenergic
ing ventricular arrhythmia than other avail- receptors.
able drugs (253). It is used to treat supraventricular tachyar-
Bretylium Tosylate (56) (Fig. 1.15, Table rhythmias and to restore normal sinus
1.8): Bretylium tosylate is a bromobenzyl qua- rhythm during atrial fibrillation and atrial
5 Antiarrhythmic Agents

Table 1.9 Class IV Antiarrhythmic Agents: Uses and Side Effects


Use Side Effects
Bepridil(8) Supraventricular arrhythmias GI, CNS," cardiovascular
Diltiazem (9) Supraventricular arrhythmias GI, CNS, cardiovascular
Verapamil(11) Supraventricular arrhythmias GI, CNS, hepatic
"CNSadverse effects include headache, dizziness, nausea, vomiting, diarrhea, flushing, weakness, rash, and syncope.

flutter (264,265). Dofetilide is formulated as a the propagation of an electrical impulse


hydrochloride salt and is administered orally. through the AV node (270). By decreasing this
Azimilide (60) (Fig. 1.15, Table 1.8): This influx, the calcium channel blockers slow con-
agent is a novel class I11 antiarrhythmic agent duction. This, in turn, slows the ventricular
that has been shown to block both the slow rate. Furthermore, many of the calcium chan-
activating and rapidly activating components nel blockers also have the ability to block so-
of the delayed rectifier potassium current dium channels, which is not surprising be-
(266).Structurally it is unlike other molecules cause of the close homologies in the amino acid
in this class, containing both imidazolidione sequences of calcium channels and sodium
and piperazine moieties. Azimilide has re- channels.
cently received much attention and is being Drugs in this class also decrease SA node
evaluated for its ability to treat atrial flutter, automaticity, depress myocardial contractil-
atrial fibrillation, and paroxysmal supraven- ity, and reduce peripheral vascular resistance.
tricular tachycardia (267-269). The prototype drug in this class is verapamil.
A list of uses and side effects for compounds in
5.8 Class IV: Calcium Channel Blockers
this class are shown in Table 1.9.
All of the calcium channel blockers in this Verapamil(l1) (Fig. 1.16, Table 1.9): Vera-
class of agents-verapamil, diltiazem, and be- pamil blocks the influx of calcium ions across
pridil-also possess antianginal activity and cell membranes (271). Structurally it is not
are also covered in detail under antianginal related to other antiarrhythmic drugs. It is
agents and vasodilators in this chapter. With formulated as a hydrochloride salt and is
respect to cardiac arrhythmias, these agents readily soluble in water. Verapamil is used to
affect calcium ion flux, which is integral for treat supraventricular arrhythmias including

Diltiazem (9)
Bepridil (8)

Figure 1.16. Chemical structures of class IV antiarrhythmic agents: calcium channel blockers.
44 Cardiac Drugs: Antianginal, Vasodilators, and Antiarrhythmics

Adenosine (61) Digoxin (62)

Figure 1.17. Chemical structures of miscellaneous antiarrhythmic agents.

atrial tachycardias and fibrillations (272) and diltiazem, it slows the heart rate by prolonging
is administered both orally and through intra- both the effective refractory periods of the
venous injection. atria and ventricles, and the refractory period
The mechanism of action of this compound of the AV node (275).
arises from the blockade of calcium ion chan- It is formulated as a hydrochloride salt and
nels, which inhibits the influx of extracellular is orally available. It is used in treating AV
Ca2+ across the cell membranes of myocardial
- reentrant tachyarrhythmias and in the man-
cells and vascular smooth muscle cells. Its ac- agement of high ventricular rates secondary
tivity is voltage dependent, with receptor af- to atrial flutter or fibrillation (276,277).
finity increasing as the cardiac cell membrane 5.9 Miscellaneous Antiarrhythmic Agents
potential is reduced; and frequency depen-
dent, with an increase in aMinity resulting Two antiarrhythmic agents that do not fall
from an increase in the frequency of depolar- within the Singh and Vaughan Williams clas-
izing stimulus. sification are shown in Fig. 1.16 and briefly
Verapamil is rapidly metabolized to at least described in the text.
12 dealkylated metabolites. Norverapamil, a Adenosine (61) (Fig. 1.17): Adenosine is
major and active metabolite, has 20% of the chemically unrelated to other antiarrhythmic
cardiovascular activity of verapamil, and drugs. It is soluble in water but practically in-
reaches .~ l a s m aconcentrations that are al- soluble in alcohol. For the treatment of ar-
most equal to those of verapamil within 4-6 h rhythmias, it is administered through intrave-
after administration. nous injection.
Diltiazem (9) (Fig. 1.16, Table 1.9): Dilti- Adenosine reduces SA node automaticity,
azem is a benzothiaze~ine. derivative and is slows conduction time through the AV node,
formulated as a hydrochloride salt. It may be and can interrupt reentry pathways. It is used
administered orally or through injection. to restore normal sinus rhythm in patients
Similar to verapamil, diltiazem inhibits the with paroxysmal supraventricular tachycar-
influx of Ca2+ during the depolarization of dia, including Wolff-Parkinson-White Syn-
cardiac smooth muscle. This decreases atrio- drome (278-281).
ventricular conduction and prolongs the re- Digoxin (62) (Fig. 1.17):Digoxin belongs to
fractory period. Therapeutically, this drug the family of compounds known as the cardiac
prolongs AV nodal refractoriness and exhibits glycosides. The natural glycosides are isolated
effects on AV nodal conduction such that the from various plant species: Digitalis purpurea
heart rate during tachycardias is reduced. Dil- Linne, Digitalis lanata Ehrhart, Strophan-
tiazem also slows the ventricular rate during thus gratu, or Acokanthea schimperi.
atrial fibrillation or atrial flutter (273-274). Digoxin inhibits sodium-potassium ATPase,
Bepridil (8) (Fig. 1.16, Table 1.9): Bepridil which is responsible for regulating the quan-
inhibits the transmembrane influx of Ca2+ tity of sodium and potassium inside cells. In-
into cardiac and vascular smooth muscle. Like hibition of this enzyme results in an increase
6 Future Trends and Directions 45

in the intracellular concentration of sodium (287), which act by prolonging the action po-
and calcium and a decrease in intracellular po- tential duration and the refractory period.
tassium. Because of decreased intracellular Class I11 agents lack many of the negative side
potassium concentrations, phase 4 of the ac- effects observed in other classes of antiar-
tion potential becomes more positive, which rhythmics, affect both atrial and ventricular
in turn reduces the height of phase 0. As a tissue, and can be administered orally or intra-
result, the conduction rate in cardiac cells is venously. Members of this class, such as ami-
slowed-SA node discharge and AV node con- odarone (which has proven to be a clinically
duction are slowed. It is available both orally efficient therapeutic for the treatment of a
and through intravenous injection and is used wide variety of arrhythmias) and racemic so-
to treat and prevent sinus and supraventricu- talol, have been the center of much attention
lar fibrillation, flutter, and tachycardia (282). in recent years and have led to the search for
new class I11 drugs with improved safety pro-
6 FUTURE TRENDS AND DIRECTIONS files (288). New and investigational class I11
agents that are more selective for potassium
6.1 Antiarrhythmics: Current channel subtypes include azimilide (2891,
and Future Trends dofetilide, dronedarone, ersentilide, ibutilide,
Following the discovery that lidocaine was tedisamil, and trecetilide (290).
useful for treating cardiac arrhythmias, early There have also been numerous reports on
drug discovery and development of antiar- the synthesis and evaluation of new antiar-
rhythmic agents focused on compounds that rhythmic compounds; several of these are
were structurallv" similar to lidocaine and -vos- briefly described: Matyus et al. (291) have re-
sessed similar mechanisms of action-that of ported the synthesis and biological evaluation
blocking sodium channels. This led to the ini- of novel phenoxyalkyl amines that exhibit
tial identification of lidocaine congeners such both class IB and class I11 type electrophysio-
as tocainide and mexiletine, and later to en- logical properties; Tripathi et al. (292) have
cainide and flecainide. The long standing hy- performed synthesis and SAR studies on
potheses for treating arrhythmias with so- 1-substituted-N-(4-alkoxycarbonylpiperidin-
dium ion channel blockers was based on the 1-yl)alkanes that showed potent antiarrhyth-
belief that these molecules could effectively mic activity comparable with quinidine; Bodor
prevent or suppress the onset of arrhythmias et al. (293) reported a novel tryptamine analog
andlor terminate this condition when it be- that was found to selectively bind to the heart
came persistent (283). However, the Cardiac (and within the heart to have tissue specific-
Arrhythmia Suppression Trial (CAST) (2841, ity) and to possess effects on vital signs of the
which evaluated the effects of well-established cardiovascular system that indicated antiar-
sodium channel blockers on mortality in post- rhythmic activity; Morey et al. (294) designed
myocardial patients (with frequent premature a series of amiodarone homologs that resulted
ventricular arrhythmias), dispelled this hy- in an SAR that will have implications for the
pothesis. In fact, these studies found that both future development of amiodarone-like anti-
encainide and flecainide increased mortality. arrhythmic agents; Himmel et al. (295) syn-
Since the CAST studies, other trials with thesized and evaluated the activities of thia-
mexiletine, propafenone, and moricizine (285) diazinone derivatives that are potent and
(CAST 11) (286) have also shown similar re- selective for potassium ion channels and show
sults, and a correlation between increased class I11 antiarrhythmic activity; Thomas et
mortality and the use of sodium ion channel al. (296) have developed a novel antiarrhyth-
blocking agents in post-myocardial infarction mic agent-BRL-32872-that inhibits both
patients has been established. potassium and calcium channels; and Levy et
ed on the findings of CAST and related al. (297) have described novel dibenzoazepine
, the treatment of antiarrhythmias has and 11-0x0-dibenzodiazepinederivatives that
away from class I sodium channel are effective ventricular defibrillating drug
ockers and now focuses on class I11 drugs candidates.
46 Cardiac Drugs: Antianginal, Vasodilators, and Antiarrhythmics

Along with advances in the understanding major genetic and biochemical regulators of
and development of new therapeutic agents, apoptosis in the heart. There has been a quest
the development of technological devices to for a therapeutic agent that would delay the
treat arrhythmias has also evolved. One of the onset of apoptosis in the ischemic heart. In the
most important achievements has been the future, several therapeutic interventions can
implantable cardioverter defibrillator (ICD) be developed to prolong the survival of smooth
(241). This device has been an option for treat- muscle and endothelial cells and to enhance
ing arrhythmias since the early 1980s, and in the vascular contractibility, tone, and eventu-
the treatment of ventricular tachycardia and ally delay the process of atherosclerosis (299,
fibrillation, no other therapy has been as effec- 300).
tive in prolonging patient survival (283).How- Elucidation of the phenomenon of myocar-
ever, an important point regarding ICD treat- dial preconditioning may hold the key to the
ment is that it is often used in combination development of a drug to the treatment of
with antiarrhythmic drug therapy (241). For ischemic heart disease (301).
frequent symptomatic episodes of ventricular NO is a unique moiety implicated in the
tachycardia, administration of an adjuvant regulation of various physiological processes,
drug therapy is often required to provide max- including smooth muscle contractibility and
imum prevention and treatment of life-threat- platelet reactivity. Consequently, it has been
ening arrhythmias. In particular, combination suggested that NO may have a significant car-
therapy with ICD and both P blockers and dioprotection role in hypercholesterolemia,
amiodarone have received the most attention atherosclerosis, hypertension, or inhibition of
(241). platelet aggregation. As a result, the develop-
Finally, new evidence suggests that combi- ment of selective NO synthase inhibitors will
nations of therapeutics may be more effective address potential beneficial therapeutic out-
at treating and controlling arrhythmias than comes of NO modulation to the pathophysiol-
using any single agent alone (288). In particu- ogy of these disorders (302, 303).
lar, clinical sources have indicated that the Over the past few years, a number of potent
pharmacological properties of amiodarone and asymmetric aza analogs of dihydropyrimidine
p blockers may be additive or even synergistic (DHPM), possessing a similar pharmacologi-
for treating arrhythmias (288). Details of the cal profile to classical dihydropyridine calcium
analysis of amiodarone interaction with p channel blockers, have been studied exten-
blockers in the European Myocardial Infarct sively to evaluate their molecular interactions
Amiodarone Trial (EMIAT) and in the Cana- at the receptor level. Some of the lead com-
dian Amiodarone Myocardial Infarction Trial pounds (SQ 32926 or SQ 32547) are superior
(CAMIAT) have recently been reported (298). in potency and duration of antihypertensive
Data from randomized patients in these trials activity to classical DHP analogs and compare
were analyzed by multivariate proportional favorably with second generation drugs such
hazard models and indicated that combination as nicardipine and amlodipine. This class of
therapy consisting of amiodarone and p-block- compounds (DHPM) might be the next gener-
ers led to a significantly better survival rate. ation of calcium channel blockers for the treat-
Hence, the possibility of administering combi- ment of cardiovascular diseases (304).
nation therapies will be an important aspect in Clinical administration of drugs with neg-
the future development of therapeutic tech-
-
ative inotropic activity is not desirable be-
niques for treating arrhythmias. cause of their cardiosuppressive effects, espe-
cially in patients with a tendency toward heart
6.2 Antianginal Agents and Vasodilators:
failure. Therefore, there has been a search for
Future Directions
cardioprotective agents acting through en-
There is increasing evidence of a relationship tirely different mechanisms. It has been sug-
between apoptosis and pathophysiology in gested that re-evaluation of dihydropyridine
both ischemic and nonischemic cardiomyopa- calcium channel blockers might lead to the
thies, and a large number of papers published discovery of therapeutic agents that also have
since 1997 suggest a link between some of the effects on other membrane channels. Efo-
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CHAPTER TWO

Diuretic and Uricosuric Agents


CYNTHIA A. FINK
JEFFREY M.MCKENNA
LINCOLNH. WERNER
Novartis Biomedical Research Institute
Metabolic and Cardiovascular Diseases Research
Summit, New Jersey

Contents
1 Introduction, 56
1.1 Pharmacological Evaluation of Diuretics, 62
1.2 Clinical Aspects of Diuretics, 62
2 Clinical Applications, 63
2.1 Current Drugs, 63
2.1.1 Osmotic Diuretics, 63
2.1.2 Mercurial Diuretics, 64
2.1.2.1 Pharmacology, 65
2.1.2.2 History, 65
2.1.2.3 Structure-Activity Relationship,
65
2.1.3 Carbonic Anhydrase Inhibitors, 66
2.1.3.1 History, 67
2.1.3.2 Pharmacology, 68
2.1.3.3 Structure-Activity Relationship,
69
2.1.4 Aromatic Sulfonamides, 70
2.1.4.1 Mefruside, 72
2.1.5 Thiazides, 73
2.1.5.1 Pharmacology, 73
2.1.5.2 History, 73
2.1.5.3 Structure-Activity Relationship,
73
2.1.6 Hydrothiazides, 78
2.1.6.1 Pharmacology, 78
2.1.6.2 History, 80
2.1.6.3 Structure-Activity Relationship,
80
2.1.7 Other Sulfonamide Diuretics, 81
2.1.7.1 Chlorothalidone, 81
2.1.7.2 Hydrazides of m-
Sulfarnoylbenzoic Acids, 81
Burger's Medicinal Chemistry and Drug Discovery 2.1.7.3 1-Oxoisoindolines,84
Sixth Edition, Volume 3: Cardiovascular Agents and 2.1.7.4 Quinazolinone Sulfonamides,
Endocrines 85
Edited by Donald J. Abraham 2.1.7.5 Mixed Sulfamide Diuretic-
ISBN 0-471-37029-0 O 2003 John Wiley & Sons, Inc. Antihypertensive Agents, 86
55
Diuretic and Uricosuric Agents

2.1.7.6 Tizolemide, 87 2.1.10 Aldosterone Biosynthesis Inhibitors,


2.1.7.7 Bemitradine, 89 120
2.1.8 High Ceiling Diuretics, 89 2.1.11 Cyclic Polynitrogen Compounds, 121
2.1.8.1 Ethacrynic Acid, 89 2.1.11.1 Xanthines, 121
2.1.8.2 Indacrinone, 92 2.1.11.2 Aminouracils, 122
2.1.8.3 Other Aryloxy Acetic Acid High 2.1.11.3 Triazines, 123
Ceiling Diuretics, 93 2.1.12 Potassium-Sparing Diuretics, 125
2.1.8.4 Furosemide, 94 2.1.12.1 Triamterene, 125
2.1.8.5 Bumetanide, 95 2.1.12.2 Other Bicyclic Polyaza
Diuretics, 128
2.1.8.6 Piretanide, 103
2.1.12.3 Amiloride, 130
2.1.8.7 Azosemide, 104
2.1.12.4 Azolimine and Clazolimine,
2.1.8.8 Xiparnide, 104
132
2.1.8.9 Triflocin, 107 2.1.13 Atrial Natriuretic Peptide, 133
2.1.8.10 Torasemide, 107 2.1.13.1 ANP Clearance Receptor
2.1.8.11 Muzolimine, 108 Blockers, 134
2.1.8.12 MK 447, 108 2.1.13.2 Neutral Endopeptidase
2.1.8.13 Etozolin, 109 Inhibitors, 134
2.1.8.14 Ozolinone, 110 2.1.14 Uricosuric Agents, 138
2.1.8.15 Pharmacology of High Ceiling 2.1.14.1 Sodium Salicylate, 138
Diuretics, 110 2.1.14.2 Probenecid, 139
2.1.9 Steroidal Aldosterone Antagonist, 111 2.1.14.3 Sulfinpyrazone, 140
2.1.9.1 Pharmacology, 112 2.1.14.4 Allopurinol, 141
2.1.9.2 History and Structure-Activity 2.1.14.5 Benzbromarone, 141
Relationship, 114 3 Conclusion, 142

1 INTRODUCTION nective tissues. Hyperuricemia is an adverse ef-


fect sometimes observed with diuretic treat-
Diuretics are among the most frequently pre- ment and arises from decreased extracellular
scribed therapeutic agents for the treatment of volume and increased urate reabsorption. To-
edema, hypertension, and congestive heart fail- day, a heterogeneous array of diuretic com-
ure and act primarily by inhibiting the reabsorp- pounds possessing different structures and sites
tion of sodium ions from the renal tubules in the of action are available for safe and effective
kidney. This diverse class of therapeutic agents treatment of edema and cardiovascular diseases
includes o r g a n o m e r d s , polyols, sugars, thia- including compounds that have combined di-
zides, phenoxyacetic acids, arninomethylphe- uretic and uricosuric properties. This modern
nols, xanthines, aromatic sulfonamides, pteri- era of diuretic therapy began in 1949, when sul-
dines, pyrazines, and steroids. These agents fanilamide was discovered to possess diuretic
have been classified in a variety of ways includ- and natriuretic properties (1).Since the 1950s,
ing; chemical structure, mechanism of action, significant advances have been made in the dis-
tubular site of action, magnitude of natriuretic covery of new diuretic agents and their precise
effect, and by their effect on electrolyte deple- cellular mechanism of action.
tion. Today no single classification system is The kidneys are the principal organs of ex-
commonly used; however, diuretics are often cretions within the body and perform three
grouped as loop, potassium-sparing, thiazide, major functions in maintaining homeostasis:
and osmotic diuretics.
Uricosuric agents increase the excretion of 1. Remove water, electrolytes, products of
uric acid, one of the principal products of purine metabolic waste, drugs, and other materi-
metabolism. These compounds are used in the als from the blood.
treatment of gout, a condition in which plasma 2. Possess endocrine functions; that is, secrete
levels of uric acid are elevated and, as a result, erythropoietin, renin, and 1,2,5-hydroxy-
deposits of crystalline sodium urate form in con- cholecalciferol.
1 Introduction

Proximal Distal

+ Na+ CI-
isotonic

Absence

Hypotonic
urine
- - - ----

Hypertonic
urine

Collecting
hente duct

Figure 2.1. Major transport mechanisms in the apical membrane of the tubule cells along the
nephron. G, glucose; AA, amino acids; ADH, antidiuretic hormone; ALDO, aldosterone (5).

3. Selectively reabsorb water, electrolytes, The Bowman's capsule surrounds the cap-
and needed nutrients from the urine. illary network of the glomerulus and its func-
tion is to collect the filtrate. An estimated 180
Together the kidneys weigh about 300 g, L of glomerular filtrate forms daily, which is
which is about 0.4% of the total body weight. about 60 times the total plasma content (4).
The kidneys can be divided into three major Fortunately, the reabsorption process begins
regions: the pelvis, the cortex, and the me- immediately. Approximately 99% of the water
dulla. The working unit of the kidney is the and electrolytes are reabsorbed in the renal
nephron, with each kidney containing about tubules. The glomerular filtrate is composed
1.2 million such structural units (2). There are of water, electrolytes (NH,', Na+, K t , Ca2+,
two types of nephrons: the cortical nephrons Mg2+, C1-, and HP0,2-), glucose, amino ac-
and the juxtamedullary nephrons, with about ids, and nitrogenous wastes of metabolism. It
% of the nephrons found in the human kid- actually has a profile similar to that of blood
ney being cortical nephrons (3). The funda- plasma, except it contains no blood cells and
ental components that make up the nephron little or no plasma proteins. Reabsorption of
the glomerulus and the tubules. The glo- the water and solutes occurs through the walls
merulus is composed of a convoluted capillary of the proximal and distal convoluted tubules,
network that is joined with connective tissue. in the loop of Henle, and in the collecting tu-
The diameter of the afferent arterioles is bules by active and passive transport systems
than the efferent arterioles and as a (Fig. 2.1) (5). From the use of micropuncture
the glomerular filtration pressure is es- and isolated tubule techniques, much is
ed to be about 50 mm of mercury. This known about the cellular and molecular mech-
facilitates the rapid clearance of water and a anism of tubular reabsorption. Each particu-
variety of low to medium molecular weight sol- lar segment of the nephron possesses its own
tes from the blood. characteristic ion-transport systems. In gen-
Diuretic and Uricosuric Agents

Luminal %
membraYaT, Na+ Basolateral
membrane

X = amino acids
glucose, phosphate
CA = carbonic anhydrase
HO
, + C02

Urine ( 1 Blood
Figure 2.2. Cell model of the proximal tubule.

eral, these cells all contain a rate-limiting so- luminal fluid). Carbonic anhydrase in the cy-
dium entry system on the luminal membrane, toplasm indirectly catalyzes the intracellular
which is coupled to a Na+/K+-ATPaseon the formation of protons, which keeps the Na+l
basolateral membrane for sodium removal. H+exchanger active. These excreted protons
The reabsorption process begins in the also neutralize the bicarbonate in the tubule
proximal tubules. Approximately 50-55% of to form carbonic acid. Carbonic anhydrase lo-
the filtered sodium and water along with cated in the luminal brush border dehvdrates
about 90% of the filtered amino acids, bicar- the carbonic acid to form carbon diogde and
bonate, glucose, and phosphate are reclaimed water.
here (Fig. 2.2) (5a). Glucose, phosphate, and As mentioned, chloride ion is removed from
the amino acids enter proximal tubule cells the proximal tubule by passive and active
through electrogenic cotransport with so- transport systems. As solutes are removed
dium. The major route of sodium reentry into from the tubule, the osmotic gradient facili-
this tubule cell is the Na+/H+exchanger. This tates the reabsorption of water. This effec-
transport system is also responsible for most tively increases the concentration of chloride
of the proximal tubular reabsorption of bicar- ion above that found in the lateral intercellu-
bonate and creates a favorable gradient that lar space. This space is permeable to chloride,
allows for both the active and passive trans- and it is passively absorbed across the junction
port of about 50% of the filtered chloride ion (8). Chloride can also enter the cell through a
(6). The Na+/H+ exchanger has recently been chloride-formate exchanger (Fig. 2.3) (9, 10)
cloned by a gene-transfer approach (7). A Na+l The basolateral membrane is also believed tc
K'-ATPase pump located on the basolateral contain a Naf l HC0,- cotransporter (11).
side of the proximal cell removes the sodium to About 35-40% of the filtered sodium is re
maintain a low intracellular sodium concen- absorbed in the loop of Henle. The major lu
tration (approximately one-tenth that of the mind transporter of sodium in this region o
Lumen Cell Bath

~ f ) r Kn+ A ) ~
Na+ rn 3 Na+ rn
ATP
2K+

a D
ci- rn
CI-
I - 1
Figure 2.3. Cell model of the chloride-formateexchanger in the proximal tubule. [After G. Giebisch,
J. Clin. Invest., 79,32 (19871.1

the renal tubule is the Na+/K+/2ClPelectro- citriol are the main mediators of calcium reab-
neutral cotransporter located in the thick as- sorption. They both increase distal calcium re-
cending region of the loop. The energy that absorption, although the exact mechanism is
drives the cotransporter arises from a concen- not well understood (13). Calcium is believed
tration gradient generated from the Na+/K+- to exit the cell through a Ca2+/ATPase or a
ATPase pump located on the basolateral mem- Na+/ Ca2+exchanger on the basolateral mem-
brane (Fig. 2.4). Chloride exits the basolateral brane (14, 15).
side through a chloride channel and/or electro- The collecting tubule is the last section of
neutral KC1 cotransporter (12). The potas- the renal tubule in which filtrate modification
sium that enters the cell through the Na+/ occurs. This region is responsible for 2-3% of
Kf/2C1- cotransporter can be recycled back sodium reabsorption. There are two major cell
through the lumen, through a potassium types in this region of the nephron: the prin-
channel, to keep the tubule concentration of cipal cells and the intercalated cells. The prin-
this ion high enough for the cotransporter to cipal cells are the predominant cell type, and
continue to function. The result of potassium they are responsible for sodium reabsorption
leaving the luminal membrane and chloride at and potassium secretion. Sodium enters the
the basolateral side by conductive pathways cell by way of a sodium channel and exits
generates a lumen-positive potential. This through the basolateral Na+/Kc-ATPase
positive potential drives the flow of sodium pump (Fig. 2.6). Potassium can exit this cell on
ions out through a paracellular pathway. the luminal and basolateral sides through con-
There is also a Nat/H+ exchanger on the api- ductance channels. The primary site of action
cal membrane that plays a minor role in the of aldosterone is also in the principal cells. Al-
reabsorption of sodium. dosterone increases sodium reabsorption by
The distal convoluted tubule reabsorbs ap- opening sodium channels. The intercalated
proximately 5-8% of the sodium contained in cells control hydrogen ion secretion and potas-
the glomerular filtrate. The major luminal sium reabsorption (Fig. 2.7). Protons are gen-
transporter of sodium in this region is the neu- erated in the cell by the catalytic actions of
tral sodium chloride cotransporter (Fig. 2.5). carbonic anhydrase and are transported into
A Na+/K+-ATPasepump is located on the ba- the lumen through Ht/translocating ATPase
solateral membrane to remove sodium from (16). An ATP-dependent K+/ Hf exchanger
the cell. Potassium can reenter the tubule may be responsible for potassium reabsorp-
through a barium-sensitive potassium chan- tion in times of potassium depletion (17). Two
nel. This region of the renal tubule is the site recent reviews have appeared on the molecu-
at which calcium excretion and reabsorption lar mechanisms of the actions of diuretics
are regulated. Parathyroid hormone and cal- (18, 19).
Diuretic and Uricosuric Agents

w
I
'
- Basolateral
membrane
Na+ rn

K+
4
I

Na+ L-J---'
Urine Blood

Figure 2.4. Cell model of the thick ascending loop of Henle.

w
Basolateral
membrane
Na+ +
ATP
K+
* I
CI-
---------- +

Figure 2.5. Cell model of the Urine Blood


distal convoluted tubule.
1 Introduction

Basolateral
------------ membrane

Luminal

-
membrane

Aldosterone receptor
J
Urine I I Blood
Figure 2.6. Cell model of the principal cells in the collecting tubule.

u
- ATP
H+
'\ H
HCOg
C
Basolateral
membrane

Urine Blood

Figure 2.7. Cell model of the intercalated cell of the collecting tubule.
Diuretic and Uricosuric Agents

1.1 Pharmacological Evaluation of Diuretics is a potent diuretic in contrast to the very low
diuretic activity seen with this compound in
The following three statements or generaliza- the rat.
tions are direct quotes from a study by K. H. The chimpanzee (25) has also been used to
Beyer and refer to the pharmacological evalu- evaluate certain diuretics. In this animal, the
ation of drugs in general and to diuretics in effect of the compound on uric acid excretion
particular (20). can also be studied, because apes, like hu-
The more closely one can approximate under mans, are devoid of hepatic uricase and there-
controlled laboratory conditions the physiologi- fore maintain a relatively high level of circu-
cal correlates of clinically defined disease, the lating serum urate (26). Chimpanzees have
more likely one will be able to modulate it effec- also been used in the study of uricosuric
tively. agents (25), but tests with such large animals
In vitro experiments are apt to be inadequate obviously present numerous problems. At-
and hence misleading when employed solely to tempts have also been made to block the en-
anticipate the physiological correlates of com- zyme uricase in rats by administering potas-
plex clinical situations. sium oxonate and thus to obtain higher serum
Often aberrations in function we call toxicity, uric acid levels (27).
relate more to changes a compound induced Considerable evidence is available that
physiologically than to a direct, inherent, de- demonstrates the tendency of certain benzo-
structive effect of the agent, per se, on tissues.
thiadiazine diuretics to elevate blood glucose
values in seemingly normal, as well as diabetic
Diuretics are generally evaluated in two or prediabetic, individuals (28). A method us-
species: the rat and the dog. The rat is used, as ing high doses of the compounds injected in-
a rule, in initial screening for convenience and traperitoneally into rats and determining
economy. Results obtained with diuretic blood glucose levels, as compared to control
agents in dogs, however, are generally more values, has been used to estimate a possible
predictive of the response in humans than hyperglycemic effect of diuretics (29).Today
those obtained in the rat. However, many the molecular and cellular mechanism of ac-
compounds exhibit diuretic activity in the rat tions of diuretics can also be investigated. Mi-
(e.g., antihistamines, such as tripelennamine) cropuncture and single nephron studies can
but are inactive in the dog and in humans. On provide insight into the exact site of action in
the other hand, mercurial diuretics and the renal tubule and yield information regard-
ethacrynic acid are inactive in the rat; furo- ing the specific transport mechanisms that are
semide exhibits diuretic activity in the rat only blocked by a drug.
at doses several times higher than effective 1.2 Clinical Aspects of Diuretics
doses in the dog. Various modifications of the
experimental procedures of Lipshitz et al. (21, In a healthy human subject, changes in di-
22) are frequently used to measure urine vol- etary intake or variations in the extrarenal
ume and Naf, K+, and C1- excretion (e.g., loss of fluid and electrolytes are followed rela-
male rats, fasted for 18 h, given 5 mL of 0.2% tively rapidly by adjustments in the rate of
NaCl solution/100 g body weight by stomach renal excretion, thus maintaining the normal
tube). The diuretic drugs are given by stomach volume and composition of extracellular fluid
tube at the time of fluid loading. The rats are in the body. Edema is an increase in extracel-
placed in metabolism cages and urine volumes lular fluid volume. In almost every case of
are measured at 30-min intervals over a 3- to edema encountered in clinical medicine, the
5-h period. Total amounts of Na and K' ex-
f

underlying abnormality involves a decreased


creted over the time period are determined by rate of renal excretion. One of the factors in-
flame photometry; chloride can be determined fluencing the normal relationship between the
in the Technicon Autoanalyzer using Skegg's volume of interstitial fluid and the circulating
modification of Zall's method (23). A number plasma is the pressure within the small blood
of standard diuretics have also been tested in vessels. In diseases of hepatic origin (e.g., cir-
the mouse (24). In this animal, ethacrynic acid rhosis), the pressure relationships are dis-
2 Clinical Applications

turbed primarily within the portal circulation and increased urate reabsorption from the fil-
and ascites results. In congestive heart failure, trate. Some patients may experience an attack
pressure-flow relationships may be disturbed of gout, or those with preexisting gout or ex-
more in the pulmonary or systemic circulation cessive uric acid production may experience
and edema may be localized accordingly. more frequent attacks. A number of studies
There is overwhelming evidence to indicate have shown that some patients who have un-
that the primary disturbance of the kidney is dergone long-term diuretic therapy have ele-
in its ability to regulate sodium excretion, vated blood levels of glucose and that their
which underlies the pathogenesis of edema. tolerance to glucose decreases (31). The mech-
Three approaches are available when edema anism of the diuretic-induced glucose intoler-
fluid accumulates because of excessive reab- ance is unknown. Some other side effects that
sorption of sodium and other electrolytes by are observed with diuretic treatment are in-
the renal tubules. First, one can attempt to creases in cholesterol levels in men and post-
correct the primary disease if possible; second, menopausal women (32), ototoxicity, and hy-
one can reduce renal absorption of electrolytes pomagnesemia.
by the use of drugs; and third, one can restrict
sodium intake to a level that corresponds to
the diminished renal capacity for sodium ex- 2 CLINICAL APPLICATIONS
cretion. Cardiac decompensation is one of the
most common causes of edema. Treatment 2.1 Current Drugs
consists of full digitalization, which should be
considered the primary therapeutic agent. Di- 2.1 .I Osmotic Diuretics. Osmotic diuret-
uretic drugs have a secondary though very im- ics all have several key features in common:
portant role because it has been shown that
blocking excessive electrolyte reabsorption in 1. They are passively filtered at the
the renal tubule alleviates the symptoms of glomerulus.
cardiac failure and also improves cardiac func-
2. They undergo limited reabsorption in the
tion. Diuretics are also used in the treatment
renal tubules.
of hypertension.
Diuretic therapy may lead to a number of 3. They are usually metabolically and phar-
metabolic and electrolyte disorders. In gen- macologically inert.
eral, these disturbances are mild but can be 4. They have a high degree of water solubility.
life-threatening in certain cases. Some of the
common adverse effects observed with di- These agents all function as diuretic agents
uretic treatment are hypokalemia, hyperuri- by preventing the reabsorption of water and
cemia, and glucose intolerance. Diuretics that sodium from the renal tubules. The addition of
possess a site of action proximal to the collect- a nonreabsorbable solute prevents water from
ing tubules, such as the loop and thiazide di- being passively reabsorbed from the tubule,
uretics, induce potassium loss; an average loss which in turn prevents a sodium gradient
of 0.5-0.7 meq/L generally results with long- from forming and thereby limiting sodium re-
term therapy (30). In most young hyperten- absorption. These actions hinder salt and wa-
sive patients, this reduction does not present ter reabsorption from the proximal tubules;
any problem; however, in older patients or pa- however, it has also been proposed that these
tients with preexisting heart disease, it may agents have multiple sites and mechanisms of
lead to the occurrence of ventricular arrhyth- action (33,34).
mias. Combinations with potassium-sparing Most of the osmotic diuretics are sugars
diuretics are frequently used to minimize the and polyols (Table 2.1). Mannitol (Table 2.1,
effect. Dietary potassium supplements may 5) is the prototype of the osmotic diuretics and
also be prescribed. In patients receiving long- has been studied extensively. The compound
term diuretic therapy, serum uric acid concen- is poorly absorbed after oral administration
trations increase on average 1.3 mg/L as the and is therefore administered by intravenous
result of a decrease in extracellular volume (i.v.) infusion. It is freely filtered at the glo-
64 Diuretic and Uricosuric Agents

Table 2.1 Osmotic Diuretics


No. Generic Name Trade Name Structure
(1) Ammonium chloride
(2) Glycerine Osmoglyn

(3) Glucose

(4) Isosorbide Ismotic

(5) Mannitol Osmitrol

(6) Sorbitol

(7) Sucrose

(8) Urea Ureaphil

merulus and reabsorption is quite limited. tained, and the kidneys can be protected from
The usual diuretic dose is 50-100 g given as a damage. Osmotic diuretics have also been pre-
25%solution. scribed in the relief of cerebral edema after
These compounds are not prescribed as pri- neurosurgery, to lower intraocular pressure in
mary diuretic agents to edematous patients. ophthalmologic procedures, and after a drug
One of the most important indications for the overdose to maintain urine flow.
use of mannitol is the prophylaxis of acute re-
nal failure. After cardiovascular operations or 2.1.2 Mercurial Diuretics. For approxi-
severe traumatic injury, for instance, a precip- mately 30 years, mercurial diuretics were the
itous fall in urine flow may be anticipated. Ad- most important diuretic agents. Since the i n
ministration of mannitol, under such condi- trodudion of orally active, potent, less toxic
tions, exerts an osmotic effect within the nonmercuiral diuretics, beginning in 195C
tubular fluid, inhibiting water reabsorption. A with acetazolamide, their use has greatly de
reasonable flow of urine can thus be main- clined. Today they represent only a small frae
2 Clinical Applications

tion of the injectable diuretics used, and inject-


able diuretics in turn are only a small portion
of the total diuretic market. Organomercuri-
als are generally given intramuscularly. The
ual dose is a 1-mLsolution containing 40 mg
g. In responsive, edematous patients, an in-
urine flow is evident in 1-2 h and
maximum in 6-9 h. The effect is
24 h. A loss of about 2.5%
ight represents an average response
(35). Mercury is eliminated from the body in
the urine, as a complex with cysteine. These
diuretics therefore should not be prescribed to
patients with renal insufficiency marked by chloride) was used by Paracelsus (1493-1541,
adequate excretion of the mercury-cysteine A.D.) as a diuretic. This information was lost
until the nineteenth century, when Jen-
2.1.2.1 Pharmacology. Before the develop- drassik rediscovered the use of calomel as a
nt of the loop diuretics, the organomercuri- diuretic agent (40). Calomel was an ingredient
e most potent diuretics available. of the famous Guy's Hospital pill (calomel,
her studies have found that the major ef- squill, and digitalis). Calomel exerted a ca-
fect of organomercurials appears to be in the thartic effect, and its absorption from the in-
ascending limb of Henle (36-38). Organomer- testine was unpredictable. In 1919, Vogl (41)
curials inhibit active chloride reabsorption in discovered the diuretic effect of merbaphen
the thick ascending limb of Henle. During di- (10) after p a r e n t e d administration of this
uresis, the urine contains a high concentra-
tion of chloride ion matched by almost equiv-
alent amounts of sodium ion (35). The effect of
ercurial diuretics on potassium excretion is
.complex. They depress the tubular secretion
f potassium and, for this reason, the diuresis
accompanied by significantly less potassium
loss than occurs with other diuretics that do
the secretory mechanism. How-
r, mercurials can have a paradoxical effect
potassium excretion when initial
es are low. Inhibition of organic
is seen in humans but not in the antisyphilitic agent. General use of the drug as
g. In the chimpanzee and to a lesser extent a diuretic was short-lived because of its toxic-
humans, mercurials (e.g., mersalyl, 9) have ity. However, it did lead to the synthesis of a
intense uricosuric action (39). large number of organomercurials between
Side effects, such as diarrhea, gingivitis, 1920 and 1950.
d stomatitis, can occur with or- 2.1.2.3 Structure-Activity Relationship. It is
mercurial treatment. As with other di- believed that the mechanism of action for
ics, electrolyte imbalances are common af- these diuretic agents involves the in uivo re-
long-term use (hypochloremic alkalosis, lease of mercuric ion in the renal tubules (42-
kalemia, hyponatremia). In some cases 44). This ion is thought to bind a sulfhydryl
dministration, severe hy- enzyme in the tubule membrane that is in-
actions may develop. volved in sodium reabsorption. The mercuric
The medicinal use of mer- ion reacts with a sulfhydryl group on the en-
es back to 400 B.C., when zyme and another nucleophilic group in close
ppocrates administered metallic mercury to proximity, to form a bidentate complex that
crease urine excretion. Calomel (mercurial inactivates the enzyme and, as a result, the
Diuretic and Uricosuric Agents

The nature of the X substituent affects the


toxicity of the compound, irritation at the site
of injection, and rate of absorption (45, 46).
Theophylline has been used commonly as anX
X = groups such as OH, substituent (47) or is commonly added by it-
SH NH2,COOH, self with the organomercurials. Because of its
irnidazole peripheral vasodilating effects, it can increase
absorption of the mercurial diuretics at the
Figure 2.8. Mercury-enzyme complex.
site of injection. Theophylline is also weakly
diuretic. When X is a thiol, such as mercapto-
sodium reabsorption process (Fig. 2.8). Most acetic acid or thiosorbitol, cardiac toxicity and
mercurial diuretics have the general structure local irritation are reduced (48, 49). Digluco-
( l l ) , in which Y is usually CH, and R is a methoxane (18, Mersoben) (50) should be

complex organic moiety, usually incorporating


an amide function or a urea group. All organic
mercurial diuretics thus far examined are
acid-labile in vitro. It is interesting to note
that compounds of the related structure (12)
with an unsubstituted p-carbon atom are acid- cited as a well-tolerated, potent mercurial di-
stable and do not exhibit diuretic activity. For-
uretic that does not conform to the general
mula (13)shows the structural characteristics
structure (11). Generally, mercurial diuretics
are administered parenterally; chloromero-
drin (Table 2.2, 14), which lacks a carboxylic
acid group, is orally effective but gastric irri-
tation precludes its widespread use (51).

2.1.3 Carbonic Anhydrase Inhibitors. Ac-


etazolamide (19) is the prototypical carbonic
anhydrase inhibitor. It is rapidly absorbed
from the stomach, reaches a peak plasma level
within 2 h, and is eliminated unchanged in the
urine within 8-12 h. The efficacious dose is
250 mg to 1 g daily. During continuous admin-
istration of acetazolamide, the excretion of
HC0,- leads to the development of metabolic
of mercurial diuretics and the most important acidosis. Under such acidic conditions, the di-
mercurial diuretics (14-17) are shown in Ta- uretic effect of carbonic anhydrase inhibition
ble 2.2. The R substituent largely determines is much reduced or completely absent, and
the distribution and rate of excretion of the therefore the effect of the drug is self-limiting
compound. The Y substituent, determined by (52). This is attributed to the fall in the level of
the solvent in which the mercuration is car- plasma and filtered bicarbonate, given that
ried out, generally has little effect on the prop- the latter is lost in the urine. A state of equi-
erties of the compound (45,46). Amongothers, librium is reached when the small amount of
Y substituents such as H, CH,, CH2CH20H, hydrogen ion that is secreted in spite of car-
and CH2CH20CH3have been studied. bonic anhydrase blockade is sufficient to reab-
2 Clinical Applications 67

Table 2.2 Mercurial Diuretics


No. Generic Name Trade Name Structure
(14) Chloromerodrin Neohydrin H2NOCHN-CH2CH(OCH3)CH,HgC1
(15) Meralluride USP Mercuhydrin

0
I
R = theophylline
(16) Sodium mercaptomerin Thiomerin

HO N/Y\H
H 0

0 I

R = theophylline

sorb the reduced amount of filtered bicarbon-


ate ion. Because of the development of
kaliuresis, metabolic acidosis, and their self-
limiting nature, these inhibitors are not gen-
erally prescribed for diuretic therapy. Their
most common use today is to lower intraocular
pressure in the treatment of glaucoma. There
is a great deal of current research in this area
(53-55). Two new agents are available for the
treatment of glaucoma dorzolarnide (20, Tru-
sopt) and brinzolamide (21, Azopt) (56, 57). Mann and Keilin (611, Davenport and Wil-
Carbonic anhydrase inhibitors also have some helmi (62), and Pitts and Alexander in 1945
therapeutic utility in epilepsy, congestive (63) proposed that the normal acidification of
heart failure, mountain sickness, gastric and the urine results from the secretion of hydro-
duodenal ulcers, and more recently in the area gen ions by tubular cells. They confirmed that
of osteroporosis, antitumor agents, and as di- in dogs sulfanilamide (22) renders the urine
agnostic tools (58, 59). alkaline, perhaps because of the reduction of
2.1.3.1 History. Building on earlier work the availability of Hf for secretion brought
by Strauss and Southworth in 1937 (60), about by the inhibition of the enzyme carbonic
Diuretic and Uricosuric Agents

tion of cytosolic carbonic anhydrase, to pro-


duce cellular H+ and HC0,-. The hydrogen
ion is secreted into the tubular lumen through
the Na'/ H+ exchanger, and then the Nat is
reabsorbed and enters the peritubular fluid as
NaHCO,. In the proximal tubule, the secreted
H+ combines with HC03- to form H,C03,
which is then dehydrated to CO, and H,O.
This reaction is also catalyzed by carbonic an-
hydrase located on the luminal border of the
proximal tubular cells. The carbon dioxide dif-
fuses back into the cell, where it is again hy-
drated and used as a source of hydrogen ion to
drive the H+/ Na' exchanger (see Fig. 2.2).
Carbonic anhydrase inhibitors are among the
most well understood class of diuretics. Their
anhydrase. The resulting increase in Na+ and major function is to inhibit the enzyme car-
HC0,- excretion suggested to Schwartz (64) bonic anhydrase, although they are also be-
the diuretic potential of sulfanilamide. How- lieved to decrease cell membrane permeability
ever, this was of no practical significance be- for carbon dioxide and also to inhibit glucose
cause of the very high doses required to 6-phosphate dehydrogenase (65). The admin-
achieve diuresis. istration of an inhibitor of carbonic anhydrase
2.1.3.2 Pharmacology. Carbonic anhydrase promptly leads to an increase in urine volume.
is a zinc-containing enzyme that was first dis- The urinary concentrations of HC0,-, Naf,
covered in erythrocytes by Roughton in the and K' increase, whereas the normally acidic
early 1930s. This enzyme was subsequently urine becomes alkaline and the concentration
found in many tissues, including the renal cor- of chloride ion drops. In addition, there is a fall
tex, gastric mucosa, pancreas, eye, and central in titratable acid and ammonium ion excre-
nervous system. Carbonic anhydrase cata- tion. The net effect of the inhibitors in the
lyzes the reversible hydration of carbon diox- proximal tubule is to prevent the reabsorption
ide and the dehydration of carbonic acid. of bicarbonate. This can effect volume reab-
These reactions can occur in the absence of the sorption in a number of ways:
enzyme, although the rates are too slow for
normal physiological function to occur. Nor-
1. Fewer protons are available for the H+I
mally the enzyme is present in the tissue in
Na+ exchanger on the luminal membrane;
high excess. Because of the levels of this en-
thus sodium reabsorption is slowed.
zyme in the kidney, approximately 99%of the
enzyme's activity must be inhibited for physi- 2. The passive reabsorption process in the
ological activity to be observed. To date, 14 late proximal tubule is indirectly inhibited
different isozymes or carbonic anhydrase-re- by the decreased chloride gradient.
lated proteins have been identified in humans 3. Bicarbonate effectively becomes a nonreab-
and higher vertebrates. sorbable anion and osmotically adds to the
Hydrogen ion secretion takes place in the diuretic effect.
proximal tubule, the distal tubule, and the col-
lection duct. The driving force for H+ secre- However, more than half of the bicarbonate
tion in the distal portions is the trans-tubular that passes through the proximal tubule is re-
negative potential. In the proximal tubule, absorbed in later segments of the renal tubule,
protons are actively excreted by the H+/ Na+ thus attenuating the effectiveness of this class
exchanger. The source of cellular hydrogen of diuretic agents. Carbonic anhydrase inhib-
ion is the hydration of carbon dioxide within itors also cause a significant kaliuresis, which
the proximal tubular cells catalyzed by the ac- can be attributed to the inhibition of distal
2 Clinical Applications

proton secretion and high aldosterone levels, hydrase inhibitor class. A number of struc-
which result from volume depletion. tural modifications of acetazolamide have
2.1.3.3 Strvcture-Activity Relationship. There been studied. An increase in the number of
are two main classes of compounds that in- carbons in the acyl group is accompanied by
hibit carbonic anhydrase: unsubstituted sul- retention of in vitro enzyme inhibitor activity
fonamides and metal-complexing inorganic and diuretic activity, but the side effects be-
anions (e.g. azide, cyanate, cyanide, hydrogen come more pronounced. Removal of the acyl
sulfide, etc.). The inorganic anions have groups leads to a markedly lower activity in
served as tools for better understanding of the vitro (72,73).Substitution on the sulfonamide
catalytic and inhibitory mechanism of car- nitrogen abolishes the enzyme-inhibitory ac-
bonic anhydrase inhibition. The sulfonamide tivity in vitro, but diuretic activity in animals
class has led to the development of several use- is still present if the substituent is removable
ful therapeutic agents. After the discovery of by metabolism (74). Two isomeric products,
the carbonic anhydrase inhibitory activity of (33) and (31) (methazolamide), are obtained
sulfanilamide, a variety of aromatic sulfon-
amides were found to exhibit the same type of
activity (66). Aliphatic sulfonamides were
much less active; substitution of the sulfon-
amide nitrogen in aromatic sulfonamides
eliminated the activity. Roblin and coworkers
(67,68),following the work of Schwarts (64),
investigated a series of heterocyclic sulfon-
amides. Compounds up to 800 times more ac-
tive in vitro than sulfanilamide as carbonic
anhydrase inhibitors were found. An attempt
to correlate pK, values and in vitro carbonic
anhydrase inhibitory activity in a series
of closely related, 1,3,4-thiadiazole-2-sulfon-
amides (23) was not successful (69). The rela-

on methylation of acetazolamide (19) (69).


Both these compounds are somewhat more ac-
tive in vitro than acetazolamide but offer no
advantages as diuretics over the parent
(23) R' = lower alkyl, phenyl compound.
R = H, CH&O A related sulfamoylthiadizolesulfonamide,
benzolamide (32, Table 2.4), is about five
tionship between in vitro enzyme inhibition times more active than acetazolamide. Clini-
and in vivo diuretic potency was not very pre- cal studies showed that 3 m a g p.0. of (32)
dictable because of variation in drug distribu- produces a full bicarbonate diuresis, with in-
tion, binding, and metabolism (70), especially creased excretion of sodium and potassium
when different types of aromatic or heterocy- (75, 76). Ethoxzolamide, a benzothiazole de-
clic sulfonamides were compared (Table 2.3). rivative (Table 2.4, 30) is a clinically effective
Certain derivatives of 1,3,4-thiadiazolesul- diuretic carbonic anhydrase inhibitor (77).
fonamides were among the most active in vitro The compound lacking the ethoxy group is in-
inhibitors of carbonic anhydrase, with poten- active as a diuretic when given orally to dogs,
cies several hundred times that of sulfanil- although it is a potent carbonic anhydrase in-
amide (67, 68). One of these, 5-acetylamine- hibitor in vitro (78). Dichlorophenamide (26,
1,3,4-thiadiazole-2-sulfonamide(acetazol- Tables 2.3 and 2.4). a benzenedisulfonamide
amide, 19, Tables 2.3 and 2.4) was studied in derivative, is as active as acetazolamide in
detail by Maren et al. (71) and became the first vitro as a carbonic anhydrase inhibitor and
clinically effective diuretic of the carbonic an- equally active as a diuretic (70). A large num-
70 Diuretic and Uricosuric Agents

Table 2.3 Carbonic Anhydrase Diuretics


No. Generic Name Trade Name Structure Ref.
Acetazolamide USP Neohydrin

Dichlorophenamide USP Daranide,


Oratrol

Ethoxzolamide USP Cardrase,


Ethamide

Methazolamide USP Neptazane

Benzolamide

ber of benzenedisulfonamide derivatives have uretic properties sought and ultimately found
been prepared and studied as diuretics. Some in chlorothiazide (28, Table 2.4). A key discov-
of these are very active as diuretics, although ery by Sprague (80) was that the introduction
they are weak carbonic anhydrase inhibitors. of a second sulfamoyl group meta to the first
In contrast to the compound just discussed, can markedly increase not only the natriuretic
Nai and HC0,- excretion is not increased; effect but also the chloruretic action of the
instead, an approximately equal amount of compound. This is evident when the data for
chloride ion accompanies the sodium. These compounds (22) or (24) are compared with
are described in the section on aromatic sul- compound (25) (Table 2.4). Interestingly, the
fonamides. introduction of a second chlorine substituent
as in compound (26) (dichlorphenamide, Ta-
2.1.4 Aromatic Sulfonamides. Beyer and ble 2.4) produces a compound that is consider-
Baer (791, in a study published in 1975, dis- ably less chloruretic, with an excretion pat-
cussed their early findings on the natriuretic tern that is typical of a carbonic anhydrase
and chloruretic activity of p-carboxybenzene- inhibitor.
sulfonamide (24, Table 2.4). Although the The activity of 6-chlorobenzene-1,s-did-
compound is considerably less active as a car- fonamide (25, Table 2.4) is further enhanced
bonic anhydrase inhibitor than acetazol- by the introduction of an amino group ortho to
amide, the way the kidney handled the car- the second sulfonamide group as in (27). Thus,
boxybenzenesulfonamidewas considered to be 4-amino-6-chloro-l,3-benzenedisulfonamide
more important and served to define the sal- (27) is an effective diuretic agent, with a more
2 Clinical Applications 71

Table 2.4 Dissociation of Carbonic Anhydrase Inhibitory Activity In Vitro and Renal
Electrolyte Effects in the Doga
Concentration Urine Excretion Rate
Causing 50%Inh. of Dose (peqlmin)
Carbonic Anhydrase (i.vJb
No. Structure (M) (rnglkg) Na+ Kt C1- pH

favorable electrolyte excretion pattern than


that of (25).Chloride is the major anion ex-
creted, and HCO,- excretion is low because
the urinary pH does not increase. The car-
bonic anhydrase inhibitory activity of (27) is
only about three times that of sulfanilamide
(22). The chlorine in the 6-position of (27)can
be replaced by a bromine, trifluoromethyl, or R 1 = R3 = H, R4 = CH3 or CH2CHCH2
nitro group without much change in activity; R 1 = R3 = H, R4 = CO(CH2)2.4CH3
whereas a fluoro, amino, methyl, or methoxy R 1 = R3 = R4 = CH3C0
group is less effective(81). R 1 = R3 = CH3 or Ethyl, R4 = H
Substitution on the nitrogen atoms of
4-amino-6-chloro-l,3-benzenedisulfonamide compounds with reduced oral activity. Acyla-
gives compounds (34). Methyl or ally1 substi- tion of the anilino group leads to an increase in
tution of the aromatic amino group yields activity when R, is a simple aliphatic acyl rad-
Diuretic and Uricosuric Agents

Table 2.4 (Continued)


Concentration Urine Excretion Rate
Causing 50%Inh, of Dose (keqlmin)
Carbonic Anhydrase (i.vJb
No. Structure (M) (mg/kg) Na+ K+ C1- pH
(28) 1.7 x 10-6 c 11 11 7 6.1
0.05 20 24 7 6.6

"From Ref. 70.


bC, control phase (no drug), average of two or three 10-min clearances.

ical and reaches a maximum at four to six car- diuretic activity in the rat, but the observed
bon atoms. Aromatic acyl derivatives are less activity is attributable to in uiuo dealkylation
active. Acylation with formic acid results in a of the sulfonamide function (83).
cyclized product, 6-chloro-l,2,4-benzothiadia- 2.1.4.1 Mefruside. Horstmann and cowork-
zine-7-sulfonamide-1,l-dioxide, chlorothia- ers (84) at a later date realized that 6-chloro-
zide (28, Table 2.4) (82). The compound was 1,3-benzene-disulfonarnide (25, Table 2.4)
the starting point for the development of the had shown an excretion pattern combining
thiazide diuretics, one of the most important Na+, HC0,-, and a substantial amount of
group of diuretics, which are discussed later. chloride ions, even though it was an active car-
Complete acetylation (R, = R, = R, = bonic anhydrase inhibitor. Investigation of de-
CH,CO) lowers the activity (82). Methylation rivatives substituted on the sulfonamide ni-
of both sulfonamide groups (R, = R, = CH, or trogen para to the chloro substituent led to
CH,CH,; R, = H) gives a compound that has compounds with high diuretic activity. Intro-
2 Clinical Applications

duction of a single methyl group (35)does not 7.3 x M concentration (chlorothiazide;


reduce the HC0,- excretion as compared to 1.7 x lop6M). The potency of mefmside and
(25), but disubstitution as in (36) leads to a its effect on the renal concentrating/diluting
substantially lower excretion of HCO,- and mechanisms suggest that its action is similar
improved Nat/C1- ratio because of less car- to that of the thiazide diuretics (88,89).Stud-
bonic anhydrase activity. A large number of ies in hypertensive patients also showed a
N-substituted benzenesulfonamide deriva- close correlation with the thiazide diuretics in
tives were prepared; the N-disubstituted com- terms of both desirable and undesirable ef-
pounds were of more interest because they ex- fects (90, 91).
hibited primarily a saluretic effect. A selected
number of these compounds are shown in Ta- 2.1.5 Thiazides. Thiazides are a major
ble 2.5. The threshold dose level and the in- class of diuretic agents that have been used for
crease in sodium excretion over control values over 30 years. It was this group of therapeutic
in the rat are also given. These compounds are agents that first challenged and then replaced
relatively weak carbonic anhydrase inhibitors, the mercurial diuretics that were used in the
with little effect on HC0,- excretion, particu- first half of the twentieth century. Treatment
larly those that are &substituted on the sul- of 4-amino-6-chlorobenzene-1,3-disulfonamide
fonamide nitrogen. A particularly favorable with formic acid resulted in the formation
type of substituent is the tetrahydrofurfuryl of the cyclic benzothiadiadiazine derivative,
group (41-44, Table 2.5). Activity is enhanced chlorothiazide (28, Table 2.4) (82). This com-
when the tetrahydrofuran ring bears a methyl pound was the first orally active, potent di-
substituent in the 2-position. A diuretic effect uretic that could be used to the full extent of
in rats is obtained with this compound (43, its functional capacity as a natriuretic agent
mefruside) at the low dose of 0.04 m a g . This without upsetting the normal acid-base bal-
compound has an asymmetric carbon atom; ance. Chlorothiazide is saluretic with minimal
however, the difference in diuretic activity be- side effects and fulfilled a clinical need. In ad-
tween the more active form and the racemate dition to diuretic activity, chlorothiazide and
is not of practical significance. The action of its congeners exert a mild blood pressure-low-
mefruside is characterized by a prolonged in- ering effect in hypertensive patients.
crease in the rate of excretion of NaCl and 2.1.5.1 Pharmacology. This is discussed in
water (85). The corresponding pyrrolidine de- relation to the hydrothiazides in Section
rivatives are also active diuretics (45 and 46, 2.1.6.1.
Table 2.5). 2.1.5.2 History. Thiazide diuretics arose
Studies with 14C-labeled mefruside have as an outgrowth of the carbonic anhydrase in-
shown that the compound is almost com- hibitor area, in particular from the work of
pletely metabolized in vivo to the lactone (44, Novello and Sprague. Research in this area
Table 2.5) (86). This lactone has about the was based on the conviction of Beyer (92) that
same diuretic activity as that of the parent it should be possible to find a sulfonamide de-
compound and may be responsible for much of rivative that was saluretic and that increased
the observed activity- of mefruside (84).Mefm- Na' and C1- excretion in approximately equal
side has been compared with chlorothiazide in quantities; in other words, a compound that
the dog, and the findings suggest that mefm- did not act as a classical carbonic anhydrase
side has a mechanism of action similar to that inhibitor, which increased water, Na t , and
of the thiazide diuretics without any unique HC0,- elimination. A saluretic diuretic
atures (87). Mefruside has also been studied should permit a substantial reduction of
humans at doses of 25 and 100 mg. In vol- edema without affecting the normal acid-base
teers undergoing water diuresis, the drug balance.
sed naturiuresis and chloruresis extending 2.1.5.3 Structure-Activity Relationship. An
r 20 h. Bicarbonate excretion was also in- SAR has been developed for the thiazides. The
ased, whereas the acute excretion of potas- effect of varying the 3 and 6 substituents is
was slightly increased. In vivo carbonic shown in Table 2.6. Interestingly, compound
drase studies revealed 50% inhibition at (48) (R, = H) has very little diuretic activity,
74 Diuretic and Uricosuric Agents

Table 2.5 Diuretic Activity of Some 4-Chloro-3-sulfamoyl-N-substituted


Benzenesulfonamides"
rn

~ ~ ~ 0 , s '
Increase in Na+
Excretion at 80
Threshold Dose, p.0. &kg P.0.
No. R (mg/kg) [peq/kg/6 h (Rat)]

(37) NHCH2CH(CH3)2 2.4 4615


(38) 2.1 3000

(39) -3 6 3910

(40) 18 2900

"From Ref. 84.


2 Clinical Applications 75

Table 2.6 Comparative Effects of 3- and 6-Substituted Thiazides on Electrolyte Excretion


in the Dog upon Oral Administrationa

Urinary Excretion
No. Rs R3 Na+ Kf C1- Reference
(28) C1 H ++++ + ++++ chlorothiazide
(48) H H +/- +/-
(49) Br H ++++ + ++++
(60) Me H + +/- +
(51) OMe H ++ +/- ++
(62) NO2 H +++ +I- +++
(53) NH, H +/- +/-
(54) C1 Me +++ +/- +++
(55) C1 n-Pr +++ +/- +++
(56) C1 n-C5H~~ +++ +/- +++
(67) c1 C6H5 +/- +/- +/- +/-
(58) CF3 H ++++ ++++ 93
(59) C1 CH,SBn ++++ + ++++ 96,97
(80) C1 CHC1, ++++ + ++++ 98
(61) C1 CH,(C5H9) ++++ + ++++ 98
"From Ref. 80.

whereas compounds where R, = C1, Br, or CF, H gives compounds with little activity (81).
are highly active; and alkyl groups in the 3-po- The degree of activity observed with com-
sition decrease the activity slightly. The 3-0x0 pounds bearing an acyl or alkyl group on the
derivative of chlorothiazide also has weak di- 7-sulfamoyl group is in accord with the hy-
uretic activity (80). Interchanging the chlo- pothesis that metabolic cleavage of the N-sub-
rine and sulfamoyl groups at positions 6 and 7 stituent occurs to yield the free sulfamoyl
in chlorothiazide lowers the activity. Replace- function (93, 94). In the case of N,-caproyl-
ment of the 7-sulfamoyl group by CH,SO, or chlorothiazide, urinary bioassay indicated

Table 2.7 Pyrido-1,2,4-thiadiazines1,l-Dioxidesa

No. R No. R No.


(62) H (64) H (69)
(63) Me (65) Me
(66) NH2
(67) OH
(68) C1
"From Ref. 99.
Table 2.8 Benzothiadiazine Diuretics
Clinical Dose p.0.
No. Generic Name Trade Name (mglkg) Structure Reference
(28) Chlorothiazide NF Diuril

(29) Hydrochlorothiazide

(59) Benzthiazide NF Aquatag, Exna

V
Q,

(71) Bendroflume thiazide NF Bristuron,


Naturetin

(72) Buthiazide Saltucin,


Eunephran

(73) Cyclopenthizide
that 50% of the excreted drug was present as sis they have been effective, but their thera-
chlorothiazide (70), whereas the N,-acetyl de- peutic usefulness in such cases has been un-
rivative showed only weak saluretic activity predictable. The side effects observed with
and no detectable cleavage of the acetyl group thiazide treatment can be divided into two
(95). Substitution of the ring nitrogen atoms types: hypersensitivity reactions and meta-
at position 2 or 4 with a methyl group reduced bolic complications. Some of the common
the activity and makes the heterocyclic ring metabolic complications of these diuretics are
more vulnerable to hydrolytic cleavage (81). hypokalemia, magnesium depletion, hypercal-
The introduction of a more complex substitu- cemia, hyperuricemia, and hyperlipidemia.
ent in the 3 position [e.g., R, = CH,SCH,C,H, Thiazides may also induce hyperglycemia and
(59,Table 2.611 led to a compound that was 8-10 can aggravate a preexisting diabetic state.
times more potent on a weight basis than chlo- With respect to hypersensitivity, dermatitis,
rothiazide (96, 97). Similarly, the dichlorom- purpura, and necrotizing vasculitis have been
ethyl and cyclopentylmethyl analogs (60 and 61, observed. The thiazide diuretics are available
Table 2.6) were 10-20 times more potent, re- as tablets; the wide range of dosages of the
spectively, than chlorothiazide on a weight basis individual preparations are shown in Table
when tested in experimental animals (98). A 2.8. To minimize the possibility of potassium
number of "aza" analogs of chlorothiazide, de- depletion, fixed combinations with potassium-
rived from 2-aminopyridine-3,5-disulfonamide sparing diuretics have been made available
and 4-aminopyridine-3,5-disulfonamide, have (e.g., hydrochlorothiazide-triamterene and
been prepared (62-69, Table 2.7). In general, hydrochlorothiazide-amiloride).
the activity of each compound was comparable 2.1.6.1 Pharmacology. Chlorothiazide and
with, although somewhat less potent than, that hydrochlorothiazide are the prototypes of a
of its 1,2,4-benzothiadiadiazineanalog (99).Ini- group of related heterocyclic sulfonamides
tially, the antihypertensive effect was thought that differ among themselves mainly in regard
to be a consequence of the diuretic action. It was to the dosage required for natriuretic activity.
subsequently found, however, that removal of Examples of these compounds are shown in
the 7-sulfonamide group from compounds of the Tables 2.8 and 2.9. The unique property of
chlorothiazide class eliminated the diuretic ef- these drugs is their ability to produce a much
fect but not the antihypertensive action (100, larger chloruresis associated with a greater
101). A compound of this type, diazoxide (70), is natriuretic potency than that of the carbonic
anhydrase inhibitor acetazolamide and its
congeners.
After oral administration to normal sub-
jects, hydrochlorothiazide is rapidly absorbed.
Peak plasma levels are reached after 2.6 +
0.8 h, and the drug is still detectable after 9 h.
Approximately 70% of a 65 mg dose was ac-
counted for in urine after 48 h (102). Hydro-
chlorothiazide and other benzothiazine di-
a much more effective antihypertensive agent. uretics are excreted by the kidneys both
Surprisingly, salt and water retention has been through glomerular filtration and tubular
observed with this compound (101). secretion. The latter is shared with other or-
ganic acids and is specifically inhibited by pro.
2.1.6 Hydrothiazides. The thiazide diuret- benecid. Concurrent administration of hydro-
ics have their greatest usefulness in the man- chlorothiazide and probenecid did not modify
agement of edema of chronic cardiac decom- the effects of hydrochlorothiazide on the uri.
pensation. In hypertensive disease, with or nary excretion of calcium, magnesium, and ci-
without overt edema, the thiazides have a mild trate. This combined therapy also prevented
antihypertensive effect. They are used with or abolished the increased serum uric acid lev-
caution in patients with significantly impaired els associated with the use of thiazide diuretics
renal function. In some patients with nephro-
Table 2.9 Hydrochlorothiazide Derivatives Structure-Activity Relationships of Canine Studies
Dose, Urine Natriuretic Partition
p.0. Excretion Na+ Excretion Kf Excretion Activity Coefficienta
No. Structure (P&K) (mL over 6 h) (meq over 6 h) (meq over 6 h) (Approx.) (EtherIWater)
Cont. 0 42.5-53.2 7.1-10.0 4.75

c1nN7
avg.
(28) 1250 102 18.5 6.5 1 0.08

/ NH
HzNSOz S/
\o

(72) R = CH,CH(Me), 1.3 59 13.8 4.3


20 100 20.7 6.8
(73) CHz(C&) 1.3 95 22.7 4.9 1000 10.2
(79) CH2Cl 20 83 17.2 4.0
310 131 30.6 6.1
(80) CHCl, 1.3 65 11.6 5.1 100 1.53
20 113 22.5 6.5
(81) CH,C& 74 18.6 4.0
"From ref. 79.
Diuretic and Uricosuric Agents

in varying degrees after oral administration. The metabolic fate of thiazides varies sig-
Chlorothiazide is absorbed to the extent of nificantly. Chlorothiazide and hydrochlorothi-
only about lo%, although other members of azide undergo very little metabolism, whereas
this family have a much higher bioavailability. the more lipid-soluble drug, indapamide (92,
Bile acid-binding resins, such as cholestyra- Table 2.101, undergoes extensive degradation.
mine, have been reported to bind to these Given that 90% of the sodium is reabsorbed
drugs and therefore prevent their absorption before it reaches the distal convoluted tubule,
(104). Generally these diuretics are highly the effectiveness of this class of diuretics is
plasma protein bound, primarily to albumin. limited.
Thiazides gain access to the renal tubule prin- 2.1.6.2 History. The new phase in the de-
cipally through proximal tubular secretion velopment of the thiazide diuretics was
and to a small extent by glomerular filtration. opened by the findings of de Stevens et al.
Thiazide diuretics work by inhibiting the elec- (log), that condensation of 4-amino-6-chloro-
troneutral Na+/ C1- cotransporter in the dis- 1,3-benzenedisulfonamide(27) with 1 mole of
tal convoluted renal tubules. It is believed that formaldehyde gives 6-chloro-3,4-dihydro-2H-
these agents compete for the C1- binding site 1,2,4-benzothiadiadiazin-7-sulfonamide-1,l-
on the cotransporter (105). dioxide (29), a stable crystalline compound.
As a class, the thiazides have an important This compound has been given the generic
action on potassium excretion. In most pa- name hydrochlorothiazide. It was surprising
tients, a satisfactory chloruretic and natri- that saturation of the 3,4-double bond in chlo-
uretic response is accompanied by significant rothiazide leads to a compound that is 10
kaliuresis; this is also seen in dog diuretic times more active in dogs (109, 110) and hu-
studies (see Table 2.9) (106,107). At low doses, mans (111-113) as a diuretic. Hydrochlorothi-
with some selected thiazides, a separation of azide (29) has less than one-tenth the carbonic
natriuretic and kaliuretic effects have been anhydrase inhibiting activity of chlorothiazide
observed, although at higher doses and re- (28, Table 2.4). Like chlorothiazide, it also ex-
peated administration these differences disap- erts a mild antihypertensive effect in hyper-
pear. The kaliuresis, although enhanced by tensive subjects (114).
the carbonic anhydrase activity of many of 2.1.6.3 StructureAdvity Relationship. Struc-
these compounds, is probably a consequence tureactivity relationships have been exten-
of increased delivery of sodium and fluid to the sively investigated by various groups (81,
distal segment of the nephron. Elevated se- 115-129). Substitution in the 6-position of hy-
rum uric acid levels, which may be associated drochlorothiazide follows the same rules as
with gout resulting from decreased uric acid found for chlorothiazide; that is, compounds
excretion during chronic thiazide administra- of approximately equal activity result when
tion, have been well documented (107). Cal- the substituent in the 6-position is C1, Br, or
cium excretion is decreased, and the excretion CF,. Compounds where R, = H or NH, are
of magnesium is enhanced by the administra- only weakly active. Substitution in the 3-posi-
tion of thiazide diuretics in normal subjects tion of hydrochlorothiazide has a pronounced
and in patients (103). Thiazide treatment has effect on the diuretic potency, and compounds
also been observed to increase plasma levels of that are more than 100 times as active as hy-
cholesterol and triglycerides (108). drochlorothiazide on a weight basis have been

yJNiH
H
CH:20
___)
/
H2N02S s'
04 \\o

(29)
2 Clinical Applications 81

obtained. It should be noted, however, that the


maximal diuretic and saluretic effect that can
be achieved with any of the thiazide diuretics
is of the same magnitude, although the dose
required may vary considerably.
Substituents in the 3-position of hydrochlo-
rothiazide having the most favorable effect on
activity were alkyl, cycloalkyl, haloalkyl, and
arylalkyl, all of which may be classified as hy-
drophobic in character. This is illustrated in
Table 2.9, where the structures of derivatives
of hydrochlorothiazide and the respective di-
uretic responses in the dog are shown. Table
2.8 lists the commercially available thiazide
diuretics and the respective optimally effec-
tive doses per day in humans. Beyer and Baer
(79)have studied four thiazides covering a
1000-fold increase in saluretic activity on a
log-stepwise basis from chlorothiazide to hy- though it induces primarily a saluresis, there
drochlorothiazide, to trichlormethiazide, to is a increased output of K+ and HCO,- at
cyclopenthiazide. This increase in activity ap- higher doses (70). Clinical studies have sub-
pears to correlate with their lipid solubility (in stantiated the pharmacological properties
terms of their phosphate buffer partition coef- (132-135). As with the thiazide diuretics, a
ficient), rather than their carbonic anhydrase mild antihypertensive effect was seen (136).
inhibitory effect (Table 2.9). The recommended clinical dosage is 50-200
mg daily or every other day.
2.1.7 Other Sulfonamide Diuretics. This 2.1.7.2 Hydrazides of m-Sulfamoylbenzoic
group of diuretics includes compounds that Acids. The diuretic properties of a large series
produce a pharmacological response similar to of compounds derived from 2-chlorobenzene-
that seen with the thiazide diuretics (i.e., they sulfonamide, with a wide variety of functional
are saluretics), and the maximally attainable groups in the 5-position (851, have been stud-
level of urinary sodium excretion is in the
5 same range as that of hydrochlorothiazide.
3 The compounds in this group differ in chemi-
1 cal structure; however, most of them are de-
r rivatives of m-sulfarnoylbenzoic acid.
e 2.7.7.1 Chlorothalidone. An interesting class
of compounds was developed from certain sub-
stituted benzophenones (130). Optimum di-
uretic properties were found in 3-(4-chloro-3- ied in the rat and in the dog (137).The R group
sulfamoylpheny1)-3-hydroxy-1-oxoisoindoline includes such functional groups as substituted
(82,chlorthalidone, Table 2.101, which is the iso- arnines, hydrazines, pyrazoles, ketones, ester
ric form of an ortho-substituted benzophe- groups, substituted carboxamides, and hydra-
none. The related compounds, (83)and (841,are zides. The hydrazides are the most active
ore potent carbonic anhydrase inhibitors than group of compounds. One of these, cis-N-(2,6-
chlorthalidone but are less active as diuretics dimethyl- 1-piperidyl)-3-sulfamoyl-4-chloro-
and have a shorter duration of action. benzamide (clopamide, 86, Table 2.10), has
Chlorthalidone has shown good diuretic ac- been studied in greater detail (137-139).
tivity in dogs (131) and is characterized by an A dose-related diuretic response was seen
unusually long duration of action. It is about in rats at doses of 0.01-1 mg/kg. In unanesthe-
70 times as active as hydrochlorothiazide as a tized dogs, a diuretic response was seen with
carbonic anhydrase inhibitor in vitro and, al- oral doses as low as 2 pg/kg. In anesthetized
Table 2.10 Other Sulfonamide Diuretics
Clinical Dose, p.0.
No. Generic Name Trade Name (mg) Structure Reference
84
(43) Mefruside Baycaron 25-100
I P?

Chlorthalidone Hygroton

Clopamide Aquex,
Brinaldix

Alipamide
25-100 148
Nefrolan

Diapamide Vectren

Metolazone Zaroxolyn

Quinethazone Hydromox

Indapamide Iparnix,
Natrilix
Diuretic and Uricosuric Agents

dogs, the natriuretic response observed was and antihypertensive activity in rats, dogs, and
dose related between 0.01 and 1 mgkg admin- humans after oral administration. In humans, 5
istered i.v. The drug produced a prompt in- mg of indapamide administered daily produces a
crease in urine flow and increased the excre- greater and more consistent lowering of blood
tion of sodium, potassium, and chloride. A pressure than 500 mg of chlorothiazide given
small increase in bicarbonate excretion, which daily (144). Indapamide at a daily dose of 2.5 mg
did not significantly alter plasma or urinary decreases serum potassium levels 0.5 meqL and
pH, was also noted. During maximal diuresis increases uric acid levels to about 1.0 mg/lOO
produced by hydrochlorothiazide, administra- mL. Replacement of the indoline moiety of in-
tion of clopamide had no effect on sodium ex- dapamide has also yielded compounds that pos-
cretion. Conversely, after a maximally effec- sess potent diuretic activity. The l-methylisoin-
tive dose of clopamide, hydrochlorothiazide doline analog (93)had a diuretic activity similar
was without effect, although in both cases an
additional response to furosemide and spi-
ronolactone was observed. This suggests that,
although clopamide is not a thiazide diuretic,
its natriuretic action closely resembles that of
the thiazides. The recommended clinical dose
is 10-40 mglday.
A related hydrazide, alipamide (87, Table
2.10), is an effective diuretic agent in rats, dogs,
monkeys, and humans (140). The suitable ther-
apeutic dosage in humans is 20-80 mglday. Nip
amide exhibits primarily a saluretic action; car- to that of indapamide; however, it possessed an
bonic anhydraseinhibition becomes an important improved Nat/K+ excretion ratio (145). This
fador only at high dose levels. A structureactivity compound was later found to cause blue pigmen-
study, in rats, showed that a hydroxamic acid moi- tation of the fur and some internal organs in rats
ety could replace a hydrazidegroup without loss of and mice at 500 mgkg p.o (146). The tram-pyr-
activity (141). rolidine derivative (94) has been found to be a
Similarly, diapamide (89)is an effective sal-
uretic agent in rats, dogs, and monkeys (142). In
humans, the compound is comparable to the
thiazides in terms of urine volume and electro-
lyte excretion (143). Elevated plasma urate and
glucose levels accompany chronic administra-
tion. The clinically effective dose is 500 mglday.
Indapamide (92, Table 2.101, a related sul-
phamoylbenzamide, possesses both saluretic

more potent diuretic and natriuretic agent


than indapamide in the rat, with an improved
Na+/K+ratio (147).
2.1.7.3 1-Oxoisoindolines. Interesting re-
sults have come from studies on a series of>
4-chloro-5-sulfamoyl-N-substituted phthali.,
mides (95). Maximum activity, about six times 1
that of chlorothiazide on a weight basis, was
seen when the N-substituent was a saturated
ring containing six to eight carbons. Com-
pounds in which R represented a smaller or
larger ring were less active. When R was lower
2 Clinical Applications

Table 2.11 Diuretic Activity i n Rats


of 2-Substituted 1-Oxoisoindolinesa

O
*R
N
-lc
/
HzNOzS
0

(95)
Diuretic
alkyl, decreased activity was also found. Re- Activity
duction of one of the carbonyl groups to yield (Chlorothiazide
the corresponding 3-hydroxy-1-oxoisoindoline No. R = 1)

(96, R = cyclohexyl) resulted in a 10-fold in- (88) Cyclohexyl


(98) Isobutyl
OH (99) Cyclopentyl
(100) 4-Methylcyclohexyl
(101) 3-Methylcyclohexyl
(102) 3,4-Dimethylcyclohexyl
(103) Cycloheptyl
(104) Cycloodyl
(105) Norborn-2-yl
(106) Cyclohexylmethyl
"From ref. 148.
8 crease in votencv. Com~letereduction of the
A
in vivo involvement of renal carbonic anhy-
a methylene yielded clo-
drase inhibition. As is the case with the thia-
'
I
rexolone (88, Table 2.lo), which was 300 ti.mes
more active than chlorothiazide on a weight zide diuretics, elevated serum uric acid levels
are seen, but there may well be less propensity
1 basis when tested in the rat (148). Interest- for hyperglycemia (149-151). In contrast to
ingly, reduction of the other 0x0 group to yield
the isomeric 6-chloro-5-sulfamoyl-1-oxoioin- the thiazide diuretics, insignificant amounts
I doline (97) resulted in complete loss of activ-
of the drug are excreted unchanged in humans
and in dogs. The metabolites are compounds
monohydroxylated on the cyclohexane ring.
Neither the compound nor its metabolites are
stored in the body tissues (152a).
2.1.7.4 Quinazolinone Sulfonamides. Re-
placement of the ring sulfone group in the
thiazides by a carbonyl yields quinazolinones
and dihydroquinazolinones (107) and (108),

ity. Structure-activi.ty relationships falr a


number of 2-substitu.ted 1-oxoisoindoline~ 9 are
shown in Table 2.11. Methylation or acetyla-
tion of the sulfamoyl group of clorexolone de-
creases the activity by at least a factor of 10. In
humans, clorexolone (88)is a potent diuretic.
The clinical dose is 25-100 mglday; the pat-
tern of water and electrolyte excretion is sim-
ilar to that caused by the benzothiadiazine di- respectively. These compounds produce nearly
uretics. Urinary pH and HC0,- excretion the same diuretic response as that of the par-
remain unchanged after administration of clor- ent thiazide derivatives; the dihydro-deriva-
exolone, indicating that there is no significant tives again are more active on a dosetkg basis.
Diuretic and Uricosuric Agents

tion of urate or glucose or signs of toxicity


were seen in a short-term study (159). The
recommended clinical dose is 5-20 mglday. In
hypertensive patients a double-blind study
compared a dose of 50 mg of hydrochlorothia-
zide with 2.5 and 5.0 mg of metolazone, and
similar effects on blood pressure were ob-
served. The effects on other parameters (e.g.,
body weight, electrolytes, serum uric acid, and
Substitution at R, by alkyl is disadvantageous, blood sugar levels) were also comparable (160).
in that it reverses the favorable C1- and K+ In a study in patients with nonedematous, sta-
excretion patterns seen in the few examples ble chronic renal failure, a high dosage of meto-
studied (152b). The preferred member of the lazone (20-150 mg) increased urine flow signif-
series was quinethazone (91, Table 2.10; 108, icantly. Its activity was greater than that of the
R, = Et, R, = HI, which in humans has the thiazides, which are ineffective at glomerular fil-
same order of potency as that of hydrochlo- tration rates of less than 15-20 mumin.
rothiazide with a high Nat/Kt excretion ratio. Fenquizone (109),which is structurally re
The duration of activity appears to be about lated to metolazone, has a thiazide-like diuretic
24 h (154). The recommended clinical dose is
50-200 mg/day.
A series of dihydroquinazolinones substi-
tuted in the 3-position (108, R, = aryl and
arylalkyl) were studied and it was shown that
some of these compounds are highly active di-
uretics. The more active derivatives have at
least one hydrogen in the Pposition, a primary
SO,NH, group in the 6-position, and an ortho
or para lower alkyl or CF,-substituted aro-
matic ring in the 3-position of the quinazoline
nucleus. The most interesting member of the
series is metolazone (90, Table 2.10; 108, R, = profile, but it has less of an effect than that of the
Me, R, = o-Me-C,H,) (153).Studies in normal thiazides on carbohydrate and lipid metabolism
volunteers led to the conclusion that metola- (161). In patients with mild essential hyperten-
zone exerts its effect in the proximal tubule sion, a 12-month study with fenquizone (10 mgl
and in the cortical segment of the ascending day) showed that the compound sigmficantly
limb of Henle of the early distal convoluted lowered systolic and diastolic blood pressure. Se-
tubule. The absence of significant bicarbona- rum levels of glucose, triglycerides, and choles-
triuria is evidence against carbonic anhydrase terol remained unchanged; however, uric acid
inhibition. Metolazone did not impair the abil- levels increased slightly.
ity to acidify the urine normally in response to 2.1.7.5 Mixed ~ulfamide' Diuretic-Antihy-
an oral load of NH,Cl, and it was concluded pertensive Agents. Recent attempts have been
that metolazone has no effect on the distal Ht made to combine an o-chlorobenzenesulfon-
secretory mechanism (155-157). In dogs, me- amic diuretic moiety with another molecule
tolazone was found to be excreted by glomer- with known antihypertensive activity. Mencel
ular filtration and renal tubular secretion. and coworkers synthesized compounds in
The secretory mechanism was antagonized by which they covalently joined enalaprilat, a
probenecid; however, this did not affect the known angiotensin-converting enzyme (ACE)
diuretic action of metolazone (158).A 10-15 inhibitor, to several known thiazide diuretics
mg dose of metolazone was approximately and arylsulfonarnides (162). Compound (110)
equivalent to 50 mg hydrochlorothiazide, and was found to be a potent ACE inhibitor in
the time course of diuretic action was similar vitro.In a sodium-depleted spontaneously hy-
to that of hydrochlorothiazide. No acute eleva- pertensive rat (100 mgkg i.p.1, (110)reduced
2 Clinical Applications 87

blood pressure by 41-42%.Compound (110) spontaneously hypertensive rats, oral admin-


did elevate potassium and sodium excretion in istration of 0.330 mglkg of this compound de-
the rat but it was found to be less potent than creased mean arterial pressure and induced
chlorothiazide in this model. saliuresis. The duration of action of BMY-
Fravolini and coworkers have covalently 15037-1 was similar to that of prazosine.
linked an o-chlorobenzesulfonamicdiuretic to 2.1.7.6 Tizolemide. A structurally novel
a propanolamine p-blocking pharmacophore type of sulfonamide diuretic was developed by
(163). Compounds (111) and (112) possess Lang and coworkers at Hoechst (164). Ti-
both p-adrenergic antagonist and diuretic ac- zolemide (114) was selected from a series of
tivity in the rat. At an equimolar dose, (111) compounds for further investigation. Optimal
produced a saluretic effect similar to that of activity was associated with an unsubstituted
hydrochlorothiazide, but as a p-blocker it was sulfamoyl group. In dogs the diuretic activity
weaker than cartel01and propranolol. was similar to that of hydrochlorothiazide. In-
BMY-15037-1 (113) is a chlorosulfamoyl- terestingly, tizolemide lowered serum uric
isoindolone derivative that has both diuretic acid levels in the cebus monkey, indicating a
and a-adrenoceptor antagonistic effects. In possible uricosuric effect.
88 Diuretic and Uricosuric Agents

Table 2.12 High Ceiling Diuretics


Clinical Dose,
No. Generic Name Trade Name P.O. (mg) Structure I
Ethacrynic Acid Edecrin

(117) Bumetanide Burinex,


Lunetron
1-5
fi NHBu

(118) Furosemide Lask

(119) Piretanide

(120) Xipamide Aquaphor 40-80

(121) Azosemide Luret


I

I 2 Clinical Applications 89

Table 2.12 (Continued)


Clinical Dose,
No. Generic Name Trade Name p.0. (mg) Structure
(122) Torasemide Unat, 2.5-20
Toradiur

0 0 HN

N
H

administration; however, the bioavailability is


xHCl low because of hepatic first-pass metabolism.

4-
H2N02S
HO N
2.1.8 High Ceiling Diuretics. The term
high ceiling diuretics has been used to denote
a group of diuretics that have a distinctive ac-
/ NCH3 tion on renal tubular function. As suggested
by their name, these drugs produced a peak
(114)
diuresis far greater than that observed with
other diuretics. These agents act primarily by
2.1.7.7 Bemitradine. Workers at Searle ex-
inhibiting the reabsorption of sodium in the
tended work on a series of previously de-
thick ascending loop of Henle and, thus, they
scribed tetrazolopyrimidines that were shown
are also commonly referred to as loop diuret-
to be antihypertensive agents in rats and in
humans (165, 166).A series of triazolopyrimi- ics. This class of diuretic agents holds few
dines were prepared and SC-33643 was found structural features in common. Because they
tobe the most potent. Bernitradine (SC-33643, are most alike in potency and with respect to
115) has a thiazide-like profde of diuresis but their renal site of action, they represent more
a pharmacological rather than a chemical
class of agents. The high ceiling or loop diuret-
ics now in use or currently being studied are
shown in Table 2.12.
2.1.8.1 Ethacrynic Acid. The clinical dose
of ethacrynic acid (116) lies between 50 and
200 mgtday. In long-term studies, the antihy-
pertensive effects of 100 mg of ethacrynic acid
were similar to 50 mg hydrochlorothiazide in
patients with mild hypertension (168).
Ethacrynic acid continues to be an effective
diuretic even at very low glomerular filtration
is not a sulfonamide. Bemitradine (115) was rates, and therefore is useful in the treatment
5.5 times more potent than hydrochlorothia- of patients with chronic renal failure (169).
zide after oral administration in the unanes- Ototoxicity has been reported that manifests
thetized dog (167) and significantly increased itself as transient deafness (169). Permanent
renal blood flow and glomerular filtration deafness has also been observed after treat-
rates. Bemitradine is well absorbed after oral ment with high doses of ethacrynic acid in re-
90 Diuretic and Uricosuric Agents

Table 2.13 Structure-ActivityRelationships of Ethacrynic Acid Analogs


Diuretic Activity
No. Structure (Dog i.v.1 t,,, (min)" Reference

nal failure (170).Ethacrynic acid possesses ex-


cellent oral absorption and rapid onset of
action is seen when it is administered orally or
intravenously (171). Ethacrynic acid is exten-
sively metabolized to its cysteine adduct after
oral administration. It is believed that it is this
metabolite that represents the pharmacologi-
cally active form of the drug because it has a
much greater activity than that of the parent
compound (172). About two-thirds of the com-
2 Clinical Applications

Table 2.13 (Continued)


Diuretic Activity
No. Structure (Dog i.vJ t , , (mida Reference

"t,, = time in minutes required for one-half of a standard amount of test compound to react with excess mercaptoacetic
acid at pH 7.4 and 25°C in DMF-phosphate buffer, analogous to the procedure of Duggan and Noll(176).

highly active diuretics were developed that and (124). They are highly active in the dog
were thought to react selectively with func- when administered orally or parenterally, but
tionally important sulfhydryl groups, or possi- are inactive in the rat.
bly other nucleophilic groups, that were essen- A marked increase in diuretic activity was
tial for sodium transport in the nephron. observed when chlorine was introduced ortho
These compounds generally contain an acti- to the carbonyl group of the aryl side chain.
vated double bond attached to a moiety con- Not only was the diuretic activity better, but
taining a carboxylic acid group of a type ex- the rate at which chemical addition of sulfhy-
pected to assist transport into, or excretion by, dry1 compounds across the double bond in an
the kidney. The general structure of these in vitro system increased. The presence of two
compounds is exemplified by formulas (123) chlorines in positions 2 and 3 of the phenoxy-

of X, Y = H, C1, R1, R2 = CH3C0, NOz, CN, alkyl


acetic acid further increased the activity. A able from the values found in the placebo
number of compounds corresponding to struc- group; MK 196 thus appears to be a diuretic ,
tures (123) and (124) and their diuretic activ- without uric acid-retaining properties (178).
ity in dogs are shown in Table 2.13 (173,174). In a comparison of the diuretic effects of MK
Ethacrynic acid (116) is the most interesting 196 and furosemide in normal volunteers re-
compound of this series and has been studied ceiving the drug every day for 14 days, an oral
extensively. A report on (diacylvinylary1oxy)- dose of 10 mg of MK 196 caused a gradual
acetic acids has shown that compound (130) diuretic and saluretic response, resulting in a
(Table 2.13) is approximately three times as maximal plateau during the period 4-7 h after
active as ethacrynic acid (175). The corre- drug administration followed by a slow return
sponding (acylvinylary1oxy)acetic acids (e.g., to baseline during the next 16-18 h. Although
131) are less active (176). The 4-(2-nitropro- at the doses studied (10 and 20 mg, MK 196),
peny1)phenoxyacetic acid derivatives (e.g., the maximal response to furosemide (40 mg)
132) are also three to five times as active as was always higher than the maximal response
ethacrynic acid (177). to MK 196, the total 24-h saluresis after 10 mg
Ethacrynic acid does not inhibit carbonic of MK 196 was equivalent to that produced by
anhydrase in vitro. It has a steeper dose-re- furosemide. After 20 mg of MK 196 the 24-h
sponse curve than that of hydrochlorothia- response was greater than that with furo-
zide, and the magnitude of its maximum semide (179). A double-blind pilot study was
saluretic effect is several times that of hydro- conducted to compare the antihypertensive ef-
chlorothiazide (171). The renal corticomedu- ficacy of two doses of MK 196 (10 and 15 mg)
lary electrolyte gradient, after administration with 50 mg hydrochlorothiazide in patients
of ethacrynic acid and other high ceiling di- with mild to moderate hypertension. Both
uretics, is virtually eliminated as a result of doses of MK 196 lowered blood pressure as
nearly total inhibition of Na' transport in the much as or more than 50 mg hydrochlorothi-
ascending limb of Henle (172). azide during the 24-h period after drug admin-
2.1.8.2 lndacrinone. Indacrhone or MK 196 istration (180).
(133) is an indanyl derivative of ethacrynic Clinical results indicated that MK 196 is a
highly active diuretic with a gradual onset of
action that reaches a plateau that persists for
4-7 h, then gradually returns to baseline val-
ues over the next 16-18 h. This is probably
attributable to the long half-life of this com-
pound as observed in animals. The antihyper-
tensive effects are comparable to those ob-
served with hydrochlorothiazide.
Workers at Merck discovered that annula-
tion of the unsaturated ketone side chain on
the aromatic ring yielded compounds that
acid. In clinical studies in healthy subjects retained their diuretic activity but that also
consuming a standard diet, 10 mg of MK 196 possessed uricosuric properties. Both enantio-
produced a slightly smaller diuresis than 40 mers of indacrinone possess uricosuric activ-
mg of furosemide, and MK 196 did not influ- ity but the (-)-enantiomer is the more potent
ence uric acid excretion or 24-h urate clear- diuretic (181). Clearance studies in the rat in-
ance. A single dose of 40 mg of furosemide dicated that MK 196 is a potent diuretic acting
caused uric acid retention with a significant in the ascending limb of Henle, which results
decrease of 24-h urate clearance; prolonged in significant increases in urinary excretion of
administration caused a statistically signifi- sodium, calcium, magnesium, water (182),
cant increase in plasma uric acid levels. Pro- and uric acid (183).
longed administration of MK 196 did not in- The physiological disposition of 14C-labeled
crease plasma uric acid levels, and the ratio of MK 196 was studied in the rat, dog, monkey,
urate-creatinine clearance was indistinguish- and chimpanzee. The drug was well absorbed
2 Clinical Applications

and showed minimal metabolism in the rat, rat, dog, rhesus monkey, and baboon (187).
dog, and monkey. Triphasic rates of elimina- The half-life of the compound in the rat and
tion of drug and radioactivity were observed in dog is about 2 h. In the monkey and baboon it
these three species. In dogs, the terminal half- was found to have a much longer half-life, 18
life was estimated to be about 68 h; in mon- and 40 h, respectively. In all species less than
keys, there was a longer terminal half-life of 5% of MK-473 is excreted intact in the urine.
approximately 105 h. The long terminal half- In humans, this compound is well absorbed,
life of this compound may result in part from but it undergoes extensive metabolism. Re-
binding to plasma proteins. The major route of lated compounds from this series have been
radioactivity elimination is through the feces studied for their therapeutic utility in the area
for the rat (-80%). In contrast, the monkey of brain injury (188).
and the chimpanzee eliminate the majority of 2.1.8.3 Other Aryloxy Acetic Acid High
the dose through the urine. Minimal metabo- Ceiling Diuretics. Since the discovery of
lism of MK 196 was observed in the rat, dog, ethacrynic acid and indacrinone many new
and monkey; however, in humans and in members of the phenoxyacetic acid family of
chimpanzees, there was extensive biotrans- diuretics have been reported.
formation. The major metabolite resulted A series of [(3-aryl-l,2-benzisoxazo1)-6-
from para hydroxylation of the phenyl group yloxy] acetic acids were described by Shutske
to yield [6,7-dichloro-2-(4-hydroxypheny1)-2- and coworkers at Hoechst (189).Of this group,
methyl-1-0x0-5-indanyloxyl-acetic acid (134). HP 522 (136) was found to be a potent diuretic

This metabolite accounted for more than 40%


of the 0- to 48-h urinary radioactivity; about
20% of the radioactivity was accounted for as in mice and dogs. It was found to lower plasma
unchanged drug (184,185). uric acid levels in chimpanzees after chronic
The cyclopentyl analog MK-473 (135) has administration (10 mg kg-' day-', p.0.) (190).
diuretic and uricosuric activity similar to that Workers at Merck reported on a series of
2,3-dihydro-5-acylbenzofurancarboxylic acids
(191). The 5(2-thienylcarbony1)-2-benzofur-
ancarboxylic acid derivative (137) was found
to have a higher natriuretic ceiling than that
of hydrochlorothiazide and furosemide in the

of indacrinone (133),but it also possesses sub-


stantial antihypertensive activity (186). The
physiological disposition of this compound has
been studied in several species including the
Diuretic and Uricosuric Agents

rat. Resolution of the enantiomers and testing group, showed that these compounds were ef-
in the chimpanzee revealed that the S-enan- fective diuretics. The isomeric series (140) did
tiomer is responsible for the compounds' di- not show saluretic properties (176, 193).
uretic and saluretic activity. More than 100 variously substituted deriv-
Plattner and coworkers at Abbott have dis- atives were studied pharmacologically, but
closed a series of 5,6-dihydrofuro[3,2-fl-l,2- only those that corresponded to the general
benzisoxazole-6-carboxylic acid high ceiling structure (139)exhibited outstanding saluretic
diuretics (192). Abbott 53385 (138) had di- activity. The most active was furosemide (118,
R = furfuryl; Table 2.12). In contrast to the
dihydrobenzothiadiazine diuretics, where the
substitutent in the 3-position of the heterocy-
clic ring can be varied to a considerable degree,
the requirements for high activity in the 5-sul-
famoylanthranilic acid series are much more
stringent. On parenteral and oral administra-
tion to different species and to humans, the
degree of diuretic effect elicited, as measured
by urine flow and Naf and C1- excretion, was
several times that obtainable with the thiazide
diuretics (194, 195).
In a study undertaken to explore the effect
uretic effects similar to those of furosemide in of furosemide on water excretion during hy-
the saline-loaded mouse. In the conscious dog, dration and hydropenia in dogs, it was found
it was about six times more potent than furo- that as much as 38% of filtered sodium was
semide. Resolution of the enantiomers and excreted during furosemide diuresis and both
pharmacological evaluation showed that only free water clearance (CHz0)and solute-free
the S-isomer displays the diuretic and sal- water reabsorption (TC,,,) were inhibited,
uretic activity. In humans the drug was well indicating a marked effect in the ascending
tolerated and no adverse effects were noted loop of Henle. Furosemide is largely excreted
(192b). At 20,40, 60, and 80 mg a significant unchanged in the urine, but a metabolite,
dose-related increase in urine volume, so- 4chloro-5-sulfamoylanthranilicacid, has been
dium, and chloride excretion were produced. identified (196, 197). Studies by Hook et al.
Hepatic clearance is the main route of excre- (198) and Ludens et al. (199) indicated that
tion in humans. Very little of the drug is elim- furosemide reduces renal vascular resistance
inated in the urine. and thus enhances total renal blood flow in
2.1.8.4 Furosemide. At the time work on dogs. Clinical studies in normal subjects and
ethacrynic was proceeding at Merck, Sharp & in patients with edema of various etiologies
Dohme, furosemide was being developed in have clearly shown that furosemide is an ex-
the Hoechst laboratories in Germany. Investi- tremely potent saluretic drug (200,201).
gation of a series of 5-sulfarnoylanthranilic ac- Furosemide is rapidly absorbed after oral
ids (139), substituted on the aromatic amino administration in humans. It is highly bound

HO P0
/
SOzNHz

(140)

.yl), -CHz(2-thienyl)
2 Clinical Applications

to plasma protein, primarily to albumin, with NHBu


bound fractions averaging about 97%. The
bioavailability of furosemide is 50 -70% in nor-
mal subjects; it is eliminated by renal, biliary,
and intestinal pathways; it is excreted as its
glucuronide; and it is unchanged by the renal
route.
The antihypertensive effects of furosemide
were shown to be qualitatively and quantita-
tively similar to those of chlorothiazide in non-
edematous patients with essential hyperten- proached the activity of furosemide when
sion (202). Furosemide has been reported to given i.v. (10 mg/kg in a NaOH solution) to
produce moderate diuretic response in patients dogs. Interestingly, whereas in the anthranilic
with renal disease and resistant edematous acid-furosemide series the N-furfuryl sub-
states when other diuretics (e.g., thiazides, stituent afforded outstanding activity, this
mercurials, triamterene, and spironolactone) was not the case in this series (206).
have failed. Doses up to 1.4 glday may be re- Further investigation showed that com-
quired (203, 204). Ototoxicity has also been pounds, in which R2 = -OC6H5,-NHC6H5, or
reported after large doses of furosemide (205). -SC6H5 in the generic structure (141), were
2.1.8.5 Bumetanide. In a series of studies very active diuretics; the structures and sal-
starting in 1970, Feit and coworkers in Den- uretic activity in dogs of the more active deriv-
mark investigated the diuretic activity of de- atives are shown in Table 2.14.
rivatives of 3-amino-5-sulfamoylbenzoic acid Compound (148) (bumetanide, Table 2.14)
(141). In the initial series, R2was chlorine and was the most interesting drug and has been
studied extensively (207). Further structure-
activity studies uncovered related compounds
with equally high diuretic potency; com-
pounds (150-166) (Table 2.15) are represen-
tative of the series studied. It was found that a
phenoxy group in the 4-position enhances ac-
tivity in the anthranilic acid series as well as in
the 3-amino-5-sulfamoylbenzoic acid series
(e.g., compound 150) (208). The phenoxy
group could be replaced by C,H5C0, C6H5CH2
(209), and even a directly bonded C6H5group
R, was varied widely from alkyl to substituted (210) (e.g., compounds 151, 152, and 158)
benzyl. One of the most interesting com- (Table 2.15). Interestingly, an equilibrium ap-
pounds in this series was (142) (3-butylamino- pears to exist between (151) and the corre-
4-chloro-5-sulfamoylbenzoic acid), which ap- sponding benzoyl derivative (167).
96 Diuretic and Uricosuric Agents

Table 2.14 Compounds Related to Burnetanidea


Urinary Excretionb
Dose, p.0. Volume
No. Structure (mg/kg) (mL/kg Urine) Naf

OH
NHBu

opened benzoyl derivative (167). Compounds heating (210).


(161) and (162) (Table 2.151, which do not In the 3-amino-5-sulfamoylbenzoic acid
have an amino substituent attached to the ries, the 3-amino substituent can be repla
p.0. Volume
No. Structure (mgflrg) (mL/kg Urine) Na+ Kf C1-

"From Ref. 204.


*Per6 h in dog (meqkg).

by an -OR or -SR group (155,159,162, Table terns of these compounds resemble those of
2.15) (210,211);however, oxidation of the -SR previously discussed sulfarnoylbenzoic acids.
group to -SO,R (156, Table 2.15) eliminates However, substitution of the sulfamoyl group
the diuretic activity (212). Compound (162) by the spatially and sterically similar methyl-
(Table 2.15) is one of the most potent benzoic sulfonyl group generally led to decreased po-
acid diuretics ever reported. It shows signifi- tency. Substitution of methylthio or methyl-
cant diuretic activity in dogs at 1 ~ g / k gwhich
, sulfinyl for the methylsulfonyl group reduced
represents a potency approximately five times the potency considerably (e.g., 164, Table
as high as that of bumetanide (210). In the 2.15). The anthranilic acid analog (166, Table
anthranilic acid series, the structural require- 2.15) of the highly active 3,4-substituted
ments are more exacting and the thiosalicyclic methylsulfonylbenzoic acid derivative (163)
acid analog (157, Table 2.15) is only weakly (Table 2.15) was inactive at the dose tested,
active. again confirming that the structural require-
A series of compounds in which the sulfa- ments in the anthranilic acid series are more
moyl group was replaced by a methylsulfonyl demanding.
group was also investigated (213). Many of Replacement of the chloro group in hydro-
the 5-methylsulfonylbenzoic acid derivatives chlorothiazide, by a C,H,S group (168)elimi-
showed considerable diuretic activity (e.g., nated the diuretic activity (214). Similar re-
163 and 165, Table 2.15). The diuretic pat- sults were found in the case of quinethazone
1 2 Clinical Applications
i
E

Bumetanide is a potent diuretic in dogs af-


ter both oral and intravenous administration.
It is comparable to furosemide in its type of
action and its maximum effect, but when
given orally bumetanide is approximately 100
times more active. In dogs the drug is excreted
rapidly by glomerular filtration and tubular
secretion. No metabolites were detected in
dogs (218). A parallel between bumetanide ex-
cretion and saluretic action in humans over
the total period of response has been shown
and clopamide (91,86,Table 2.10). Structural (219). The drug is a highly potent diuretic in
, modifications of bumetanide (148, Table 2.14) patients with congestive heart failure (220,
F were further explored by Nielsen and Feit 221) and in subjects with liver cirrhosis (222).
(215).It was found that the carboxyl group in Studies in humans indicate a major site of ac-
the 1-position of bumetanide could be replaced tion in the ascending limb of Henle; a signifi-
by a sulfinic or sulfonic acid group or con- cant phosphaturia induced during the period
verted to an aminobenzyl group (216) (169 to of maximum diuresis also suggests additional
action in the proximal tubule (223, 224). Bu-
metanide produces a rapid diuretic response,
with a pattern of salt and water excretion re-
sembling that of furosemide. At the time of
maximal diuresis, 13-23% of the filtered load
of sodium is excreted; urinary calcium and
magnesium also increase. As with other sul-
fonarnide diuretics, hyperuricemia occurs af-
ter prolonged therapy. The bioavailability of
bumetanide after oral administration is 72-
96% in normal subjects and diuresis usually
begins within 30-60 min. Bumetanide is
170) with retention of diuretic activity. In a highly bound to plasma protein (94-97%)and
further modification, the sulfamoyl group in thus probably gains access to its site of action
the &position was replaced by a formamido by secretion into the proximal tubule.
group (171) (217); this compound has approx- Probenecid, however, fails to alter bumet-
anide-induced diuresis in cats and in humans
(225, 226). Bumetanide is quickly eliminated
by metabolism and urinary excretion from the
body and has a plasma half-life of 1.5 h. Sev-
eral metabolites have been identified after oral
administration of 14C-labeled bumetanide in
humans (227). All of the metabolites isolated,
with exception of the conjugates, involved ox-
idation of the n-butyl side chain. About 80% of
bumetanide is excreted from the body through
the renal route, with about 50% of the drug
excreted unchanged by this route. The major
metabolite detected is the 3'-alcohol (172).
The remaining 20% of the drug is excreted by
intestinal elimination. The 2'-alcohol (173) is
imately one-tenth the activity of bumetanide. the major metabolite found in the bile and feces.
The electrolyte excretion pattern is still simi- 2.1.8.6 Piretanide. Workers at Hoechst
lar to that of bumetanide. synthesized a number of 3,4-disubstituted
Diuretic and Uricosuric Agents

In patients with hypertension, long-term


treatment with piretanide was shown to sig-
nificantly lower blood pressure (233). From
animal studies it appears that piretanide has a
direct effect on the vascular tissue, but the
mechanism is unknown (234). In the sponta-
neously hypertensive rat, chronic treatment
of drug (30 mglkg) attenuated the pressor re-
sponse to angiotensin I1 and phenylephrine
(235). Piretanide also lowered blood pressure
in the anesthetized dogs without significant
cardiac effects (236). At high doses negative
chronotropic and inotropic effects were ob-
served. These effects were not observed for
furosemide given at 1-3 mgikg.
2.1.8.7 Azosemide. Azosemide (121) is a
sulfamoyl diuretic that was developed at
Boehringer Mannheim (243). It is about five
times more potent than furosemide after i.v.
dosing, although it is equipotent after oral ad-
ministration. Azosemide is poorly absorbed in
the gastrointestinal tract and has a bioavail-
ability of about 10% (231). Clearance studies
have shown that the main site of action of
azosemide is in the loop of Henle and to a
5-sulfamoyl- benzoic acids similar to bumet- lesser extent in the proximal tubule (238). In
anide but using a new synthetic method to in- normal subjects, azosemide has a slower onset
corporate the amine group in the presence of of action than that of furosemide but at 4,8,
the other functional groups (228). Piretanide and 12 h, volume and sodium excretion levels
(119, HOE 118) is the most interesting com- were similar. The compound is extensively
pound in the series. Micropuncture and clear- metabolized and only about 2% of the drug is
ance studies indicate that the primary site of excreted unchanged after oral dosing (239).
action is in the thick ascending loop of Henle Glucuronide conjugates and 5-(2-amino-4-
(229). Tritiated piretanide was prepared and chloro-5-sulphamoylphenyl)tetrazolehave been
was found to bind to two sites in purified mem- the identified metabolites (240). Azosemide is
branes of dog kidney medulla (230). Studies stable in solutions of various pH values, rang-
have shown that a 6 mg dose of piretanide ing from 2 to 13, up to 48 h (241). The drug
provides equipotent diuresis as 40 mg of furo- shows some instability at pH 1. The recom-
semide or 1 mg of bumetanide. However, com- mended dose to treat fluid retention is 80-160
pared to furosemide and bumetanide, piret- mg orally or 15 mg i.v. The compound's poor
anide causes a lower level of potassium bioavailability and slower onset of action may
excretion and, like bumetanide and furo- limit its use when rapid diuresis is required.
semide, piretanide causes an increase in se- 2.1.8.8 Xipamide. Xiparnide (120, Table
rum uric acid levels. Piretanide is well ab- 2.12) is a derivative of 5-sulfamoyl salicyclic
sorbed in the gastrointestinal tract after oral acid (242); 4-chloro-5-sulfamoylsalicyclic acid
administration and its bioavailability is itself had previously been reported by Feit and
greater than 95% in normal subjects and in pa- coworkers (211) to be a high ceiling diuretic.
tients with renal failure (231). The kinetics of Investigation of esters; aliphatic, cycloaliphatic,
absorption were studied in normal volunteers aromatic, and heterocyclic amides; ureides; and
that were immobilized (232). The drug was hydrazides of 4-chloro-5-sulfamoylsalicyclicacid
found to be absorbed directly from the stomach showed that 4-chloro-2',6'-dimethyld-sulfa-
when administered through a gastroscope. moylsalicylanilide is the most active derivative.
2 Clinical Applications

Table 2.16 Analogs of Xipamidea


--

H~NO~S'
Urine Volume in Rats Urine olume
No. R Group
Control
(120)

f Replacement of the C1group by Br, F, or CF, led ingly, the 2,6-dimethylpiperidino- derivative
tocompounds with lower activity. The effects of (183, Table 2.16) related to clopamide (86) is
% modification of the anilide group are shown in inactive (243).
Table 2.16 (242).Compounds methylated on ox- Xipamide is active in rats and dogs after
ygen andlor nitrogen are less active. Interest- oral or intravenous administration. Applica-
Diuretic and Uricosuric Agents

Table 2.16 (Continued)

54- H

Urine Volum I Rats Urine Volume in Rats


No. R Group (mLikg at 1 mg/kg) (mL/kg at 100 mglkg)
0.60b 31.2

-Q
(179)

H3C

:>
H3C

H3C
"From Ref. 242.
bDose(in m&g) that increases 5-h urine volume by 50%.

mgtkg i.v. in the dog, accompanied weak car anhydrase inhibitor (E


nuous infusion of 5% mannitol so- 1.1 x 10 and is comparable to su
lition, led to a diuretic effect starting 40 min nilamide (ED,, = 1.3 x l o p 5M ) o
postinjection. After 2 h, a diuretic effect chlorothiazide (ED,,- - = 2.3 X l o p 5M (Tal
could still be detected. An antihypertensive 2.4) (245).
effect in spontaneous hypertensive rats was Xipamide is aksorbed quickly from the
observed after 1 mglkg p.o (244).Xipamide is trointestinal tract and has a bioavailability
2 Clinical Applications

about 73%. At its therapeutic plasma level, it


is 99% bound to plasma proteins (246).
Twenty-four hours after oral administration,
about 50% of the drug is excreted unchanged
and about 25% as its glucuronide conjugate.
The plasma radioactivity half-life of 35S-xip-
amide was 5 h after intravenous administra-
tion and between 5.8 and 8.2 h after oral ad-
ministration (247).
The diuretic profile of xipamide is mixed; it
behaves both as a loop diuretic and as a thia-
aide diuretic. In normal volunteers, doses of
0.5 mg/kg p.0. of xipamide are more effective
than 0.5 mglkg of chlorothalidone. In patients
with mild to moderate hypertension, xipamide
at 20 to 40 mglday p.0. is as effective as 1 mg excellent oral absorption, rapid onset of ac-
bumetanide or 50 mg of hydrochlorothiazide. tion, and short duration of effect. The magni-
Xipamide produces a maximal natriuresis and tude of diuresis produced by the compound is
kaliuresis similar to that of furosemide. The similar to that seen with furosemide and
diuretic effects of xipamide last for about 24 h, ethacrynic acid. The renal sites of action are
with the maximum effect occurring during the interpreted to be the proximal tubule and the
first 12 h (248).In an evaluation in patients ascending limb of Henle (254, 255). Interest-
with edema of cardiac origin, xipamide was ingly, a study of rats and dogs indicated that
found to be an effective diuretic at 40 mg/day triflocin has no propensity for evoking hyper-
p.0. Serum potassium levels were slightly low- glycemia (256).The drug was studied in nor-
ered (249).In rats, serum potassium depletion mal volunteers and found to be a markedly
could be avoided, and an equilibrated potassium potent natriuretic agent; free water clearance
balance was achieved in a 13-day study by com- (CHzo)was inhibited during water diuresis
hing xipamide with triamterene (250). and solute-free water reabsorption (TCHzo)
Xipamide is generally well tolerated. Mild reduced hydropenia, indicating a major site of
upper gastrointestinal symptoms are the most action in the ascending limb of Henle. In addi-
frequently reported side effects (2-4%) (251) tion a fall in the glomerular filtration rate of
and fatigue (3-8%) (252).Although ototoxic- 10-15% was found at doses of 1 g given orally
ity has been seen with salicyclic acid and with (257).Long-term toxicity studies revealed ad-
furosemide, studies with xipamide in guinea verse effects; clinical studies were therefore
pip did not reveal any ototoxicological prop- discontinued (258). In rats, doses of 100 and
erties (253). Xipamide does cause small in- 200 mg kg-' day-' caused no adverse effects
creases in serum urate and blood urate con- (259).Doses of 2000 and 5000 mg kg-' dayp1
centrations and occasional increased glucose generally caused death within 1 week.
and plasma lipid levels in diabetic individuals 2.1.8.10 Torasemide. Torasemide (122) is
a pyridylsulfonylurea, high ceiling diuretic
2.1.8.9 Triflocin. The discovery of triflocin that is structurally similar to triflocine (184).
sulted from a study of derivatives of Its site of action is the Na+/ 2C1-/ K+ cotrans-
ic acid for possible anti-inflammatory porter located in the loop of Henle. It also
. Compounds incorporating a nicotinic blocks chloride channels located on the baso-
moiety unexpectedly exhibited diuretic lateral side of the thick ascending limb (260).
flocin is structurally a novel and Torasemide is about 8-10 times more potent
cacious diuretic agent, capable of in dogs and in humans, has a longer duration
promoting the excretion of as much as 30% of of action, and causes less potassium excretion
the sodium chloride filtered at the glomerulus. than furosemide.
It was effective in the rat, rabbit, guinea pig, Torasemide is quickly absorbed from the
dog, and monkey. Triflocin is characterized by gastrointestinal tract after oral administra-
Diuretic and Uricosuric Agents

tion and has a bioavailability of about 90%. It uretics because it contains neither a sulfon-
is highly bound to plasma protein (>95%)and amide nor a carboxyl group. It has a pK, value
has a half-life of about 2 h after intravenous of 9.2 and is very lipophilic. Clearance studies
dosing and 3 h after oral dosing in humans in dogs indicate that muzolimine does not in-
(261).The compound undergoes extensive me- crease the glomerular filtration rate, but has a
tabolism in several species. In rats, less than saluretic effect similar to that of furosemide,
1%of the drug is excreted unchanged: most is induced by inhibition of tubular reabsorption
excreted as a variety of hydroxylated metabo- in the ascending limb of the loop of Henle
lites (262). In humans, only 20% of the (267).
unchanged drug is excreted in the urine. Its Micropuncture studies in rat kidneys
volume of distribution in humans was deter- showed that muzolimine was effective only
mined to be 0.2 L/kg (263). In normal volun- when given as a peritubular perfusion and not
teers torasemide was administered orally in when administered intraluminally, in con-
doses ranging from 10 to 100 mg (264). At the trast to furosemide and bumetanide, which
highest doses (80 and 100 mg) some volun- were effective when applied either peritubu-
teers complained of knee, calf, and foot larly or intraluminally (267). Renal Nat/K+-
cramps. These were generally short in dura- ATPase activity in vitro is inhibited only at
tion. In hypertensive patients, a 2.5-20 mg high concentrations: Mg2+-ATPase activity
dose of torasemide is effective. Torasemide was not affected (268).
was first introduced into the market in 1993 in Muzolimine is rapidly absorbed after oral
Germany and Italy by Boehringer Mannheim. administration and is estimated to have a bio-
It is available in 2.5, 5, 10, and 20 mg tablets availability of greater than 90%. The plasma
and 10 mg/2 mL ampules for injection. Re- protein binding is 65%, which is lower than
cently, some additional analogs of torasemide many of the other high ceiling diuretics, and
were reported where the sulfonyl urea was re- this may be the reason that the drug is effec-
placed with other isosteric groups including tive in patients with advanced renal failure
sulfonyl-thiourea, cyanoguanidine, and 1,l- (269). Muzolimine also has a half-life of 10 to
diaminonitroethylene (265). These analogs 20 h. It undergoes extensive metabolism in the
had diminished diuretic activity in respect to liver; its major route of excretion is through
torasemide. the bile (270), with only about 10% of the drug
2.1.8.11 Muzolimine. Workers at Bayer being excreted unchanged.
synthesized a series of 1-substituted pyrazol- Preliminary studies with muzolimine in
5-ones. Some of the compounds prepared in patients showed that the drug is a high ceiling
this series were disclosed to be highly active diuretic with an onset of action and a peak
diuretics. Muzolimine (Bay g2821, 185) was diuresis similar to that of furosemide. The du-
selected for further study (266). ration of action was 6 to 8 h a s compared to 3 to
5 h after furosemide; 40 mg of muzolimine was
more potent than 40 mg furosemide in all pa-
rameters investigated (271). In normal volun-
teers, the threshold dose was 10 mg and the
dose response curve for sodium was practically
linear for doses up to 80 mg (272). Acute water
diuresis and hydropenic studies carried out in
seven normal volunteers suggested that
muzolimine acts in the proximal tubule and in
the medullary portion of the ascending limb of
the loop of Henle (273). In July 1987, muzoli-
mine was withdrawn from the market for tox-
icological reasons (polyneuropathy).
2.1.8.12 MK 447. Through screening prc-
The structure of this compound differs con- cedures, workers at Merck found that 2-ami-
siderably from that of other high ceiling di- nomethyl-3,4,6-trichlorophenol(186) (274)
2 Clinical Applications

After oral administration, MK 447 is rap-


idly absorbed from the gastrointestinal tract.
It has a plasma half-life in rats and dogs of 1
2 HN J - + and 7.5 h, respectively. In humans, the half-
life is 4-8 h (281). The compound undergoes
extensive metabolism in rats, dogs, and hu-
C1 mans. The major metabolite identified in rat
and dog urine is the 0-sulfate conjugate (282).
(186) In humans, 17% of the activity of radiolabeled
MK 447 is ascribed to the 0-sulfate conjugate.
played significant saluretic-diuretic prop- The major metabolite in human urine was ten-
erties. Exploration of structure-activity rela- tatively identified as the N-glucuronide. It is
tionships showed that alkyl substitution, pref- believed that the 0-sulfo derivative is an ac-
erably a-branched, in position-4 and halo tive metabolite of MK 447 and may be respon-
substitution in position-6 resulted in greatly sible for the salidiuretic activity observed
anced activity. Substitution of the nitro- (283,284). MK 447 also possesses anti-inflam-
and oxygen with groups resistant to hy- matory activity. It is believed that the drug's
olysis greatly reduced the saluretic effects of anti-inflammatory activity is attributed to its
ese compounds (275). Also, reorientation of ability to inhibit the endoperoxide PGG,
e 2-(aminomethyl) group from the position (285).
o to the phenolic hydroxyl group to the 2.1.8.13 Etozolin. During the investiga-
ta and para positions results in loss of ac- tion of a series of Cthiazolidones, some of
ty (276). Optimal activity was displayed by which have choleretic properties (286),a num-
ino-methyl-4-(1,l-dimethylethyl)-6-iodo- ber of compounds with high ceiling diuretic
en01(MK 447, 187 (277). The 5-aza analog activity were found (287). Compound (188)

yNH2 (187)

layed similar activity to MK 447 in the rat,


was less active in the dog (278).
(188) R = ethyl
(189) R = methyl

(piprozoline)is a choleretic compound without


The saluretic effects of MK 447 in rats and diuretic activity. Compound (189)(etozolin)is
are generally superior, both qualitatively a highly active diuretic with weak choleretic
d quantitatively, to those of earlier high properties. Minor deviations from structure
g loop diuretics. MK 447 was more effec- (189) lead to a loss of diuretic activity. The
than furosemide at 0.1-10 mgkg p.0. in different pharmacodynamic properties of
and dogs (279). A study in normal volun- (188)and (189)could not be traced to thermo-
rs confirmed the high potency of the com- dynamic factors but rather must be related to
nd. Despite copious diuresis and natriure- closely defined receptor interactions (286).
, no significant change in the elimination Long-term toxicity studies in rats and dogs
of potassium was observed (280). In hu- have shown that etozolin is well tolerated, and
s, a 100 mg dose is equipotent to 80 mg of that it has a wide margin of safety (288). Stud-
osemide, although its duration of action is ies in rats and dogs indicate that the com-
pound is a potent saluretic agent, with a rela-
tively slow onset of action and prolonged in dogs it is less potent. In subjects with nor-
activity. The maximal diuretic effect of etozo- mal renal function, a 800 mg dose of the drug
lin lies between that of the thiazides and furo- increased the excretion of water, chlorine,
semide. Antihypertensive effects occur in the magnesium, potassium, and sodium without
spontaneous hypertensive rat, DOCA, and altering creatine clearance (294). In normal
Goldblatt rats. Etozolin does not appear to in- volunteers, a dose of 400 mg of etozolin was
fluence glucose tolerance in rats and dogs; equipotent to 75 mg of a thiazide diuretic;
these results are of particular interest because 1200 mg was 2.8 times more effective than the
the tests were carried out in animals that had 75 mg of the thiazide. Diuresis starts within
been treated with high doses of drug for 18 and 1-2 h after dosing, reaches a peak after 2-4 h,
12 months, respectively (289). and then gradually decreases over the next 6 h
Clearance and micropuncture studies have (295). Because of the long-lasting effect of eto-
shown that an initial dose of 50 mgkg i.v. fol- zolin, the compound would seem indicated for
lowed by 50 mg kg-' h-I i.v. results in a mark- the treatment of cardiac and renal edema as
edly increased urinary flow and sodium excre- well as for the treatment of hypertension
tion, combined with a decreased glomerular (294). In hypertensive patients after a period
filtration rate. Reabsorption in the proximal of 2 weeks, treatment with 400 mg of etozolin
tubule is not affected significantly; however, daily significantly reduces systolic and dia-
fluid and electrolyte reabsorption in the loop stolic blood pressure. The drug was introduced
of Henle is definitely decreased. Although eto- in 1977 as Elkpain.
zolin differs chemically from furosemide and 2.1.8.14 Ozolinone. Ozolinone (1901, as
ethacrynic acid, it appears to share the same stated above, is the major metabolite of etozo-
site of action in the nephron (290). line, which is formed by enzymatic cleavage of
Absorption and metabolic studies with 14C the ethyl ester. It is reported to be more potent
etozolin in rats, dogs, and humans indicate and less toxic than etozoline (296). Resolution
that at least 90% is absorbed after oral admin- of the enantiomers and examination of their
istration in humans. Etozolin is approxi- biological activity revealed that the (-)-enan-
mately 45% bound to plasma proteins. In the tiomer possesses the diuretic activity (298).
rat, blood levels could be described with a two- The (+)-enantiomer possesses little diuretic
compartment body model, the absorption half- activity and actually antagonized furosemide
life was 0.6 h, and the elimination half-life was activity (295). Ozolinone has a half-life of 6-10
determined to be approximately 6 h. In hu- h in humans (299) and has a plasma protein
mans the elimination half-life was 8.5 h; the binding of 35%.
blood levels followed with high probability a 2.1.8.15 Pharmacology of High Ceiling Di-
one-compartment body model (291). uretics. It is currently believed that the renal
The main metabolite of etozolin is the free site of action for these diuretics is the Na+IK+I
acid ozolinone (190) and its glucuronide. 2C1- cotransporter located in the thick as-
cending loop of Henle. By binding to the chlo-
ride site of the cotransporter, these drugs
inhibit the reabsorption of sodium, thus pro-
moting their diuretic action (300). The car-
boxyl group common to many of these diuret-
ics is essential for the binding activity, and it
has only been successfully replaced by a sul-
fonamide. Most of these drugs are readily
absorbed in the gastrointestinal tract. For ex-
ample, furosemide and bumetanide have bio-
availabilities of 65 and loo%, respectively.
Generally these compounds are secreted from
Other metabolites have also been detected the blood to the urine through the organic acid
(292, 293). In rats, etozolin is a more potent transport system in the proximal tubule and
diuretic than hydrochlorothiazide, although travel through the renal tubule to their more
Clinical Applications

distal site of action. Increased potassium ex- trial has now vastly heightened interest
cretion and the elevation of plasma uric acid within this class of compound (304).The study
levels, as was observed with the thiazides, are looked at the effect of adding aldactone (spi-
also seen with the loop diuretics. These diuret- ronolactone, 191) as a potential therapy for
ics also increase calcium and magnesium ex- reducing death in patients with heart failure.
cretion. The calciuric action of these agents It also defied current thinking, in that spi-
has led to their use in symptomatic hypercal- ronolactone should not be administered in
cemia (301). Many of the hypersensitivity and conjunction with an ACE inhibitor because of
metabolic disorders seen with the thiazides the possibility of hyperkalemia and the mis-
are also seen with loop diuretics. The develop- conception that, by using an ACE inhibitor,
ment of transient or permanent deafness is a aldosterone will be as effectively blocked as
serious but rare complication observed with angiotensin 11. Within the trial, investigators
this class of agents. It is believed to arise from compared a standard treatment regimen of an
the changes in electrolyte composition of the ACE inhibitor and a diuretic, with or without
endolymph (302). It usually occurs when blood digoxin added to this regimen, plus (191) or
levels of these drugs are very high. placebo in patients suffering with severe heart
failure. The RALES study, conducted in 15
2.1.9 Steroidal Aldosterone Antagonist. Spi- countries, was a randomized, double-blind,
ronolactone (191) is the most extensively placebo-controlled trial in which 1663 patients
with systolic left ventricular dysfunction were
enrolled; these patients were classified as ei-

@
i / - %
,
.llllllll
ther Class I11 or Class IV, a classification for
the most severe of cases as defined by the New
York Heart Association (NYHA). Although
originally scheduled to conclude in December
1999, the trial was halted 18 months early,
given that the results were statistically and
clinically significant and failure to terminate
the trial would have been unethical.
Among the 1663 patients there were 386

A
0
deaths (46%,n = 841) in the placebo group
and 284 deaths (35%,n = 822) in the spirono-
0 lactone (191) group; this figure represented a
30%decrease in mortality. Also observed dur-
(191) ing the trial were 336 placebo-treated and 260
spironolactone-treated patients who had at
tudied aldosterone antagonist. It promotes least one nonfatal hospitalization, represent-
diuresis by competing with aldosterone at the ing 753 hospitalizations for the placebo group
receptor sites responsible for sodium ion reab- and 515 for the spironolactone group. This
tion and best clinical results have been represents a 30%decrease in nonfatal cardiac
ained from those patients suffering from hospitalizations. Also of significance within
hosis and nephrotic syndrome, in whom the study were two observations of differences
e aldosterone secretion rate is very high. Af- between the placebo and spironolactone-
r administration for several weeks to hyper- treated groups. It should be noted that within
tensive patients, the compound exhibits a both groups the average blood pressure levels
odest antihypertensive effect; however, in during the course of the study were normal
ormotensive subjects no reduction in blood and unchanged and that the incidence of hy-
ressure is seen. perkalemia was no different, as defined by po-
Most recently, spironolactone (191) has tassium ion concentration [Kt]>> 6 meq/L.
i been the subject of clinical investigations in What did differ was the mean plasma [Kfl,
I heart failure (HF) (303). Indeed, the Random- which was 0.2-0.3 meqh higher in the (191)-
! ized Aldactone Evaluation Study (RALES) treated group as compared to the placebo and
Diuretic and Uricosuric Agents

there was a 10% incidence of gynecomastia

11
within the spironolactone-treated group com-
pared to 1.5% in the placebo group. Thus,
given the widespread use of potassium supple-
mentation and a difficult explanation by the /wo
.,+\
.11l11111

antiandrogen effect, it is difficult to under-


stand the exact benefit that spironolactone
had in this trial. However, it is clear that these
findings suggest that the gold-standard treat-
ment for severe heart failure should now in-
clude an aldosterone receptor antagonist. The
RALES trial also helped to confirm that aldo-
0
"r 0
O'

sterone plays in important role in the patho- (194)


physiology of heart failure. It also directs re-
search and development strategies toward a
more effective treatment option based on aldo- rently, eplerenone (194) appears to be the de-
sterone blockade. sired specific antimineralocorticoid that is
Consequently, within this context and es- required (305). It is currently in Phase 111,hav-
pecially with respect to the side-effect profile ing been licensed from Ciba-Geigy (now Novar-
of spironolactone (19 l),the equally efficacious tis) by G. D. Searle. The efficacy and tolerability
and far more selective compounds (192-194) of eplerenone (194) has been evaluated in a clin-
can now be considered as potential cardiopro- ical setting involving 417 patients suffering mild
tectant and antihypertensive therapies. Cur- to moderate hypertension, in which it was found
to have a profile similar to that of spironolactone
(191) (306). In a related dose study involving
321 patients with heart failure (NYHA, 11-IV),
(194) was compared again with (191) in con-
junction with standard therapy (ACE inhibitor,
diuretic, andlor dixogin) (307).It was found that
blood natriuretic peptide decreased significantly
with either active treatment after 12 weeks and
the urinary aldosterone and renin levels in-
creased compared to placebo in patients receiv-
ing doses of 50 mg/day or higher of (194). Inter-
estingly, the incidence of hyperkalemia was
significantly higher with 100 mglday eplerenone
when compared to spironoladone (12.0 versus
8.7%); however, the testosterone in male pa-
tients increased more with spironolactone than
with eplerenone. The latter effed was probably
attributable to a feedback mechanism in re
sponse to blockade of the androgen receptors by
(191).
Thus, although the use of antialdosterone
agents would now appear to be part of good clin-
ical practice for heart failure, it may well tran-
spire that its most widespread use will be as a
cardioprotectant for patients who have hyper-
tension. Expectations are that eplerenone (194)
will be marketed in 2002.
2.1.9.1 Pharmacology. The adrenal cortex
is responsible for the biosynthesis of a number
113

Glucocorticoids
Mineralocorticoids

C19

Cholesterol
C27

Cholesterol

Corticosterone Cortisol Estradiol

Aldosterone Esterone
Figure 2.9. Pathway of adrenal steroidogenesis. 11 = llp-hydroxylase;17 = l7a-hydroxylase;18 =
18-hydroxylase; 21 = 2la-hydroxylase.

through sodium and water retention, whereas


cortisol (a glucocorticoid) is thought to impart
its effects through its incomplete metabolism
within target tissues. The aldosterone content
in the adrenal gland is 1-2 pg from which it is
secreted at a rate of 70-250 pglday, yielding
plasma levels between 5 and 100 pg/mL, indi-
cating that the adrenal does not store the hor-
mone but is capable of rapidly synthesizing it
when needed. Greater than 85% of this hor-
mone is metabolized by first pass through the
liver; thus, its degradation is intrinsically
linked to hepatic blood flow. Reduction in he-
patic blood flow is very possible in heart fail-
ure, which would then generate a vicious circle

iandrosterone sulfate (DHEA), and cop creases the extracellular volume through so-

glomerulosa, zona fmciculata, and zona which itself then leads to a decrease in cardiac
lark, respectively. The overproduction output. Recent evidence has been amassed

), or cortisol all have the potential to (308-310). It would now appear that a direct

eralocorticoids and produce their effects plasma levels of aldosterone and mortality.
Diuretic and Uricosuric Agents

These data also indicate that the detrimental cribed to the compounds, given that they had
effects of aldosterone in HF can be attributed no effect in adrenalectomized animals unless
not only to the increased load on the heart by aldosterone or another mineralocorticoid was
way of sodium retention but also: (1)hypoka- administered before the spirolactone (3141,
lemia and hypomagnesemia with concomitant and also because the spirolactone produced
promotion of arrythmias; and (2) increased the same effect as impaired aldosterone syn-
sympathetic tone arising from baroreceptor thesis (318). The first compound of interest
desensitization. which blocks neuronal nor- was 3-(3-oxo-17~-hydroxy-4-androsten-17-a-
epinephrine reuptake and increases myocar- yl)-propanoic acid lactone (196), which when
dial toxicity from catecholamines and myocar-
dial fibrosis. Thus, aldosterone blockade
appears to be logical in the treatment of heart
failure and potentially hypertension, thus
serving as a cardioprotectant strategy.
Aldosterone binds to a cytoplasmic recep-
tor from the basolateral side located in the
", .~~1111ll

pJp
principal cells of the collecting tubule. Trans-
location of this hormone-receptor complex to
the nucleus leads to the generation of specific
transport proteins. These proteins then di- 0 / (196)
rectly or indirectly increase reabsorption of so-
dium and the excretion of potassium (311).
The renin-angiotensin-aldosterone system administered subcutaneously showed the de-
(RAAS) is an important regulatory system for sired aldosterone antagonistic effect. Subse-
the modulation of arterial blood pressure to- quent studies established the importance for
gether with fluid and electrolyte homeostasis. both the five-membered spirolactone and the
A reduction in renal blood flow stimulates re- 3-keto-A4 a,p unsaturated enone system
nin production and excretion from the juxta- within the A-ring. Interestingly, the diaste-
glomerular cells of the kidney and into the sys- reoisomer possessing the opposite configura-
temic circulation. This enzyme converts tion of the spirolactone at C17 was devoid of all
angiotensinogen to angiotensin I; thereafter activity (318), and the 19-normethyl deriva-
angiotensin-converting enzyme (ACE) con- tive (197) was more active than (196) in rats.
verts this into angiotensin 11. Angiotensin I1is
a potent vasoconstictor that also stimulates
the production of aldosterone. Thus, with the
use of ACE inhibitors it was thought - that al-
dosterone production would be modulated;
however, it would now appear that there is an
escape phenomenon possibly leading to in-
creased plasma levels of the hormone rather
&i -
111111111

than the anticipated decrease (312). Hence,


blockade of aldosterone and its action through
antagonism at the mineralocorticoid receptor /
0
(MR) or through the inhibition of its biosyn-
thesis (see Section 2.1.10) is necessary to re- (197)
duce elevated levels of the hormone.
2.1.9.2 History and Structure-Activity Rela- During experiments conducted with com-
tionship. During the late 1950s Cella et al. re- pound (197), when it had been administered
ported the synthesis and structure-activity re- to rats maintained on a low sodium diet, it was
lationships in a series of steroidal-spirofused found that compensation for the renal loss of
lactones t h a t possessed aldosterone antago- sodium was mediated through upregulation of
nist activity (313-317). This activity was as- aldosterone secretion (319). Likewise, sodium
Clinical Applications

the urine (320). The 19-normethyl deriva-

a (322, 323), and hepatic cirrhosis (323,


. Patients with cardiac failure did not re-
d well. The effect of (197) is determined
he degree to which sodium reabsorption is
trolled by aldosterone; consequently, it is
t well tolerated in cases of untreated Addi-

sodium diet (326). Because the compound


a direct effect on the kidney by way of
ition of aldosterone, sodium and chloride
excretions increase and potassium, hydro-

rent from that of most other diuretics


increase potassium ion loss and hence
to hypokalemia. Both (196) and (197),
en administered parenterally, showed bet-

was encountered through incorpora-


saturation at positions 1,6, or both
ultaneously (198-200) (316), an enhance-

lactone; aldactone), has undergone extensive


clinical trials. Inversion of the 7-thioacetyl
group into the P-configuration reduced both
nt was also seen when a thioacetyl group oral and parented activity by 90% (327).
incorporated into the l a orientation Introductions of methyl groups at the 2,4,
1).This compound itself was then super- 6, 7, and 16 positions as well as a keto- or
ed through incorporation of the same thio- hydroxyl incorporation at 11 position or a
tyl moiety, however, into the 7 position of fluoro at the 9 position did not improve the
steroid nucleus, interestingly again with compound profile (327,328). The spirolactams
a-orientation. This latter compound, 343- (202-203) have also been synthesized (329,
-7a-acetylthio-l7P-hydroxy-4-androsten- 330) but have little aldosterone antagonist ac-
I -yl)-propanoicacid lactone (191; spirono- tivity (331).
Diuretic and Uricosuric Agents

The metabolism of spironolactone has been


studied in detail (332-334); several metabo- compound is detected in the urine and it is
lites have been isolated from the urine of nor- known to induce CYP450s within the liv
mal subjects, indicating that the compound is thus possibly altering the metabolic profile
subject to elimination, oxidation, and rear- coadministered drugs. The compound is a1
rangements (204-208). After oral adminis- greatly plasma protein bound (95%).
tration, about 70% of the drug is absorbed In further efforts (336) to gain water s
(335); however, extensive first-pass metabo- bility the y-hydroxy potassium carbo
lism and enterohepatic circulation greatly re- was prepared (easily generated upon sap0
duces circulating levels. No unmetabolized cation of 199) and it was found to be appro
2 Clinical Applications

Table 2.17 Steroidal Aldosterone Antagonists


No. Generic Name Trade Name Structure Reference
(191) Spironoladone Aldadone 316

0
I

0
(194) Epoxymexrenone Eplerenone

0
(209) Potassium Soldactone 336
canrenoate

0
(210) Potassium 0 337
prorenoate

mately equipotent with spironolactone (191).


e
It was equally efficacious when dosed orally or
parenterally; however, it was found to be inef-
/

(MC).Thus, potassium canrenoate (209; sol-


dactone; Table 2.17) is a specific antagonist of
mineralocorticoids with pharmacodynamic
fective in the absence of mineralocorticoids properties similar to those of spironolactone.
118 Diuretic and Uricosuric Agents

Table 2.17 (Continued)


No. Generic Name Trade Name Structure Reference
(211) Potassium
mexrenoate

was significantly higher than that of spirono-


lactone. Prorenoate (210) also produced
greater natriuresis but this effect was not sig-
nificant. Interestingly, the in vivo experiment
converts the hydroxy carboxylic acid salt to
the spironolactone (338).
As previously mentioned, the 7a-thioacetyl
derivatives were found to possess increased
bioavailability, and within these latter open
lactone potassium salt derivatives, enhanced
activity was now seen through the inclusion of
a 7a-carbalkoxy group. The result was potas-
sium mexrenoate (211) (339), a water-soluble
compound whose oral activity was better than
Cyclopropanation of the A6 double bond of that of its spirolactone variant (Table 2.17).
(209) generated potassium prorenoate (210), Two further cyclopropyl-containingderiva-
a water-soluble steroid with the ability to an- tives within these series have been high-
tagonize the sodium-retaining and, when ap- lighted as potent analogs that possess lower
parent, potassium-dissipating effects of the relative binding affinities for the progesterone
mineralocorticoids (337). Within the aldoste- and androgen receptors (PR and AR) com-
rone-treated dog model, the latter compound pared to that of spironolactone. This desired
was three times more potent than (191); how- progression toward more selective compounds
ever, it was similarly relatively inactive at the is the result of the side-effect profile of spi-
renal level in the adrenalectomized rat with- ronolactone, which is a factor that also lirnita
out MC replacement. Further investigations its utility. These two derivatives both contain
showed that the compound possessed no more the p-configured cyclopropane of the A15 dou-
than 2% of the natiuretic activity of hydro- ble bond. Mespirenone (212) is an analog of
chlorothiazide in the intact animal. Clearance (200) and possesses three times more potency
studies within dogs showed a direct renal tu- within the adrenolactomized rat treated with
bular interaction between (210) and aldoste- glucocorticoid or d-aldosterone (340) and six
rone (337). The relative potency of (210) and times more potency within normal volunteers
(191) was compared in a double-blind, bal- (341),and ZK 91587 (213) is twice as potent as
ance, cross-over study in normal subjects spironolactone in the former model (342).
(338). The compound (210), as related to ele- Modification of the steroid nucleus has also
vation of urinary log[sodium/potassium ion] been attempted, and the resulting compounds
ratio and as related to potassium retention examined for mineralocorticoid-blocking ac-
Clinical Applications

Although cyclopropanation has thus far


been seen to be advantageous, within a series
of progesterone derivatives it was seen to abol-
ish the desired antialdosterone activity (344).
Progestereone blocks aldosterone at high '
doses, and this effect was further enhanced
through the introduction of oxygenation
within the steroid D-ring, specifically at C15;
indeed. insertion of unsaturation at A1 or A6
also enhanced activity, as exemplified by
(216).However, cyclopropanation of the C6-C7
double bond yielded (217),a compound of
greatly reduced antialdosterone activity. In-
0 terestingly, and not analogously to the proges-
terone series, oxygenation of spironolactone

en removed (214)retained the sodium-


properties to a degree (343).During
extensive studies a series of naphthylcyclo-
ketones were prepared. The a-hy-
-ketone(215)showed the best properties,
chloro- and fluoro-derivatives were
Diuretic and Uricosuric Agents

within the D-ring reduced activity to approxi- 2.1.1 0 Aldosterone Biosynthesis Inhibitors.
mately 14%of that of the parent (345);however, The alternative approach to an antialdoste-
this was at C16 and not C15 as before (218). rone diuretic rather than through blockade of
aldosterone and its action through antago-
nism at the mineralocorticoid receptor (MR) is
to inhibit the biosynthesis of aldosterone
within the adrenal zona glomerulosa. The pre-
viously described spironolactones in addition
to their effects on the aldosterone receptor
have also been shown to have an effect directly
on aldosterone biosynthesis (350). This obser-
vation was made in the adrenal tissues of so-
dium-depleted rats at 10-4-10-5 M concen-
trations. It is believed that spironolactone,
carenone, and potassium carenoate inhibit the
mitochondrial llp- and l&hydroxylase activ-
ity and hence prevent aldosterone synthesis
Workers at Ciba-Geigy also introduced ox- (351). Spironolactone may even inhibit 21-hy-
ygenation within the steroid nucleus, al- droxylase (352); however, it is believed that
though in an alternative fashion, through ep- the major site of action of these steroids is the
oxide formation (346). They prepared a series aldosterone receptor because the systemic
of 9,ll-a-epoxysteroids, which generated as a concentrations needed to affect biosynthesis
result of this modification derivatives of spi- are greatly elevated when compared to those
ronolactone (1921, prorenone (193), and required for the receptor antagonist activity.
mexrenone (194). In general the epoxide func- There are few small molecules that exhibit
tionality had only a small effect on the binding the inhibition of aldosterone through inhibi-
of these compounds to the MR, and in vivo at a tion of its biosynthetic pathway, one such com-
dose of 3 mgkg all these derivatives were pound ofwhich is (219).Metyrapone (219)has
twice as potent as (191). However, most inter-
estingly, these compounds were shown to have
decreased affinity for the PRs and ARs, now
exhibiting selectivities between 10- and 500-
fold (347). Indeed, it is the binding to these
latter receptors that accounts for the side ef-
fects of spironolactone (1911, evidenced by
menstrual irregularities in women and gy-
necomastia in men (348).
Equally interestingly, when comparing
eplerenone, epoxymexrenone (194) to spirono- undergone extensive biological profiling and
lactone (191),and their respective in vitro bind- clinical trials (353). In moderate doses it
ing affinities to their in vivo potencies, there is a blocks llp-hydroxylation within the steroid
marked difference; (194) has 5% the afKnity for nucleus, thus inhibiting the biosynthesis and
the MR and 100% potency in vivo when com- secretion of cortisol, corticosterone, and alde
pared to (191) (349). A contributing factor to sterone. However, the consequential in-
this is undoubtedly the fact that (194) is mini- creased secretion of 1l-deoxycorticosterone,a
mally plasma-protein bound as compared to potent salt-retaining hormone, negates the ef-
(191), which is 95% plasma-protein bound. fects of reduced aldosterone secretion. An-
However, potentially compounding this is the other synthetic small molecule that can in-
metabolic fate of eplerenone: it has not been well hibit the biosynthesis of aldosterone is
studied in either rat or human and consequently CGS16949A (220) (354). This interesting
these data could aid the explanation of these dif- property was shown during profiling studies
ferences seen in vivo. carried out with the aromatase inhibitor. The
Clinical Applications

CN
(220)

bitory effect is specific within the aldoste-


ne biosynthetic pathway, in that the com-
inhibits the second hydroxylation step
11)of C18, the angular methyl group of
sterone, and hence the subsequent pro-
duction of aldosterone. Thus when compared
to (219)and its inhibitory effects attributed to
llp-hydroxylase inhibition, this latter ap- proposed that the pharmacological basis for
proach would appear to be more specific and their mechanism of action is adenosine recep-
selective. Closely related to this compound tor antagonism (356). Adenosine produces an-

r
and implicitly its generic structure is a series tidiuretic and antinatriuretic responses in
of compounds (exemplified by 221) covered in several species. These effects can be competi-
tively antagonized by theophylline (357).
There are four adenosine receptor subtypes,
L
A,, 4,, A,,, and A, (358). It is believed that
the renal actions of adenosine are the result of
its stimulation of the adenosine A, receptor
(359). It has been shown that compounds that
antagonize the A, receptor exhibit diuretic ef-
fects (360, 361). Compounds that are adeno-
\ N
(221)
sine 4 antagonists do not show any diuretic
or natriuretic properties (362).
A series of 8-substituted 1,s-dipropylxan-
thines were reported by Suzuki and coworkers
a patent filed by Yamanouchi Pharmaceuti- (363). Many of these compounds were potent
d ds (355), these bicyclic imidazoles are also adenosine A, receptor ligands with diuretic ac-
t claimed as aldosterone biosynthesis inhibitors tivity. One of the most potent analogs was
d ding the identical cytochrome P450 en- 8-(dicyclopropylmethy1)-1,3-dipropylxanthine
d zyme, as previously detailed. (224; K,,A, = 6.4 nM).This compound also
1-
1- 2.1 .I 1 Cyclic Polynitrogen Compounds
a 2.1.11.1 Xanthines. The diuretic actions of
f- lxanthines, such as caffeine (222) and
1- eophyline (223), have been known for more
I- a century. They have been of limited clin-
is utility because of their low potency, devel-
% ment of tolerance after repeated adminis-
es ation, and side effects such as psychomotor
1e ffects and cardiac stimulation. It has been
Diuretic and Uricosuric Agents

increased urine volume and sodium excretion 370, 371). From stop-flow methods and lith-
in the rat after oral administration. ium clearance studies, it appears that the site
8-Cyclopentyl-l,3-dipropylxanthine (225) of action of this drug is in the proximal tubule
was also reported to be a potent selective aden- (372). KW-3902 had little effect on renal he-
osine A, receptor antagonist with diuretic ac- modynamics. More recently, CVT-124, 1,3-
tion (363). At 0.1 mg/kg, i.v., (225) signifi- dipropyl-8-[2-(5,6-epoxy)norbornyl]xanthine
(227) has been reported to be a very potent

cantly increased urine volume and sodium


;:" HN 'N

0P N - F ' .
excretion in the rat with no significant change
in potassium excretion (364).The tubular site
of action is thought to be in the proximal /N+
Pr 0
tubule.
8-(Noradamantan-3-y1)-1,3-dipropylxan- (227)
thine (KW-3902, 226) has been described as
and selective A, antagonist [K,, A, = 0.45 nM;
K,, A,, = 1100 nM;human (373)l. In anesthe-
tized rats CVT-124 (0.1-1 mg/kg) resulted in a
dose-dependent increase in urine flow and so-
dium excretion (374). No changes in heart rate
or blood pressure were observed. In conscious
chronically instrumented rats, the adminis-
tration of CVT-124 led to a significant increase
in urine and sodium excretion without affect-
ing renal hemodynamics or potassium excre-
tion (375). The natriuretic effects of this com-
being a potent adenosine A, receptor antago- pound have been demonstrated in normal
nist (K,, A, = 1.1 nM) (365). In saline-loaded volunteers and in humans with congestive
norma1 rats, a 0.001-1 mgkg oral dose of KW- heart failure (376).
3902 caused a significant increase in urine vol- 2.1.11.2 Aminouracils. During an exten-
ume and sodium excretion, kith little effect on sive study of compounds related to the xan-
potassium excretion (366). In the saline- thines, it was discovered that certain of the
loaded conscious dog, KW-3902 exhibited a intermediate substituted 6-aminouracils were
longer-lasting natriuresis than that of either orally active diuretics in animals (377). The
furosemide or trichlormethiazide (367). It also 1,3-disubstituted derivatives of 6-aminouracil
induced less hypokalemia and hyperuricemia (228) are diuretics, whereas the monosubsti-
in rats when compared to furosemide or tri- tuted compounds are not. The l-n-propyl-3-
chlormethiazide (368). No attenuation in its ethyl derivative, which was the most potent
pharmacological action was observed after re- diuretic in the series, was unsuitable for clin-
peated oral dosing (0.1 mg kg-' day-') for 24 ical use because of gastrointestinal side ef-
days (369). KW-3902 possessed renal protec- fects. Compounds that were investigated
tive effects against glycerol-, cisplatin-, and clinically were l-allyl-3-ethyl-6-aminouracil,
cephaloride-induced acute renal failure (366, aminometradine (229, Table 2.18), and the
Clinical Applications

HzN ixr I
R1

(228)

ethallyl-3-methyl analog (230, Table 2.18).


nical studies in edematous subjects indi-
d that mercurial diuretics usually have a
ater and more reliable effect (378).
At the time of their development, they rep-
ed some advance over the xanthines,
ey were displaced by more effective oral pounds in the series were 2-anilino-s-triazine
etics within a relatively short time. (234, amanozine) and 2-amino-4-(p-chloroani-
2.1.1 1.3 Triazines. Recognition of the tri- 1ino)-s-triazine (235, chlorazanil). The di-
ines as a class of diuretic agents stemmed uretic activity of the last two compounds was
the work of Lipschitz and Hadidian confirmed in dogs (384, 385) and in humans
, who tested a group of compounds of this (386, 387). The m-chloro isomer of chlorazanil,
in rats [e.g., melamine (231) and formo- 2-amino-4-(m-chloroani1ino)-s-triazine, was a
potent, orally effective diuretic in rats, dogs,

r 'N
and humans (388,389)and may be significantly
more active than chlorazanil, with an enhanced
saluretic effect. 2-Amino-4-(p-fluoroani1ino)-

R1 AA N NHR2
(231) R l = NH2, R2 = R3 = H
s-traizine was twice as active as chlorazanil
(386).Replacement of the halogen in chloraza-
nil with acetyl, carbethoxy, or sulfamoyl
groups reduced activity (390), but replace-
ment with alkylmercapto groups led to a two-
(232) R 1 = R2 = R3 = H fold increase in activity (391). Both the inci-
(233) R 1 = H, R2 = R3 = Ac dence and degree of crystalluria in dogs were
greater with alkylmercapto compounds than
mine (232)l. Formoguanamine was ef- with chlorazanil, but the oral toxicity in mice
ive orally as a diuretic in humans (3801, was reduced (391).
subsequent clinical studies revealed side The only triazine to achieve any degree of
such as crystalluria (379) and poor Na f

clinical use is chlorazanil. It has a more vro-


-
on (3811,which precluded its further use. nounced effect on water excretion than on
dural variant, prepared in an attempt to Na+ and C1- (387) and has little effect on K+
the side effects, was diacetylformo- excretion, which is probably linked to a lack of
ne (2331, which was active as an oral marked enhancement of Nat excretion. Be-
in dogs (382) but still caused crystallu- cause diuresis is not accompanied by changes
inadequate Na+ excretion. The extraor- in glomerular filtration rate (392), the drug
potency of the triazines in rats does not probably exerts its action through inhibition
over into the dog, and the compounds are of tubular reabsorption. The effects of deoxycor-
moderately active in humans. ticosterone and chlorazanil on Na+ and K+ ex-
ng other derivatives of formoguana- cretion are mutually antagonistic, which may
e, those with other substituted amino mean that the natriuretic and diuretic proper-
s had a particularly favorable diuretic ties of the drug are attributed to inhibition of
in rats (383). The most potent com- Na+ reabsorption in the distal segment (393).
124 Diuretic and Uricosuric Agents

Table 2.18 Cyclic Polynitrogen Compounds


No. Generic Name Trade Name Structure Reference
(223) Theophylline In combination with diaminoethane 2:l -
(aminophylline)

I
(229) Aminometradine Mictine 376

0
(230) Amisometradine Rolicton

P 376

0
(235) Chlorazanil Daquin, 382
Diurazine,
Orpisin

HN

C1
(240) Triameterene Dyrenium 396

(249) Amiloride Collectril 0 -

N
(255) Clazolimine 433

HN
triazines, in particular chlorazanil, have dines in a simple rat diuretic screening proce-
n used clinically mainly in Europe. Interest , dure (396). One compound, 2,4-diamino-6,7-
this type of compound declined with the ad- dimethyl-pteridine (2371, showed sufficient
of the more effective thiazide diuretics.

2.1.12 Potassium-Sparing Diuretics. There


three structurally different classes of po-
ium-sparing diuretics: steroids, pyrazines,
pteridines. The steroidal aldosterone an-
nists and inhibitors have been discussed
dions 2.1.10 and 2.1.11. These diuretic
m reabsorption mainly in
principal cells of the collecting tubules.
e aldosterone antagonists interact with the diuretic activity to encourage further investi-
sterone cytoplasmic receptors, which in- gation of the diuretic potential of the pteri-
dion causes a series of events leading to dines. A number of related 2,4-diaminopteri-
um excretion and potassium reabsorption. dines were studied, but only (237) showed
pyrazines and pteridines, on the other good activity in both the saline-loaded and sa-
inhibit the actions of the sodium chan- line-deficient rat. Changes in the 2,4-diamino
mind side of the princi- part of the molecule resulted in a marked de-
11s. These compounds are thought either crease in diuretic activity (397).
or to switch them from A class of related pteridines, of which 4,7-
n to a closed state (394). As a result, the diamino-2-phenyl-6-pteridine-carboxamide
cell membrane becomes hyperpolar- (238) is the prototype, has been investigated.
ithelial potential is de-
he driving force for po-
to exit the luminal side by way of
sium channels, thus decreasing renal po-
age, the human body
potassium. A calcula-
of potassium is intra-
ar and only about 2% is in the extracel-
compartment. Therefore, removal or
ion of a small amount of potassium to
extracellular pool is very evident (395).
1.12.1 Triamterene. The triamterene ring
m is found in many naturally occurring This derivative is active in both the saline-
unds, such as folic acid and riboflavin. loaded and sodium-deficient rat, but in con-
compounds are important in the regu- trast to (237), it causes substantial potassium
n of metabolism in humans. The observa- loss in the sodium-deficient rat. In structure-
that xanthopterin (236) was capable of activity studies, particular attention was di-
ing renal tissue led Wiebelhaus, Wein- rected toward modification of the carboxamide
, and associates to test a series of pteri- function (397399).
One of the more interesting compounds was
4,7-diamino-N-(2-morpholinoethy1)-2-phenyl-6-
pteridinecarboxamide (239). In pharmacologi-
cal investigations (400), this compound was an
orally active diuretic agent, generating about
the same maximum degree of response in dogs
as did hydrochlorothiazide. The urinary excre-
tion of Na+ and C1- was markedly enhanced,
with minimalaugmentation of K t excretion and
Diuretic and Uricosuric Agents

Table 2.19 2.4.7-Triamino-6-substituted

I
NH2
Diuretic Diuretic
Activity in Activity
Saline-Loaded in Sodium-
little effect on urine pH. Onset of action was R6 Ratb Deficient Ratc
rapid, with the greatest saluretic effect occur-
ring within 2 h of oral administration to saline- Phenyl
loaded dogs. The compound showed diuretic ac- (triamterene) 3 3
tivity in both normal and adrenalectomized rats, 2-Me-C6H4 2 1
3-Me-C6H, 1 1
which, with the absence of K+ retention, indi-
-
cated that aldosterone antamnism is not a ma- 2-F-C6H4
jar component of its saluretic activity. 4-F-C6H4
A consideration of the structural features 4-MeO-C6H4
of 2,4-diamino-6,7-dimethylpteridine(237) 4-Ph-C6H4
and 4,7-diamino-2-phenyl-6-pteridinecarbox- 2-Fury1
amide (238) led to the investigation of 2,4,7- 3-Fury1
triamino-6-phenylpteridine (triamterene, 240) 2-Thienyl
as a potential diuretic agent (397). 3-Thienyl
4-Thiazolyl
2-Pyridyl
3-Pyridyl
4-Pyridyl
H
Methyl
CH(CHJ2
Butyl
A3-Cyclohexenyl

*Rating scheme for saline-loaded rat assay: maximum


The compound was very potent in the sa-
line-loaded rat, and in the sodium-deficient rat
it not only caused a marked excretion of so-
dium but simultaneously decreased Kf excre-
<22% = 0; 22-45%= 1; 46-69%= 2, and >69% = 3.
"Rating scheme for sodium-deficient rat assay: maxi- i
tion. In structure-activity studies of com-
pounds related to triamterene, replacement of
one of the primary amino groups by lower al-
kylamino groups led to compounds that re-
tained triamterene-like diuretic activity. More
extensive changes generally led to substan-
tially less active compounds. Table 2.19 lists
the activities of some 2,4,7-triamino-6-substi-
isomers. The p-hydroxyphenyl analog of tri-
amterene, a metabolite of the latter. is essen- I
tuted pteridines. The activity of triamterene is
very sensitive to substitution of the phenyl crease activity by reducing the rate of metab- I
group with only small changes possible if di-
uretic activity is to be retained. The p-tolyl
compound, for example, is only about half as of triamterene is replaced by a heterocyclic nu-
- I
2 Clinical Applications

cleus, the size of the group appears to be im- ing the degree of binding and establishing the
portant and high activity is seen only in the correct orientation of the molecule at the re-
case of small, nonbasic groups. The low activ- ceptor site.
ity of compounds containing basic centers in Triamterene (240) is a potent, orally effec-
this position, such as thiazole and pyridine, tive diuretic in both the saline-loaded and so-
may be rationalized by assuming that the ba- dium-deficient rat, and is accompanied by no
sic centers are highly solvated and are, in ef- increase in potassium excretion. Also, the ef-
fect, large substituents. The Balky1 analogs fect of aldosterone on the excretion of electro-
are active diuretics; however, size is impor- lytes in the adrenalectomized rat are com-
tant. Although good activity is seen in the 6-n- pletely antagonized by triamterene. Similar
butyl homolog the isopropyl and cyclohexenyl results were obtained in dogs, and it appeared
derivatives have only modest activity. Isomers that the compound might be functioning as an
of triamterene were also studied; the 7-phenyl aldosterone antagonist (401). Initial clinical
isomer was one of the most potent K' blockers studies (402. 403) established the natriuretic
found in the pteridines, even though it is only properties of triamterene in humans in cases
awe& natriuretic agent. The 2-phenyl isomer when aldosterone excretion might be at an el-
is very similar to triamterene in its biological evated level, and evidence was obtained for
properties. Among pyrimidopyrimidines re- inhibition of the nephrotropic effect of aldo-
lated to triamterene, 2,4,7-triamino-5-phe- sterone. However, triamterene possessed na-
nylpyrimido[4,5-dlpyrimidine (241) was in- triuretic activity in adrenalectomized dogs
and rats (404-406) and in adrenalectomized
patients (407); this was inconsistent with an
aldosterone antagonism mechanism. Thus, al-
though triamterene reverses the end results of
aldosterone, its activity does not depend on
the disulacement of aldosterone. The com-
a

pound acts directly on the renal transport of


sodium. Stop-flow studies in dogs pointed to
an effect on the distal site of Naf/ K+ ex-
change, and there was no evidence for a prox-
imal renal tubular effect (408). Triamterene
acts at the apical cell membrane in the collect-
vestigated in some detail. It resembles (238), ing tubule where it blocks sodium channels
in that it does not block potassium excretion in and thus leads to reduced potassium excretion
the sodium-deficient rat. (409). It is also believed to act at the peritubu-
The structure-activity relationships of lar side as well (410).
pteridine diuretics may be rationalized by as- The overall effect of triamterene on electro-
suming that the pteridines bind to some active lytes is to increase moderately the excretion of
site at two points (397). The more important Na+ and, to a lesser extent, of C1- and HC0,-,
site involves a basic center of the drug, which and to reduce K' and NH,+ excretion (403,
in triarnterene may be N-1, N-8, or both. 411,412). Triamterene is a more active natri-
Groups that decrease the base strength of the uretic agent than spironolactone and is well
pteridine nucleus reduce activity. The other absorbed after administration of a single oral
site probably involves the phenyl substituent dose of 50-300 mg/day (402,403). It is about
of triamterene and may be hydrophobic in na- 50-60% bound to plasma proteins. Triam-
ture. There appear to be critical size limita- terene is extensively metabolized and only
tions at the site, as shown by the change in about 3-5% of the drug is excreted unchanged
activity in relation to methyl substitution. Be- in the urine (413). The compound undergoes
cause compounds such as 2,4,7-triaminopteri- hepatic hydroxylation and sulfate ester forma-
at of dine are active, the phenyl group is not a pri- tion. The sulfate ester is also biologically ac-
roup mary requirement for activity and apparently tive and is excreted in the urine. After intra-
:nu- acts in a reinforcing capacity, such as increas- venous administration of triamterene in
Diuretic and Uricosuric Agents

humans, the concentration of the sulfate ester


was 10 times that of the parent compound,
and it has been concluded that the pharmaco-
logically active form of the drug in humans is
the ester (414). Triamterene is also metabo-
lized to its glucuronide adduct, which under-
goes biliary elimination. The duration of ac-
tion in humans is about 16 h (415).
An increased diuresis ensued when triam-
terene was administered to patients who were
receiving the aldosterone antagonist spirono-
lactone (416, 4171, thus further emphasizing
the fundamental differences in the mecha-
nism of action of these two drugs. Triam-
terene potentiates the natriuretic action of the
thiazides while reducing the kaliuretic effect
(416),and other clinical studies with this com-
bination showed that normal serum potas-
sium levels could be maintained without po-
tassium supplements (417). The natriuretic
potency of triamterene does not approach that
of the thiazide diuretics, and the main value of
the drug would appear to be its use in combi-
nation with thiazides in clinical situations in
which Kf loss is a problem. Triamterene
should not be prescribed to patients with renal
insufficiency or hyperkalemia, nor should it be
administered concurrently with potassium
supplements. Triamterene increases serum
uric acid levels and cases of hyperuricemia
have been reported. Some other side effects
that are observed are skin rashes, gastointes-
tinal disturbances, hyperkalemia, weakness,
and dry mouth. tion induced by other diuretics is not modified
2.1.12.2 Other Bicyclic Polyaza Diuret- by DS 210. The diuretic effect is lost in adre-
ics. A group of workers at Takeda synthesized nalectomized rats and restored by cortisol
and studied the diuretic activity of a large se- treatment (419). DS 511 (243) has shown di-
ries of polynitrogen heterocycles (418). The uretic activity comparable to that of hydro-
ring systems prepared are shown in Table chlorothiazide in rats, dogs, and humans, and
2.20, documenting the extensive effort made seems to have a unique mode and site of action
in this investigation. Of the 219 compounds in the nephron (420).
studied in this series, two compounds, DS 210 Hawes and coworkers at the University of
(242) and DS 511 (2431, were selected for Saskatchewan prepared a series of 2,3-disub-
more extensive evaluation. stituted 1,B-naphthyridinescontaining a phe-
The compounds were initially screened in nyl group at the 3-, 4-, or 7-positions (421). In
rats, and hydrochlorothiazide was used as the this study, compounds containing a phenyl
reference compound. DS 210 (242) produces a group at the 7-position had no diuretic activ-
maximal natriuretic effect similar to that of ity. Compounds (244) and (2451, which con-
hydrochlorothiazide in rats without affecting tained a phenyl group at the 4-position, have
potassium excretion. It shows additive activ- similar diuretic and natriuretic properties
ity with hydrochlorothiazide, acetazolamide, when compared to those of triamterene. These
amiloride, and furosemide. Potassium excre- compounds also lack kaliuretic properties.
Diuretic and Uricosuric Agents

activity. Methylation of both the amide and


m i n e nitrogens gave a compound with simi-
lar diuretic activity but with a poorer Na+/ K+
ratio.
Hester and coworkers at Upjohn prepared
a series of 1-(2-amino-1-phenylethy1)-6-phe-
nyl-4H-[1,2,4]triazolo[4,3-a] [1,4]-benzodiaz-
epines and evaluated their diuretic activity
(424). Several of these compounds possessed
diuretic and natriuretic activity, after oral ad-
ministration to rats, with no kaliuretic activ-
ity. The most potent benzodiazepine in this
series is (247); however, it is considerably less

Parish and coworkers synthesized a num-


ber of 2-pyrido[2,3-dl-pyrimidin-4-ones
(422).
1,2-Dihydro-2-(3-pyridy1)-3H-pyrido>[2,3-dl-
pyrimidin-4-one (246) was a potent diuretic

potent than hydrochlorothiazide in the con-


scious rat. At 10 mg/kg, (247) begins to show
significant diuretic and saliuretic activity. Hy-
drochlorothiazide begins to show significant
activity at 0.3 mg/kg; however, the efficacy of
the two compounds appears to be similar.
2.1.12.3 Amiloride. An empirical approach
agent in the rat, although it was not as potent was taken by a group at Merck, Sharp &
as hydrochlorothiazide. An oral dose of 81 Dohme seeking compounds with no or mini-
mgkg in the rat resulted in potassium excre- mal kaliuretic effects. Screening procedures
tion levels that were the same as those of the indicated that N-amido-3-amino-6-bromopy-
saline controls; the sodium and chloride ion razinecarboxamide (248), a compound avail-
excretion levels were the same as those of the able through previous work in the folic acid
saline controls; and the sodium and chloride series, was of interest. The introduction of an
ion excretion levels had doubled. Monge and amino group in the 5-position markedly in-
coworkers at the University of Navarra fur- creased the sodium and chloride excretion:
ther studied the structure-activity relation- N-amidino-3,5-diamino-6-chloropyrazine-car-
ships in this series (423). Placement of nitro or boxamide (amiloride, 249) was among the
amino groups at the 6-position resulted in most promising in animals and in humans (174).
compounds that had marginal or no diuretic The N-amidinopyrazinecarboximides produce
b
2 Clinical Applications
$
i
6-position was detrimental to the antikali-
uretic, pharmacodynamic, and pharmacoki-
netic properties of this class. A number of the
N-substituted 3,5-diamino-6-chloropyrazine-
2-carboxamides were found to be as potent
as triamterene. The N-(dimethylaminoethyl)
amide analog (251) was more potent than tri-

amterene as a diuretic, natriuretic, and anti-


a pronounced diuresis in normal rats and kaliuretic agent after oral administration to
! leave potassium excretion unaffected or re- the rat. The compound was well absorbed and
[
pressed. In the adrenalectomized rat they an- was excreted without significant metabolism.
: tagonize the renal actions of exogenous aldo-
Workers at ICI have synthesized amiloride
sterone, DOCA, and hydrocortisone. In dogs, analogs that have diuretic activity combined
the compounds are less potent, but the rela- with calcium channel-blocking activity or
tive activity in the series is the same as those p-adrenoceptor blocking activity. ICI 147798
in rats. (252) is a single-molecule derivative of amilo-
Structure-activity relationships in this se- ride that was discovered to possess both di-
ries have been investigated in considerable de- uretic and p-adrenoreceptor blocking proper-
tail and some representative compounds from ties (430). At doses of 1-20 mgkg p.o., the
these studies are listed in Table 2.21. The ac- natriuretic activity of ICI 147798 was similar
tivities of the compounds were determined on to that of hydrochlorothiazide in dogs; at 1
the basis of their DOCA-inhibitory activity, mg/kg it produced significantly less kaliuresis
which closely paralleled the diuretic activity in than did hydrochlorothiazide. ICI 147798
intact rats and dogs (425-427). blocked adrenergic receptors in vitro and in
Workers at Ciba investigated the replace- vivo, and it also inhibited isoproternol-in-
ment of the acylguanidine moiety with a 1,2,4- duced tachycardia in rats, guinea pigs, cats,
oxadiazol-&amino- group (428). This com- and dogs. ICI 206970 (253), another analog of
pound, CGS 4270 (250),has a similar profile to amiloride, possessed diuretic and calcium
that of amiloride in rats and dogs. channel-blockingactivity (431). In the dog, af-
ter oral administration, ICI 206970 was less
potent than hydrochlorothiazide with respect
to its diuretic and saliuretic effects. In con-
trast to hydrochlorothiazide, no significant
changes were observed in plasma potassium
levels after 14 days of dosing.
The structural similarity of amiloride and
triamterene (249 and 240, Table 2.18) and
their similar biological actions have raised the
More recently, Ried and coworkers pre- question of whether the pteridines are, in fact,
pared a number of amiloride analogs that were closed-ring versions of the N-amidinopyrazine-
, modified at the 2- and 6-positions (429). In carboxamides. The open-chain analogs of tri-
/
i
general, replacement of the chlorine at the amterene and the bicyclic analogs of amiloride
132 Diuretic and Uricosuric Agents

Table 2.21. DOCA Inhibitory Activity in Adrenalectomized Rats of Some


N-Amidino-3-aminopyrazinescarboxamidesa

R,
H H H H H
H
H
CH3
CH3
H
H
H
H
H
H
H H H H MeNH
H H H H (Me),CHCH,NH
H H H H (Me),CNH
H
H H H H PhNH
CH3
CH3
3,4-Cl2C6H6CH2
H
H
H H H H OH
H H H H OMe
H
H H H H SMe
CH3C0
H
"From Refs. 426-428.
bThis score is related to the dose of each compound that produces a 50% reversal of electrolyte effect from the adrmnis-
tration of 12 pg of DOCA to adrenalectomized rats and is scored as follows: 10 pg (++ +), 10-50 pg (++ +), 51-100 pg (+ t),
101-800 pg (+), >SO0 pg (+I-).

that have been studied are generally less ac- In the usual dosage, amiloride has no im-
tive than the drugs themselves. Triamterene portant pharmacological actions except those
is a weaker base (pKa = 6.2) than amiloride related to the renal tubular transport of elec-
(pKa = 8.67). Amiloride as its hydrochloride is trolytes. Clinically, it is used extensively in
readily water soluble, whereas triamterene is combination with hydrochorothiazide.
slightly soluble. After oral administration, ap- 2.1.12.4 Azolimine and Clazolimine. A se
proximately 50% of amiloride is absorbed in ries of imidazolones was studied by a group at
humans (432). It is approximately 23% bound Lederle Laboratories in their search for anon-
to plasma proteins and is not metabolized in steroidal antagonist of the renal effects of min-
humans. It is excreted in the urine mainly un- eralocorticoids. Azolimine (254) and clazoli-
changed. mine (255) were the most interesting in this
2 Clinical Applications

adrenaledomized, deoxycorticosterone-treated
rats and sodium-deficient rats (433). Similar
effects were found for clazolimine (434). The
compound may be useful in combination with
HN the classical diuretics as an aldosterone antag-
onist diuretic in humans.
(254) R =H
(255) R = C1 2.1.1 3 Atrial Natriuretic Peptide. Atrial
natriuretic peptide (ANP, 256) is a 28-amino
series. Azolimine antagonized the effects of acid peptide that is released into circulation
mineralocorticoids on renal electrolyte excre- from the heart after atrial distension and in-
tion in several animal models. Large doses of creases in heart rate. It is synthesized and
azolimine produced natriuresis in adrenalec- stored in specific atrial secretory granules as a
tomized rats in the absence of exogeneous 126-amino acid precursor molecule. ANP ex-
mineralocorticoid, but its effectiveness was erts natriuretic, diuretic, and vasorelaxant
greater in the presence of a steroid agonist. In properties upon administration and sup-
conscious dogs, azolimine was effective only presses renin and aldosterone levels (435,
when deoxycorticosterone was administered. 436). Through interaction with its receptor it
Azolimine significantly improved the urinary promotes the generation of cGMP by guanyl-
Na+/Kf ratio when used in combination with ate cyclase activation. The pharmacological
thiazides and other classical diuretics in both properties produced by this peptide suggest

ASP-MET-ARG-GLY-GLY-PHE-CYS-SER-SER-ARG-ARG-LEU-SER
ARG
/ \S-S
\ILE-GLY--ALA-GLN-SER-GLY-LEU-GLY-CYS-ASN-SER-PHE-ARG-TYR126
\

(256)
Diuretic and Uricosuric Agents

that it mav" be beneficial in the treatment of much of the current research in this area has
several cardiovascular disorders. The thera- been focused on methods of potentiating the
peutic potential of ANP, however, is limited by activity of ANP in vivo by preventing its deg-
its poor oral absorption and extremely short radation (436).
biological half-life of less than 60 s in the rat 2.1.13.1 ANP Clearance Receptor Block-
and only a few minutes in humans (437,438). ers. Several groups have prepared ligands for
The mechanism of action for the natri- the ANP c-receptor that have prolonged the
uretic activity of ANP has been the subject of t,,, of ANP. SC 46542 {des-[Phe106, Gly107,
much research over the past few years. It is Ala115, Gln1l61ANP (103-126)) is a biologi-
believed that ANP-induced natriuresis results cally inactive analog of ANP that has similar
from its effect on renal hemodynamics. ANP is affinity for the c-receptor as ANP (450). In the
able to increase the glomerular filtration rate normal, conscious rat and spontaneously hy-
significantly (439,440). This effect seems to be pertensive rat, however, SC46542 did not sig-
brought about by vasodilation of the afferent nificantly increase immunoreactive ANP con-
arterioles with vasoconstriction of the efferent centrations in plasma.
vessels. This effect increases glomerular cap- Two linear peptides, Ma,-rat-ANP,-,,-
illary pressure and, therefore, the glomerular NH, and naptoxyacetyl isonipecotyl-Arg-Ile-
filtration rate. Further studies have shown Asp-Arg-Ile-NH,, were shown to increase
that ANP may also inhibit sodium reabsorp- plasma immunoreactive ANP concentrations
tion in the collecting tubules and duct (441). in anesthetized rats (451). In response to the
ANP has also been demonstrated to inhibit infusion of these compounds, a significant in-
both basal and angiotensin-stimulated secre- crease in glomerular filtration rate and so-
tions of aldosterone in vitro in adrenal prepa- dium excretion was observed.
rations and in vivo after infusion in animals Infusion of C-ANP,-,, was also shown to
and humans (442-444). Because ANP-in- increase plasma immunoreactive ANP con-
duced natriuresis occurs very rapidly, the reduc- centrations in anesthetized and conscious rats
tion in aldosterone secretion contributes to a (452). C-ANP4-23increased the urinary excre-
longer-term modulation of sodium excretion. tion of water and sodium in the conscious
ANP is eliminated from the circulation bv " DOCAlsalt-hypertensive rats when adminis-
way of two major pathways. Studies have tered i.v. (453).
shown that ANP is eliminated from the circu- More recently, workers at AstraZeneca
lation by enzymatic degradation. The enzyme have reported on new series of nonbasic ANP
most responsible for its degradation is neutral c-receptor antagonists (454). They have made
endopeptidase (NEP, EC 3.4.24.11) (445,446). modifications to C-ANP,,, and AP-811(257),
NEP is a zinc metallopeptidase that cleaves which have retained good affinity for the
the a-amino bond of hydrophobic amino acids. c-receptor and have improved physical proper-
Other enzymes in the renin-angiotensin and ties. Either of the arginines of AP-811could be
kallikrein-kinin systems have also been shown replaced with alanine.
to degrade ANP. 2.1.13.2 Neutral Endopeptidase Inhibitors.
ANP is also removed from circulation Originally, NEP inhibitors were designed and
through a receptor-mediated clearance path- studied for their analgesic properties, given
way (448). ANP clearance receptor (c-recep- that this enzyme was known to degrade en-
tor) can be found in several tissues, including kephalins. When it was discovered that NEP
kidney cortex, vascular, and smooth muscle also degraded ANP, many of the known NEP
cells (448, 449). ANP binds to this receptor, inhibitor compounds, such as thiorphan (258)
and then this receptor-ANP complex is inter- and phosphoramidon (2591, were evaluated
nalized. ANP is transported to the lysosome, for their potential diuretic and cardiovascular
where it undergoes extensive hydrolysis. The activities. Both thiorphan and phosphorami-
clearance receptor is recycled to the cell's sur- don increased the half-life of exogenous ANP
face, where it can repeat this process. in the rat (455). Phosphoramidon, when in-
Because infusion of ANP was shown to pro- fused into rats with reduced renal mass, sig-
duce several potentially therapeutic benefits, nificantly increased diuresis, natriuresis, and
2 Clinical Applications

'OH
H
'N' -N.
H

dition, there is also an increase of plasma en-


dothelin-1 levels (463, 464) that may also ex-
plain the limited effectiveness of this class.
In recent years, there have been many re-
ports on new potent inhibitors of NEP. Many
of these compounds are di- or tripeptides that
contain a group that binds to the zinc atom in
the active site of the enzyme. There are four
different classes of NEP inhibitors: thiols, car-
boxylates, phosphoryl-containing, and hy-
droxamates.
Thiorphan was the first reported thiol in-
hibitor of NEP. Both the R- and S-enantio-
mers of thiorphan have the same enzyme in-
hibitory potency, IC,, = 4 nM. Extensive
structure-activity relationship studies have
shown that it is possible to replace the glycine
residue with an 0-benzyl serine and still re-
tain potency (465). This compound, ES 37
(260), has an IC,, for NEP of 4 nlM and is a

1 glomerular filtration rate (456). Thiorphan


g9 was also shown to increase sodium excretion
i in anesthetized and conscious normal rats

1I (457).

i beenSeveral selective inhibitors of NEP have


evaluated in clinical trials and have been
found to have little or no efficacy in lowering
md pressure (458-460). It is believed that
NEP inhibition may slow the metabolism or
1 clearance of angiotensin I1 (460-462). In ad-
Diuretic and Uricosuric Agents

potent inhibitor of angiotensin-converting en-


zyme (ACE), IC,, = 12 nM. Reduction of the
phenyl ring also affords a potent NEP inhibi-
tor, IC,, = 32 nM.
Investigators at Squibb replaced the gly-
cine in thiorphan with an aminoheptanoic
acid (466).This compound, SQ 29072 (261), is

(471). The compounds also led to a significant


natriuresis in the initial 24 h of treatment.
This effect attenuated over time.
A number of carboxyl-containing NEP in-
(261) hibitors were prepared by workers at Scher-
ing-Plough and Pfizer. Candoxatrilat (TJK
a potent NEP inh itor with an IC,, value o
- ~
69578,264) was a potent NEP inhibitor (Ki =
26 nM. When administered intravenously
(300 pgkg) to a conscious SHR, SQ 29072 pro-
duced a modest diuretic and natriuretic re-
sponse (467). In the DOCNsalt-hypertensive
rat, when equidepressor doses of SQ 29072
and ANF(99-126) were administered, there
was a prolonged urinary excretion of sodium.
Another structurally related analog, SQ 28603
(2621, was also reported to be a potent and

(264) R = H, candoxatrilat

2.8 X lo-' M )designed by workers at Pfizer


(472). When given i.v. to mice, it increased en-
selective inhibitor of NEP. When infused in dogenous levels of ANP and produced diuretic
conscious, DOCNsalt-hypertensive rats, SQ and natriuretic responses. When the prodrug
28603 caused an increase in plasma ANP con- candoxatril(265) was administered to human
centration and in sodium excretion and signif- subjects, doses of 10-200 mg caused a rise in
icantly lowered mean arterial pressure (468). basal ANP levels. Natriuresis was observed
Workers at Schering-Plough also prepared only at the highest dose (473).
thiorphan-type analogs. SCH 42495 (263) ele- Workers at Schering-Plough also prepared
vated plasma ANF concentrations in animal a number of carboxyl-containing NEP inhibi-
models (469, 470). In a study with eight pa- tors. SCH 39370 (266) was discovered to be a
tients with essential hypertension, plasma potent NEP inhibitor (IC,, = 11 nM) and,
ANF levels increased (+123%,P < 0.01) and when administered to rats with congestive
later remained elevated (+34%, P < 0.01) heart failure, it caused an increase in urinary
2 Clinical Applications

potent NEP inhibitor (IC,, = 2 nM), is a nat-


ural competitive inhibitor produced by Strep-
tomyces tanashiensis. Vogel and coworkers re-
volume and plasma ANP levels (474, 475). In
ported that phosphoryl-Leu-Phe (269) is a
an ovine heart failure model, SCH 39370,
when given as a bolus injection, caused signif-
icant natriuresis and diuresis (476). A struc-
turally related compound, SCH34826 (267),

potent NEP inhibitor (IC,, = 0.3 nM) (481).


This is about an order of magnitude more po-
tent than thiorphan.
Workers at Ciba reported on a series of po-
tent phosphorous-containing inhibitors of
NEP (482). CGS 24592 (270) had an IC,,
produced a significant natriuretic effect in
DOCNsalt-hypertensive rats (477). In normal
volunteers maintained on a high sodium diet
for 5 days, SCH 34826 promoted a significant
increase in sodium, calcium, and phosphate
excretion (478).
Hydroxamates form strong bidentate li-
gands to zinc. Compounds containing this
functional group are potent NEP inhibitors.
The prototype of this class is RS-kelatorphan.
This compound strongly inhibits NEP (IC,, =
1.8 nM) and aminopeptidase N(ANP) (IC,, =
380 nM) (479). The SS-isomer of kelatorphan, (270) R = H
RB 45 (268),is a more selective NEP inhibitor (271) R = phenyl
[IC,, = 1.8 nM, IC,, = 29,000 nM (APN)]. In
rats, when given at 10 mgkg i.v., RB 45 in- value of 1.6 nM. The racemic analog CGS
creased the half-life of ANP (480). 24128 (IC,, = 4.3 nM, NEP) increased plasma
Phosphoryl-containing inhibitors also in- ANP immunoreactivity levels by 191% in rats
teract strongly with zinc and are potent NEP administered exogenous ANP(99-126) (483).
inhibitors. Phosphoramidon (2591, which is a CGS 24128 also potentiated the natriuretic ac-
Diuretic and Uricosuric Agents

(272) (273) (274)


Xanthine Uric acid Allantoin

Urea H H

+ - H 2 N ~N N
~ N~ H 2

OCHCOOH
(276) (275)
Glyoxylic acid Allantoic acid
Figure 2.10. Purine metabolism.

tivity of exogenous administered ANP(99- as the monosodium salt, which is also very
126). Because of the poor bioavailability of highly soluble and tends to form supersatu-
CGS 24592, a series of prodrugs were investi- rated solutions.
gated. CGS 25462 (271) provided significant Uric acid forms from purines, which are lib-
and sustained antihypertensive effect in the erated as a result of enzymatic degradation of
DOCNsalt-hypertensive rat after oral admin- tissue and dietary nucleoproteins and nucleo-
istration. tides, but it is also formed by purine synthesis
(484). When the level of monosodium urate in
2.1.1 4 Uricosuric Agents. In humans, one the serum exceeds the point of maximum sol-
of the principal products of purine metabolism ubility, urate crystals may form, particularly
(i.e., uric acid) is implicated in several human in the joints and connective tissues. These de-
diseases such as gout. Guanine and adenine posits are responsible for the manifestations
are both converted to xanthine (272); oxida- of gout. Serum urate levels can be lowered by
tion, catalyzed by xanthine oxidase, yields uric decreasing the rate of production of uric acid
acid (273). In humans. uric acid is the excre- or by increasing the rate of elimination of uric
tory product and most of it is excreted by the acid. The most common method of reducing
kidney. In most mammals, uric acid is further uric acid levels is to administer uricosuric
hydrolyzed by uricase to allantoin (274), a drugs, which increase the rate of elimination
more soluble excretory product. Allantoin, in of uric acids by the kidneys.
turn, is further degraded to allantoic acid 2.1.14.1 Sodium Salicylate. The uricosuric
(275) by allantoinase, and then to urea and properties of sodium salicylate (277, Table
glyoxylic acid (276) by allantoinase (Fig. 2.10). 2.22) were noted before 1890, and its use con-
Uric acid is not the major pathway of nitrogen tinued through 1950. As late as 1955, sodium
excretion in humans. Instead, the ammonia salicylate was used for the long-term treat-
nitrogen of most amino acids, the major nitro- ment of gout (485). For adequate uricosuric
gen source, is shunted into the urea cycle. Uric activity, however, salicylate must be adminis-
acid is mostly insoluble in acidic solutions, al- tered in doses greater than 5 glday, often re-
though alkalinity increases its solubility. At sulting in serious side effects, so that its usage
the pH of blood (pH 7.44), uric acid is present has gradually declined.
2 Clinical Applications 139

able 2.22 Uricosuric Agents


Generic Name Trade Name Structure Reference
Salicylic acid

Probenecid Benemid

Sulfinpyrazone Anturane

N-N

Allopurinol Zyloprim
60 OH

Benzbromarone Desuric,
Minuric,
Narcaricin

2.1.14.2 Probenecid. Probenecid (278, Ta- carinamide (279) in normal subjects and in
ble 2.22) was developed as a result of a search gouty subjects (487). Carinamide had been in-
for a compound that would depress the renal troduced as an agent for increasing penicillin
tubular secretion of penicillin (486) at a time blood levels by blocking its rapid excretion
when the supply of penicillin was limited. Rec- through the kidney. Its biological half-life was
ognition of the uricosuric properties of probe- relatively short, and the search for compounds
necid resulted from prior experience with the with a longer half-life that would not have to be
uricosuric effects of the related compound administered so frequently led to probenecid.
Diuretic and Uricosuric Agents

Probenecid is insoluble in water, but the


sodium salt is freely soluble. In the treatment
of chronic gout, a single daily dose of 250 mg is
given for 1 week, followed by 500 mg adminis-
tered twice daily. A daily dose of up to 2 g may
be required.
2.1.14.3 Sulfinpyrazone. Despite the ther-
apeutic efficacy of phenylbutazone (281) as an

/
R-N
I

(280) R = H, Methyl, Ethyl, Propyl

In a study of a series ofN-dialkylsulfamoyl-


benzoates (2801, Beyer (488) found that as the
length of the N-alkyl groups increased, the re-
nal clearance of the compounds decreased.
This most likely results from the enhanced anti-inflammatory and uricosuric agent, its
lipid solubility imparted by the longer alkyl side effects were severe enough to preclude its
groups, which would account for their com- continuous use in the treatment of chronic
plete back diffusion in acidic urine. Optimal gout. Evaluation of several chemical conge-
activity was found in probenecid, the N-dipro- ners indicated that the phenylthioethyl analog
pyl derivative. The structure-activity relation- of phenylbutazone (282) had promising anti-
ship of probenecid congeners and that of other
uricosuric agents has been reviewed in detail
by Gutman (487).
Normally, a high percentage of the uric acid
filtered by the glomerulus is reabsorbed by an
active transport process in the proximal tu-
bule. It is now clear that the human proximal
tubule also secretes uric acid. as does the Drox-
imal tubule of many lower animals. small
doses of probenecid depresses the excretion of
uric acid by blocking tubular secretion,
whereas high doses lead to greatly enhanced
excretion of uric acid by depressing proximal
reabsorbtion of uric acid (489).
Probenecid is completely absorbed after
oral administration; peak plasma levels are
reached in 2-4 h. The half-life of the drug in
plasma for most patients is 6-12 h. The drug is inflammatory and uricosuric activity (490).A
85-95% bound to plasma proteins. The small metabolite, the sulfoxide pyrazone (283), ex-
unbound portion is filtered at the glomerulus; hibited enhanced uricosuric activity (491,
a much larger portion is actively secreted by 492). Interestingly, the corresponding sulfone
the proximal tubule. The high lipid solubility (284) does not appear to be a metabolite (490).
of the undissociated form results in virtually Sulfinpyrazone lacks the clinically striking
complete reabsortion by back diffusion unless anti-inflammatory and analgesic properties of
the urine is markedly alkaline. phenylbutazone.
(pK, = 2.8)
and readily forms soluble salts. Evaluation of a
number of congeners indicated that a low pK,
and polar side chain substituents favor urico-
suric activity (493) and increase the rate of
renal excretion (494).The inverse relationship
between uricosuric potency and pK, has also
been confirmed in a number of 2-substituted
analogs of probenecid (285) (probenecid R =
(273). Allopurinol (286) and its primary me-
tabolite, alloxanthine (287).- ,are inhibitors of
xanthine oxidase.
Inhibition of the last two steps in uric acid
biosynthesis by blocking xanthine oxidase re-
duces the plasma concentration and urinary
excretion of uric acid and increases the plasma
levels and renal excretion of the more soluble
(285) R = OH, C1, NO2 oxypurine precursors. Normally, in humans
the urinary purine content is almost solely
H; 278). All three compounds were consider- uric acid; treatment with allopurinol
- results in
ably stronger acids than probenecid. Evalua- the urinary excretion of hypoxanth'ine, xan-
tion in the Cebus albifrons monkey indicated thine, and uric acid, each with its independent
that these compounds were about 10 times as solubility. Lowering the uric acid concentra.
potent as probenecid when compared on the tion in plasma below its limit of solubility fa.
basis of concentration of drug in plasma (495). cilitates the dissolution of uric acid deposits,
In small doses, as seen with other urico- The effectiveness of allopurinol in the treat.
suric agents, sulfinpyrazone may reduce the ment of gout and hyperuricemia that results
excretion of uric acid, presumably by inhibit- from hematogical disorders and antineoplas-
ing secretion but not tubular reabsorbtion. Its tic therapy has been demonstrated (497-499).
uricosuric action is additive to that of proben- For the control of hyperuricemia in gout,
ecid and phenylbutazone but antagonizes that an initial daily dose of 100 mg is increased
of the salicylates. Sulfinpyrazone can displace weekly at intervals by 100 mg. The usual daily
to an unusual degree other organic anions maintenance dose for adults is 300 mg. -

that are bound extensively to plasma protein 2.1.14.5 Benzbromarone. Benzbromarone


(e.g.,sulfonamidesand salicylates), thus alter- (288) is a benzofuran derivative that has been
ing their tissue distribution and renal excre-
tion (489, 496). Depending on concomitant
medication, this may be a clinical asset or lia-
bility.
For the treatment of chronic gout, the ini-
tial dosage is 100-200 mg/day. After the first
week the dose may be increased up to 400 mg/
day until a satisfactory lowering of plasma
uric acid is achieved.
2.1.14.4 Allopurinol. Allopurinol(286)does
not reduce serum uric acid levels by increasing
renal uric acid excretion; instead it lowers
plasma urate levels by inhibiting the final reported to lower serum urate levels in ani-
steps in uric acid biosynthesis. mals and human studies. In normal and hyper-
Uric acid in humans is formed primarily by uricaemic subjects, benzbromarone reduced
xanthine oxidase-catalyzed oxidation of hypo- serum uric acid levels by one-third to one-half
xanthine and xanthine (272) to uric acid (500, 501). In comparison with other urate-
Diuretic and Uricosuric Agents

lowering drugs, 80 mg of micronized or 100 mg Four side effects were noticed after the
of nonmicronized benzbromarone had equal widespread and prolonged use of the thiazide
urate-lowering activity to 1-1.5 g of probene- diuretics:
cid or 400-800 mg of sulfinpyrazone (500,
502). 1. potassium depletion
The mechanism of the urate-lowering ac- 2. uric acid retention
tivity of benzbromarone appears to be attrib- 3. hyperglycemia
utable to its uricosuric activity. In rats, benzo-
4. increased plasma lipids
bromarone inhibited urate reabsorption in the
proximal tubules when given at 10 mgkg i.v.
Potassium depletion has been encountered
(503). In isolated rat liver preparation, benz-
most frequently. The kaliuretic effect of the
bromarone inhibits xanthine oxidase in vitro
thiazides can be compensated for by supple-
but not in viuo (504). In humans, this com- mentary dietary potassium; nevertheless, re-
pound only weakly inhibits xanthine oxidase search was directed toward the development
and no increase in urinary excretion of xan- of potassium-sparing diuretics. Arniloride
thine or hypoxanthine was observed (505). Af- (19651, spironolactone (19591, and triam-
ter oral administration, about 50% of benzbro- terene (1965) were discovered as a result of
marone is absorbed. The drug undergoes this effort; these compounds are weak diuret-
extensive dehalogenation in the liver and is ics, however, and are generally used in combi-
excreted mainly in the bile and feces. For con- nation with other diuretics (e.g., hydrochlo-
trol of gout the usual therapeutic dose is 100- rothiazide).
200 mg daily. Benzbromarone has few side ef- The next step was the discovery of the high
fects and is usually well tolerated. ceiling or loop diuretics [e.g., ethacrynic acid
(1962), furosemide (1963), and bumetanide
(1971)],which are shorter acting and more po-
3 CONCLUSION tent than the thiazide diuretics. They too have
the same potential side effects as the thia-
The development and therapeutic use of di- zides. One advantage of the loop diuretics is
uretic agents constitutes one of the most sig- their efficacy in chronic renal insufficiency,
nificant advances in medicine made during the particularly in cases with low glomerular fil-
twentieth century. Continuous progress has tration rates.
been made during this time on the develop- A large volume of highly technical informa-
ment of safer and more effective diuretic tion has been published over the past 15 years
agents. Between 1920 and 1950, a large num- regarding this therapeutic area. More sensi-
ber of organic mercurials were prepared and tive analytical techniques have been devel-
evaluated as diuretics. Because of the lack of oped, so that data regarding bioavailability
oral activity and toxicity of these compounds, and pharmokinetics are now available for di-
research efforts were focused on the develop- uretics that are currently prescribed and that
ment of orally effective nonmercurial diuret- are in development. Advances in renal and
ics. The carbonic anhydrase inhibitors, devel- ion-transport research have led to a more pre-
oped in 1950 and later, were orally active but cise understanding of the cellular mechanisms
upset the acid-base balance and could be given of actions of the various classes of diuretic
only intermittently. The thiazide diuretics, agents. This has aided in the design of newer,
developed in the late 1950s, represented a true more effective agents.
advance in the treatment of edema. They were Diuretics introduced into more recent clin-
remarkably nontoxic and effective in most ical studies include (1) newer, more potent
cases. It very soon became apparent that not loop diuretics such as torasemide and azos-
only were they effective diuretics, they were emide, (2)development of uricosuric diuretics,
also useful in the treatment of hypertension (3)newer-generation sulfamoyl diuretics, and
by themselves or in combination with other (4) development of neutral endopeptidase in-
antihypertensive drugs. hibitors.
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Medical Practice, 9th ed., Section 1(6),Wil- millan, New York, 1975, p. 352.
liams & Wilkins, Baltimore, 1973. 500. T . Y u , J. Rheumatology, 3,305 (1976).
485. F. G. W. Marson, Lancet, 11,360 (1955). 501. F . Matzkies, F. Berg, and R. Minzlaff,Fortsch.
486. K. H. Beyer, H. F. Russo, E. K. Tillson, A. K. Med., 95, 1748 (1977).
Miller, W . F. Venvey, and S. R. Gass, Am. J. 502. N. Zoller, W . Dofel, and W . Grobner, Klin.
Physiol., 166, 625 (1951). Wochensch., 48,426 (1970).
487. A. B. Gutman, Adv. Pharmacol., 4,91 (1966).
503. R. Kramp, Eur. J. Clin. Inuest., 3,245 (1973).
488. K. H. Beyer, Arch. Int. Pharmacodyn., 98, 97
(1954). 504. R. Kramer and M. Muller, Experientia, 29,391
489. P. Brazeau in L. S. Goodman and A. Gilman, (1973).
Eds., The Pharmacological Basis of Therapeu- 505. J. Broehuysen, M. Pacco, R. Sion, L. Demeu-
tics, 5th ed., Section VIII, Macmillan, New lenaere, and M. vanHee, Eur. J. Clin. Pharm.,
York, 1975, p. 860. 4, 125 (1972).
CHAPTER THREE

Myocardial Infarction Agents


GEORGE E. BILLMAN
RUTH A. ALTSCHULD
The Ohio State University
Columbus, Ohio

Contents
1 Introduction, 156
2 Pathophysiology of Myocardial Infarction, 156
2.1 Coronary Occlusion, 156
2.1.1 Apoptosis versus Necrosis, 157
2.1.2 Preconditioning, 157
2.2 Malignant Arrhythmias, 157
2.2.1 Role of Cytosolic Free Calcium, 157
2.2.2 Calcium and Arrhythmia Formation, 159
2.2.3 Extracellular Potassium Accumulation
During Myocardial Ischemia, 162
2.2.4 Extracellular Potassium and Cardiac
Arrhythmias, 163
2.3 Ventricular Remodeling, 164
3 Treatment for Myocardial Infarction, 164
3.1 Pain Relief, 164
3.2 Thrombolysis, 165
3.2.1 Streptokinase, 165
3.2.2 Plasminogen Activators, 165
3.2.3 Anticoagulants, 166
3.2.4 Glycoprotein IJMIIa Receptor
Blockers, 166
3.3 Treatment of Arrhythmias Induced by
Myocardial Ischemia, 167
3.3.1 Classification of Anti-Arrhythmic
Drugs, 167
3.3.2 Calcium Channel Antagonists, 168
3.3.3 Verapamil, 168
3.3.4 Diltiazem, 169
3.3.5 Nifedipine, 170
3.3.6 Flunarizine, 170
3.3.7 Magnesium, 171
3.3.8 Mibefradil, 171
33.9 SodiudCalcium Exchanger
Antagonists, 173
Chemistry and Drug Discovery 3.3.10 Calcium Channel Agonists, 175
ne 3: Cardiovascular Agents and 3.3.11ATP-Sensitive Potassium Channel
Antagonists, 176
Abraham 3.3.12 P-adrenergic Receptor Antagonists, 179
O 2003 John Wiley & Sons, Inc. 3.4 Prevention of Remodeling, 180
Myocardial Infarction Agents

3.4.1 Ace Inhibitors, 180


3.4.2 Glucose/Insulin/Potassium,181
4 Summary and Conclusions, 181

1 INTRODUCTION cipitate coronary artery occlusion. Regardless


of the initiating- event, tissue downstream
Acute myocardial infarction was called the from an occlusion is deprived
- of arterial blood
quintessential disease of the 20th century (1). with its life sustaining oxygen and nutrients,
Before the introduction of coronary care units, and metabolic wastes accumulate. The lack of
short-term in-hospital mortality was approxi- oxygen inhibits mitochondria3 oxidative phos-
mately 30%. Coronary care units halved mor- phorylation, the major source of the adenosine
tality in the early 1960s, primarily because of triphosphate (ATP) used to power excitation-
the use of P-adrenergic antagonists, continu- contraction coupling and maintain intracellu-
ous electrocardiography (ECG) monitoring, lar homeostasis. The affected muscle cells are
and direct current defibrillators. The advent
briefly able to regenerate ATP from the high
in the 1980s of thrombolytic therapy for dis-
energy phosphate storage pool, phosphocre-
solving occlusive blood clots again halved mor-
atine, but with no-flow ischemia, this high en-
tality, but in the past few years, incremental
improvements in reperfusion therapy have ergy phosphate store is depleted within min-
produced only small further reductions in utes, and ATP begins to decline. This is
mortality. Acute myocardial infarction re- accompanied by the accumulation of the ATP
mains the most important cause of death in breakdown products, adenosine diphosphate,
the United States (I),and post-infarction re- adenosine monophosphate, and inorganic
modeling in survivors is contributing to the phosphate, which activate glycogenolysis and
growing congestive heart failure epidemic of anaerobic glycolysis (3). An increase in circu-
the late 20th and early 21st centuries. lating catecholamines also accelerates glyco-
There have been four classical goals in the gen breakdown and glycolytic flux (4).
pharmacologic treatment of an acute myocar- Anaerobic glycolysis can generate some
dial infarction: (1)pain relief, (2) reperfusion ATP, but the conversion of glycogen to lactic
acid yields only a small percentage of the en-
and maintenance of vessel patency, (3) pre-
ergy that could otherwise be obtained from the
vention and treatment of arrhythmias, and (4)
prevention of post-infarction ventricular re- complete oxidation of glycogen's glucose moi-
eties to carbon dioxide and water. As a result,
modeling, a leading cause of congestive heart
the tissue becomes energy starved and con-
failure (1). A fifth goal has begun to emerge,
tractile function declines. This down-regula-
i.e., the prevention of reperfusion damage fol-
tion of contractility may help preserve the
lowing successful thrombolysis, percutaneous
limited energy reserves of the ischemic myo-
intervention (e.g., angioplasty) to open an oc-
clusion, or coronary artery bypass surgery. cardium, but there continues to be a mismatch
between energy production and consumption.
The lack of blood flow also allows for the
2 PATHOPHYSIOLOGY O F MYOCARDIAL buildup of metabolic wastes, particularly lac-
INFARCTION tic acid and amphiphilic fatty acid metabolites,
and the tissue becomes acidotic. The increased
intracellular H+ concentration favors intra-
2.1 Coronary Occlusion
cellular Na+ accumulation through the sar.
The typical myocardial infarction begins with colemmal Na+/Ht exchanger and this, in
the rupture of an atherosclerotic plaque (2). A turn, favors excess Ca2+accumulation by the
thrombus or blood clot forms at the site and reverse mode of the electrogenic sarcolemmal
over time fills the lumen of the coronary ar- Na+/Ca2+exchanger (5, 6). Intracellular free
tery, interfering with or abolishing blood flow. Ca2+ concentrations gradually increase, and
Thromboemboli and vasospasm may also pre- cytosolic Ca2+ overload activates proteasee
2 Pathophysiology of Myocardial infarction

(7-9)and lipases (10-12), which, in turn, de- The need to treat the myocardium with a
grade important cellular components. preconditioning agent before a sustained isch-
emic period has limited the clinical usefulness
2.1.1 Apoptosis versus Necrosis. If the tis- of this process to elective ischemia such as that
sue downstream from a coronary occlusion is associated with cardiac surgery. If a precondi-
, affected cells will eventually tioning-like effect could be achieved pharma-
tissue. This im- cologically at reperfusion, it undoubtedly
ac pump function because would have a beneficial effect.
diac myocytes are terminally differentiated
2.2 Malignant Arrhythmias
d have very limited ability to replicate. The
the onset of thrombosis As noted above, myocardial ischemia provokes
ti1 myocyte death varies depending on the abnormalities in the biochemical homeostasis
egree of myocardial ischemia and on the con- of individual cardiac cells. These intracellular
ctile state of the myocardium. Some occlu- changes culminate in the disruption of cellular
ere can be intermittent electrophysiologic properties, and life-threat-
ckage (13). There can ening alterations in cardiac rhythm, such as
so be considerable individual variation in ventricular fibrillation, frequently occur. Var-
e extent of native coronary collateral blood ious chemical substances have been proposed
of the myocardial tis- as possible causative factors in the genesis
on can remain viable of ventricular fibrillation during myocardial
potentially salvageable for up to 12 h in ischemia, including catecholamines, amphi-
philic products of lipid metabolism, various
Cells in tissue that is not reperfused even- peptides, cytosolic calcium accumulation, and
which initiates an in- increases in extracellular potassium (25-30).
matory response and scar formation. Re- The following sections shall focus on the role
t studies indicate that some cells in the that changes in cellular calcium and potas-
ct border zone and some that are success- sium play in the induction of cardiac arrhyth-
reperfused before necrotic cell death may mias during myocardial ischemia.
equently die through programmed cell
h or apoptosis (14-16). Although many of 2.2.1 Role of Cytosolic Free Calcium. Under
ave been detected in normal conditions ventricular muscle cells
area may be non- maintain resting levels of cytosolic calcium ap-
ore abundant but proximately 5000 times lower than the extra-
h smaller in size than the contractile car- cellular calcium concentration (31). Several
to prevent cardio- important regulatory mechanisms are respon-
ocyte apoptosis should reduce infarct size. sible for maintaining the low cytosolic calcium
levels vital for normal cardiac function. In
In experimental brief, calcium influx is restricted by voltage-
s of ischemia and sensitive calcium channels that are activated
sion before a 30-90 min coronary oc- by the cardiac action potential and regulated
on reduce the size of the subsequent in- by intracellular messengers (e.g., phosphory-
ct (17). This "preconditioning" phenome- lation) (31-33). Calcium is also extruded from
n has been the subject of intense the cell by an electrogenic Na+/Ca2+ ex-
licated by a vari- changer (forward mode, 3 Na+ in, 1 Ca2+ out)
of pharmacologic agents that activate pro- and sarcolemmal Ca2+ adenosine triphos-
kinase C (18-20) and/or the mitochon- phatase (ATPase). Inside the cell, a second
ATP-sensitive potassium channel (21- Ca2+ ATPase pumps calcium into the lumen
In reperfused rat hearts, ischemic of the sarcoplasmic reticulum. These systems
nditioning reduced apoptosis by inhibit- rapidly decrease the elevations in cytosolic
eutrophil accumulation and down-regu- free calcium concentration brought about by
g the expression of the pro-apoptotic pro- excitation and induce relaxation during dias-
tole. In addition, mitochondria can take up
158 Myocardial Infarction Agents

calcium, and a number of calcium-binding pro- sequestration (46). These reactions culminate
teins also serve to buffer intracellular calcium in increased calcium entry into cardiac cella
levels (31). Under steady-state conditions, cal- and increased uptake and release from intra-
cium influx across the cell membrane (primar- cellular stores. The activation of myocardial
ily through L-type calcium channels) during P2-adrenergicreceptors can also contribute to
systole is matched by an equal calcium efflux cytosolic calcium increase induced by sympa
(mediated by the Na+/Ca2+exchanger and to thetic neural activation. Until recently, my
a lesser extent by the sarcolemmal Ca2+ AT- cardial P-adrenergic receptors were thou
Pase) during diastole. As a result, there is no to be primarily of the pl-adrenergic recept
net increase or decrease in intracellular free subtype (47,48). However, it is now apparent
calcium concentration. Disturbances in this that ventricular myocytes also contain func
intracellular calcium homeostasis can pro- tional p2-adrenergic receptors, which may b
foundly alter a variety of cellular functions, come particularly important in cardiac dise
including the myocyte electrical stability. (47-51). For example, pl-adrenergic recep
Intracellular calcium rises dramatically density decreases as a consequence of he
with the induction of ischemia, exceeding peak failure, whereas the number of p,-adrener
systolic calcium levels within 5-10 min after receptors remains relatively constant (51).
ischemia onset (3437). Myocardial ischemia such, the failing heart becomes more depen
may provoke large increases in cytosolic cal- dent on p,-adrenergic receptors for inotrop
cium both directly (by alteration of the cellu- support. The activation of p,-adrenergic re
lar calcium homeostatic mechanisms) and in- ceptors (using the selective agonist, zinterol)
directly (by activation of the autonomic has, in fact, been shown to provoke sign&
nervous system). Myocardial ischemia pro- cantly greater increases in calcium transient
foundly affects the autonomic regulation of amplitude in myocytes isolated from animals
the heart (38). Coronary artery occlusion elic- susceptible to ventricular fibrillation than in
its reflex increases in cardiac sympathetic ac- myocytes obtained from animals resistant to
tivity, accompanied by reductions in parasym- malignant arrhythmias (50). This activation
pathetic tone (38-40). In fact, Billman and of the p2-adrenergic receptors produced large
co-workers (39, 40) demonstrated that acute increases in the calcium current with little or
myocardial ischemia provoked larger in- no increase in whole cell CAMP or phospho-
creases in sympathetic activity, coupled with lamban phosphorylation (52).Thus, p,-adren-
greater reductions in cardiac vagal tone, in an- ergic receptor activation may elicit a localized
imals subsequently shown to be susceptible to CAMP-independent increase confined to the
ventricular fibrillation. sarcolemma.
Alterations in autonomic regulation trigger a-Adrenergic receptor stimulation of the
a cascade of intracellular events that ulti- heart results in activation of a phospholipase
mately increase cytosolic calcium levels. Re- that hydrolyzes phosphatidyl inositol into t
lease of catecholamines from sympathetic second messengers, diacylglycerol and inositd
nerve terminals activates a-, PI-, and P2-ad- trisphosphate (53). Inositol trisphosphate fa-
renergic receptors on cardiac myocytes. Stim- cilitates calcium release from the sarcoplas
ulation of the PI-adrenergic receptor activates reticulum, whereas diacylglycerol activa
adenylyl cyclase, which in turn, increases cel- the important regulatory protein, protein
lular levels of cyclic adenosine monophos- nase C (PKC).Thus, a- and p-adrenergic stim-
phate (CAMP)(41).This cyclic nucleotide acti- ulations act synergistically to increase cyto
vates a CAMP-dependent protein kinase lic calcium during ischemia.
(PKA) that phosphorylates a variety of pro- Conversely, parasympathetic nerve activ
teins, including the voltage-dependent cal- tion, which decreases during ischemia, op
cium channel (33) and the calcium release poses the action of sympathetic nerve stimul
channel of the sarcoplasmic reticulum tion, reduces CAMPlevels, and increases leve
(42-45). It also phosphorylates the sarcoplas- of cyclic guanosine monophosphate (c
mic reticulum Ca2+-ATPase inhibitor, phos- (54). cGMP, in turn, decreases the open ti
pholamban, relieving its inhibition of calcium of calcium channels independently of chan
2 Pathophysiology of Myocardial Infarction

in CAMPlevels (55) and activates a sarcolem- pulse conduction, or a combination of both


mal calcium pump (56). Parasympathetic (59). Elevations in cellular calcium induced by
stimulation, therefore, lowers intracellular myocardial ischemia, as described above, can
calcium and halts the response to sympathetic produce abnormalities in these cardiac electri-
stimulation. Thus, the alterations in auto- cal properties and thereby trigger malignant
nomic function elicited by myocardial isch- arrhythmias.
emia would tend to favor the accumulation of It is well established that during coronary
cytosolic calcium. artery occlusion the resting potential of isch-
Myocardial ischemia also directly alters emic cardiac tissue becomes progressively less
t several of the important calcium regulatory negative than the resting potential of sur-
- pathways noted above. As ischemia pro- rounding non-ischemic tissue (59). The spread
- gresses, cellular ATP levels decline. As a con- of this injury current tends to depolarize the
e sequence of ATP depletion, several energy-de- surrounding tissue. Under normal conditions
r pendent functions are impaired. Sodium ventricular cells do not display a spontaneous
.t (Na+)/potassium(Kt) ATPase (sodium pump) rhythm; when such cells become partially de-
lc can no longer function properly, and cellular polarized, however, they may display an auto-
Ls Na* levels increase (31). Increased Na' re- matic rhythm (59-61). This ischemia-induced
1- verses the normal direction of the Na+/Ca2+ ectopic rhythm is critically dependent on cal-
ic exchanger so that sodium is extruded and cal- cium entry and can be abolished by lowering
e- cium is taken up by the cell (31). In addition, extracellular calcium (61) or exposing the car-
9 the Ca2+ ATPases (calcium pumps) of the sar- diac cells to a calcium channel antagonist (60).
fi- colemma and sarcoplasmic reticulum are im- Therefore, some forms of ventricular ectopic
nt paired so that less calcium is pumped out of automaticity seem to depend on a slow inward
11s the cell or into the sarcoplasmic reticulum calcium current.
in during diastole (relaxation is delayed). The As noted above, myocardial ischemia also
to net result of this impairment of cellular cal- results in elevations of cytosolic calcium,
on cium homeostatic mechanisms and enhanced which in turn, have been shown to provoke
'ge sympathetic outflow to the heart is a signifi- oscillations in membrane potential (62, 63).
or cant rise in cytosolic calcium levels (35-37, These oscillations or fluctuations in mem-
no- 57). These increases in cytosolic calcium brane voltage are known as afterdepolariza-
en- could, in turn, provoke alterations in ion tions because their generation critically de-
zed fluxes across the sarcolemma that ultimately pends on the presence of a preceding action
the culminate in malignant ventricular arrhyth- potential (59). There are two types of afterde-
mias (see below). Indeed, Billman et al. (58) polarizations: delayed afterdepolarizations
the indirectly demonstrated that cytosolic calcium (DADS)that occur after repolarization of the
lase may be elevated in animals particularly sus- preceding action potential and early afterde-
two i
ceptible to ventricular fibrillation. They found polarizations (EADs) that occur either at the
iitol t that calcium-dependent kinase activity was plateau (phase 2) or later during repolariza-
! fa- significantly greater in tissue obtained from tion (phase 3) of the cardiac action potential
lmic animals that developed life-threatening ar- (59, 64). When the amplitude of the afterpo-
ates rhythmias during myocardial ischemia than tential is large enough to reach threshold, re-
1 ki- in tissue obtained from animals resistant to petitively sustained action potentials are gen-
tim- arrhythmia formation. Specifically, calcium- erated. This form of ectopic automaticity is
;oso- calmodulin-dependent phosphorylation was known as triggered activity, because it does
two- to threefold higher in ventricular tissue not occur unless preceded by at least one ac-
tiva- obtained from animals that had ventricular tion potential. These abnormal afterdepolar-
, OP- fibrillation compared with animals that did izations, particularly EADs, can also enhance
nula- not develop arrhythmias during ischemia. the electrical heterogeneity between neigh-
evels boring regions of the myocardium (64). The
iMP) 2.2.2 Calcium and Arrhythmia Formation. resulting differences in repolarization can
time Disturbances in cardiac rhythm may result lead to the formation of new action potentials
from perturbations in impulse generation, im- through electrotonic (passive electrical) inter-
Myocardial Infarction Agents

actions between areas that have repolarized larizations have been recorded in isolated
(i.e., recovered excitability) and those regions cardiac cells or tissue in response to interven-
that have not (i.e., remain depolarized). This tions that favor calcium loading (hypoxia, co-
latter mechanism represents a form of re-en- caine, catecholamines, digitalis, calcium
trant excitation (see below). Thus, under ap- nel agonist BAY K 86441, and each can
propriate conditions, afterdepolarizations suppressed by calcium channel antagonist
could provide both the trigger (premature ec- and the intracellular calcium chelator
topic beats) and the substrate (electrical het- BAPTA-AM (51, 58, 62, 63, 71, 72).
erogeneity, non-uniform repolarization) for The initiation of ventricular fibrillatio
the initiation and propagation of the lethal
may depend on inward movement of calciu
arrhythmias.
(73). Ryanodine, a plant alkaloid that rende
The membrane currents responsible for
the sarcoplasmic reticulum leaky and unabl
these oscillations remain to be fully eluci-
dated. However, it is generally agreed that to retain normal amounts of calcium (74, 751,
DADs result from spontaneous calcium re- suppresses cytosolic calcium oscillations but
lease from the sarcoplasmic reticulum and a fails to prevent electrically induced ventricu-
calcium-activated inward depolarizing cur- lar fibrillation in isolated rabbit hearts (73).In
rent (65). At least three candidates have been contrast, verapamil and nifedipine, L-type cal-
proposed to carry this inward current: Naf/ cium channel blockers, terminate ventricular
Ca2+exchanger current, a Ca2+-activated C1- fibrillation (73). In related studies, Billman
current, and a Ca2+-activated non-selective (76-78) demonstrated that several organic
cation current (66-70). Accumulating evi- (verapamil, flunarizine, nifedipine, diltiazem,
dence favors the Na+/Ca2+exchanger current mibefradil) and inorganic (magnesium M$+])
as the most important current for DAD forma- calcium channel antagonists prevent malig-
tion. Schlotthauer and Bers (66),in an elegant nant ventricular arrhythmias induced by isch-
series of studies, showed that caffeine-induced emia. Conversely, the L-type calcium channel
DADs resulted almost entirely from the Na+/ agonist, BAY K 8644, induced ventricular fi-
Ca2' exchanger current, and also that only brillation in animals resistant to the develop
small (40 f l changes in cytosolic calcium ment of arrhythmias (76). Ryanodine failedto
were necessary to provoke the afterdepolar- prevent malignant arrhythmias despite large
izations. Thus, one would predict that drugs reductions in peak cytosolic calcium, indicated
that selectively inhibit this exchanger should by corresponding reductions in contractile
also prevent arrhythmias induced during isch- force development (79). These data strongly
emia (see below). In a similar manner, EADs suggest that calcium influx across the sarm
are known to result when repolarization has lemma, rather than calcium release from the
been prolonged as the result of either decreas- sarcoplasmic reticulum, may be critical for the
ing the outward potassium current, increasing induction of ventricular fibrillation.
the inward current (either sodium or calcium), Calcium also may contribute to changes in
or some combination of changes in these cur- impulse conduction. Conduction abnormali
rents (64). However, it has proven to be diffi- ties may result from simple conduction b l d
cult to ascertain which of the individual cur- or more complex forms of re-entry (59). In the
rents is responsible for these membrane normal heart, action potentials generated '
oscillations during the prolonged repolariza- the sinus node terminate after the sequent
tion. It is now clear that reactivation of both activation of the atria and the ventricles,
the sodium and L-type calcium channel con- cause the surrounding tissue has become
tribute significantly to the upstroke of the os- fractory or non-excitable after depolarizati
cillation, whereas the inward mode of the If, however, the impulse conduction is slo
Na'/Ca2+ exchanger current plays an impor- in one region of the heart and the surroun
tant role in the initial delay in repolarization tissue has repolarized, it may be possib
(64). As was noted for DADs, EADs are criti- re-excite the surrounding tissue before
cally dependent on elevations in cytosolic cal- next impulse is conducted from the sinus
cium (64). Both early and delayed afterdepo- gion. This phenomenon, known as re-entr
2 Pathophysiology of Myocardial Infarction

excitation, is responsible for the generation of channel antagonists can diminish calcium ac-
extrasystoles (re-entrant arrhythmias). Cal- cumulation and thereby improve conduction
cium channel antagonists exert their most ob- in ischemic hearts. Verapamil reduces,
vious effects on the conduction of action po- whereas BAY K 8644 exacerbates, the slowing
tentials through the atrioventricular (A-V) of ventricular conduction induced by global
node. Because A-V nodal tissue generates ischemia in the isolated rabbit heart (87).
slow-response (i.e., calcium-dependent) action In contrast to ordered re-entrant circuits,
potentials, calcium antagonists prolong A-V calcium may contribute significantly to ran-
conduction time and refractory period (80, dom or irregular re-entrant circuits. Random
81). These actions attenuate the ventricular re-entry is characterized by multiple irregular
response to rapid atrial arrhythmias (atrial pathways that change continuously, produc-
flutter or fibrillation) and terminate su- ing an unpredictable, chaotic conduction pat-
praventricular tachycardias in which the A-V tern. Ventricular fibrillation is the epitome of
node forms part of the re-entrant circuit (80). random re-entry. A major factor contributing
The effects of calcium on conduction abnor- to ventricular fibrillation, particularly during
malities in the ventricles are, however, equiv- myocardial ischemia, is a spatial dispersion or
ocal. Ordered or simple re-entrant arrhyth- nonuniformity of the refractory period (59);
mias in which the i m ~ u l s eis conducted in a
>
this allows impulse conduction to become
finite and well-circumscribed loop may occur fragmented during ensuing heartbeats and
in an ischemic heart (59). Re-entrant arrhyth- thus sets the stage for random re-entry. Dis-
mias require decremental conduction and uni- persion of refractory periods results, at least
directional block as preconditions for arrhyth- in part, from disturbances in action potential
mia formation (59). Conduction velocity in duration, which can be recorded as alterations
cardiac tissue depends on the rate of depolar- in the S-T segment (electrical alternans)
ization (dVldt,, or V), and action potential (59, 88-91). For example, Lee et al. (36)
amplitude, factors primarily mediated by the showed that alterations in the amplitude of
fast sodium channels (33). As noted above, calcium transients accompanied correspond-
myocardial ischemia results in depolarization ing changes in action potential duration. The
of the resting membrane potential, which may pattern of alternans was stable at a given re-
lead to inactivation of sodium channels (59, cording site but varied from site to site in a
82). Consequently, conduction velocity de- given preparation. They concluded that "the
creases, and unidirectional block may occur. alternans behavior of the calcium transients
In acute ischemia, many conduction distur- in a particular region is independent of the
bances that produce re-entrant arrhythmias behavior of other regions, which results in
are not mediated by slow-response action po- spatial heterogeneity of the calcium transients
tentials but rather by reduced sodium entry during ischemia" (36). Calcium channel an-
through fast channels (83). It is therefore not tagonists have been shown to reduce calcium
surprising that calcium channel antagonists transient and electrical alternans (92) and the
are not effective against ordered re-entrant spatial dispersion of refractory period from
arrhythmias (81, 84). However, conduction the endocardium to epicardium during isch-
velocity also depends on a low electrical resis- emia (93).
. . These data indicate that nonhomo-
tance between cells (59, 82). As ischemia geneity of refractory periods may result from a
progresses, intracellular Ca2+ and hydrogen calcium-mediated oscillation of action poten-
(Hf) increase (31,35-37,57,85). High concen- tial duration, and in turn, form a substrate for
trations of these ions reduce conductance irregular re-entry.
across the gap junctions, which form the low In summary, abnormalities in cellular
electrical resistance pathway that facilitates Ca2+ may contribute significantly to the de-
cell-to-cell coupling (86). Thus, during later velopment of malignant ventricular arrhyth-
stages of ischemia or in chronically ischemic mias by inducing various forms of ectopic au-
hearts, conduction disturbances may result tomaticity, by changing conduction, or by a
from the uncoupling of cardiac cells due to the combination of both automaticity and conduc-
cellular accumulation of calcium. Calcium tion disturbances. If, for example, an extrasys-
Myocardial Infarction Agents

tole occurs in a region of nonuniform refrac- coupled with anion (lactate or inorganic phos-
tory period, irregular re-entrant pathways phate) conductance to balance transmem-
and ventricular fibrillation may result. brane charge (97). The latter hypothesis
stipulates that potassium efflux results sec-
2.2.3 Extracellular Potassium Accumulation ondarily to the movement of intracellularly
During Myocardial Ischemia. In addition to generated anions during ischemia to balance
changes in cytosolic calcium as described charge movement as these negatively charged
above, myocardial ischemia will elicit pro- ions diffuse across the sarcolemma. Thus, po-
found changes in extracellular potassium. The tassium efflux would result from a passive re-
resulting depolarization of the surrounding distribution of potassium ions in response to
tissue, decreases in action potential duration, the net inward current resulting from anion
and nonuniformities of repolarization (as well efflux, rather than from an active ion-anion
as refractory period) could all contribute to linked process. Weiss et al. (107) have tested
the induction of the life-threatening arrhyth- this hypothesis. In particular, the contribu-
mias associated with myocardial ischemia. It tion of inorganic phosphate and lactate ion to
is now generally accepted that disruptions in potassium efflux during ischemia and hypoxia
coronary blood flow elicit both rapid increases was investigated. They found that under a va-
in extracellular potassium and reductions in riety of conditions, a major component of cel-
action potential duration. Harris and co-work- lular potassium loss was not related to the ef-
ers (94, 95) were the first to show that extra- flux of these anions. They concluded that this
cellular potassium rises dramatically after "non-anion-coupled" potassium efflux during
coronary artery ligation, correlating with the metabolic inhibition was most likely to result
onset of ventricular arrhythmias. They fur- from an increase in membrane potassium
ther demonstrated that intracoronary injec- conductance.
tions of KC1 provoked electrocardiographic A growing body of evidence suggests that
changes and triggered ventricular arrhyth- ischemically induced potassium accumulation
mias similar to those induced by myocardial and the corresponding reductions in action po-
ischemia (94,951. They proposed that changes tential duration result primarily from the
in extracellular potassium represented a ma- opening of ATP-sensitive potassium channels.
jor factor in the development of malignant ar- Using the patch clamp technique, Trube and
rhythmias during ischemia. In recent years, a Hescheler (108) were the first to record single
number of studies using ion selective elec- ATP-sensitive potassium channel activity.
trodes to measure potassium activity directly Noma (104) and Hescheler et al. (109) further
have largely confirmed these earlier observa- demonstrated that reductions in cellular ATP
tions (96-98). Extracellular potassium has induced by cyanide exposure evoked an out-
been found to increase within the first 15 s ward potassium current. They, therefore, pro-
and reach a plateau within 5-10 min after the posed that the activation of an ATP-sensitive
interruption of coronary perfusion (96,97,99, potassium channel might be responsible for
100). Furthermore, regional differences or in- the reductions in action potential duration in-
homogeneities of potassium accumulation duced by hypoxia. Several studies have since
were recorded, accompanied by corresponding further implicated the activation of this cur-
differences in ventricular electrical activity rent in the changes in cardiac action potential
(98-100). The increase in extracellular potas- and extracellular potassium accumulation
sium results primarily from increases in po- during myocardial ischemia (110-120). The
tassium efflux rather than from decreased po- ATP-sensitive potassium channel inhibitor,
tassium influx due to inhibition of the Natl glibenclarnide, for example, has been shown
K+-ATPase (101-103). Several mechanisms either to attenuate or abolish reductions in
have been proposed to explain the enhanced action potential duration in hypoxic myocytes
potassium efflux, including an increased po- (115, 1171, isolated cardiac tissue (110, 112,
tassium outward conductance due to the di- 113, 116, 120), and regionally or globally isch-
rect activation of one or more potassium chan- emic hearts (118, 121, 122). This sulphonyl-
nels (104-106) or a passive potassium efflux urea drug has also been shown to reduce ex-
Pathophysiology of Myocardial Infarction

tracellular potassium accumulation induced very similar to those induced by myocardialisch-


ischemia (110-112,114). Conversely, ATP- emia. Conversely, glibenclamide attenuated the
sensitive potassium channel agonists (pinaci- S-T segment elevations induced by the occlusion
4,cromakalim) exacerbated ischemically in- of the left anterior descending coronary artery
juced reductions in action potential duration, (134). Similar results were also obtained in con-
p well as promoted extracellular potassium scious dogs. Billman et al. (135) demonstrated
,accumulation (110, 115-120, 122-125). How- that either glibenclamide or the cardioselective
ever, the ATP-sensitive potassium channel is ATP-sensitive potassium channel antagonist
activated only at low ATP concentrations with HMR 1098 attenuated ischemically induced S-T
half-maximum suppression of channel open- segment changes. These drugs also prevented
$ingat20-100 pilf (104,126,127), yet intracel- ischemically induced increases in the descend-
Mar concentrations are normally much ing portion of the T wave (an index of the trans-
higher (5-10 mM). Furthermore, cytosolic mural dispersion of repolarization) (136). Fi-
ATP levels remain in the millimolar range for nally, myocardial ischemia failed to alter the
=thefirst 10 min of hypoxia, well after potas- ECG of mice in which the Kir 6.2 gene (the gene
mum accumulation begins (103,128). The role responsible for the pore forming subunit of the
that this channel plays in the response to myo- cardiac ATP-sensitive potassium channel) (137)
cardial ischemia has therefore been ques- had been removed (138).Furthermore, the large
tioned. Recently, a number of investigators changes in the ECG that were induced by liga-
have shown that, because of the high density tion of the left coronary artery in the wild-type
control mice could be suppressed by prior treat-
a small increase in the open-state proba- ment with the cardioselective ATP-sensitive po-
(<I% of maximum) was sufficient to tassium channel antagonist HMR 1098 (138).It
tential duration during isch- therefore seems likely that the activation of the
a (129-131). For example, Faivre and ATP-sensitive potassium channel contributes
dlay (130) found during patch clamp stud- significantly to alterations in cardiac electrical
ocytes that the opening of stability induced by myocardial ischemia, lead-
30 channels (less than 1% of the popula- ing to the formation of malignant arrhythmias.
rovoked a 50% reduction in action poten-
ation. They concluded that "physiologi- 2.2.4 Extracellular Potassium and Cardiac
levant activity of the KATp channel in Arrhythmias. As noted above, cardiac arrhyth-
membrane is confined to a very small mias can result from either abnormalities in
ntage of the possible cell KATpcurrent, and impulse generation or impulse conduction.
intracellular ATP would not have to fall The extracellular accumulation of potassium
far before the opening of KATpchannels induced by myocardial ischemia can provoke
d influence cardiac excitability." Deutsch et these perturbations in cardiac electrical activ-
the potassium current in- ity. The accumulation of extracellular potas-
by hypoxia in an intact rabbit papillary sium promotes the depolarization of the tissue
that during hypoxia, a sig- surrounding the ischemic regions, as noted
action potential duration above. The flow of this injury current (electro-
e) occurred when tissue tonic current flow between ischemic and nor-
approximately 25%.They con- mal cells) has been implicated as a potential
s in cellular ATP cause for the initiation of premature ventric-
required to induce major changes in car- ular beats. Under normal conditions, ventric-
electrical properties. ular cells do not display a spontaneous
rhythm. However, when the cells are partially
depolarized an automatic rhythm can be pro-
duced (59). Coronel et al. (99) demonstrated
an increased excitability of normal tissue near
the border of the ischemia, a region of the
-sensitive potassium channel opener, pin- heart in which extracellular potassium con-
dil, elicited elevations in the S-T segment centrations were also increased.
Myocardial Infarction Agents

Changes in action potential duration in- 2.3 Ventricular Remodeling


duced by alterations in potassium efflux can In addition to sudden death from malignant
also provoke abnormalities of impulse conduc- arrhythmias, an important negative conse-
tion. As noted above, increased potassium ef- quence of myocardial infarction is the develop-
flux from ischemic tissue triggers a reduction ment of chronic congestive heart failure. A
in action potential duration. A major factor clinical history of myocardial infarction in-
contributing to ventricular fibrillation, partic- creases the age-adjusted risk of developing
ularly during myocardial ischemia, is a disper- heart failure roughly 10-fold, and the 5-year
sion or inhomogeneity of the refractory period survival rate for patients diagnosed as suffer-
(59). This allows for the fragmentation of im- ing from congestive heart failure is only about
pulse conduction during ensuing beats. Re-
30% (148). When viable myocardium is lost to
cent evidence suggests that a major factor con- an infarct, the added contractile demands on
tributing to the dispersion of refractory period
the remaining myocardial tissue elicit a series
is regional differences in action potential du-
of responses that produce hypertrophy, inter-
ration (59).As previously noted, the activation
stitial fibrosis, and remodeling of the residual
of the ATP-sensitive potassium channel pro-
muscle mass. These processes can at first nor-
duced large reductions in action potential du- malize the depressed cardiac output, but with
ration (110-113, 115-120, 122), which are a sufficient long-standing strain on the heart,
inhibited by glibenclamide (115-120, 122) there is decompensation, dilatation of the ven-
but exacerbated by ATP-sensitive potassium tricles, and eventual heart failure (148).
channel agonists (110,124,139-141).A differ- Ventricular remodeling begins within the
ential ATP sensitivity of the ATP-dependent early hours of an acute myocardial infarction
potassium channel has also been reported be- (149). First there is infarct expansion because
tween endocardial and epicardial cells during of changes in the extracellular matrix and
ischemia, such that smaller reductions in ATP
myocyte apoptosis. Dilatation of the nonin.
were necessary to activate potassium channels farcted tissue occurs later, but much of this
located in epicardial tissue (142). As a result, occurs during the first few days post-infarct.
an inhomogeneity in extracellular potassium At 1 week post-infarction, left ventricular end
and refractory period, as well as a gradient in systolic volume may be 80% above normal and
action potential duration, was recorded be- is dependent on infarct size and patency of the
tween the epicardial and endocardial tissue infarct-related artery (149). The increase in
(142-144). Nonuniformities in refractory pe- myocardial wall stress as a result of ventricu.
riod could set the stage for the formation of lar dilatation activates both the plasma and
irregular reentrant pathways and ventricular intracardiac renin-angiotensin system (148).
arrhythmias. In a similar manner, the ATP- Angiotensin has been directly implicated
sensitive potassium channel antagonist glib- ventricular remodeling and may also incre
enclamide was shown to attenuate ischemi- norepinephrine release, further increas
cally induced reductions in the refractory the metabolic demands on injured but still
period (145,146). In contrast, activation of the able myocardium (149). An important goal
ATP-sensitive potassium current with pinaci- the management of myocardial infarction is
dil elicited a marked dispersion of repolariza- minimize this maladaptive response.
tion and refractory period between the epi-
cardium and endocardium, leading to the
development of extrasystoles (147). These ef- 3 TREATMENT FOR MYOCARDIAL
fects could be abolished by glibenclamide INFARCTION
(147). Thus, the activation of the ATP-sensi-
tive potassium channel could contribute sig-
3.1 Pain Relief
nificantly to the induction of malignant ar-
rhythmias by changing impulse generation Relief of pain is important for the patient wi
(depolarization induced changes in automatic- an acute myocardial infarction. Pain and
ity), conduction (refractory period dispersion), iety heighten adrenergic responses, and t
or a combination of both. in turn, are arrhythmogenic and place a
Treatment for Myocardial Infarction

ds on the heart. Mor- the first few hours (2,153). In the GISSI trial,
e sulfate is the drug of choice in the thrombolytic therapy (streptokinase; see be-
ted States (1).It has the added benefit of low) begun within the first hour decreased in-
etic outflow to the hospital mortality by 51%, therapy begun
e, could indirectly within 3 h reduced mortality by 26%,and that
mand and the result- begun after 3-6 h reduced mortality by 20%
ionic changes associated with myocardial (2,153). In the LATE trial, tissue plasminogen
emia. Morphine is used judiciously, how- activator (t-PA) administered 6-12 h after the
n of pain is often onset of symptoms reduced 35-day all-cause
ssful thrombolysis, mortality by 27% (2). In the EMERAS study,
whereas persistent pain signifies the need for streptokinase administered after 6-12 h gave
tional intervention (1).Intravenous nitro- an 11% nonsignificant reduction of in-hospital
erin is also used for the treatment of per- mortality (153). It should be noted that com-
ent chest pain in some patients with acute parisons among the various large clinical tri-
myocardial infarction. Nitroglycerin is a pro- als are complicated by differences in adjunc-
drug that provides nitric oxide (NO), an acti- tive therapies, treatment endpoints, and
vator of smooth muscle guanylyl cyclase, the patient selection criteria.
enzyme that produces cGMP (150).Activation
of a cGMP-dependent protein kinase initiates 3.2.1 Streptokinase. Streptokinase and
a cascade of reactions that ultimately result in anisoylated plasminogen streptokinase-
smooth muscle relaxation. In particular, veno- activated complex (APSAC) are effective
dilation reduces preload and thereby lessens thrombolytic agents (154). Streptokinase is
metabolic demand. NO may also inhibit plate- a 47,000-dalton protein produced by hemo-
let aggregation, but the therapeutic signifi- lytic streptococci. It forms a 1:l noncovalent
a c e of this effect in acute myocardial infarc- complex with circulating plasminogen, pro-
tion is uncertain (150). ducing a conformational change that facili-
tates the cleavage of the inactive 790 amino
acid plasminogen at arginine 560 to form
he dissolving of an occlusive free plasmin. Plasmin is a relatively nonspe-
ood clot, has become standard treatment for cific protease that digests fibrin clots and
myocardial infarctions associated with S-T other plasma proteins, including several co-
gment elevations. Restoration of blood flow agulation factors. APSAC is a streptokinase-
evaluated angiographically in terms of plasminogen complex prepared in vitro from
I hieving thrombolysis in myocardial infarc- purified acylated human lys-plasminogen
ades (151). TIM1 grade 0 and purified streptokinase. The acyl group
L
de flow; grade 1 indicates must be hydrolyzed in vivo before activa-
! contrast penetration and incomplete tion; this allows time for the plasminogen to
r
lling; grade 2 indicates a patent infarct bind to fibrin before activation by streptoki-
acifications of the entire nase.
F ayed contrast filling or
)
indicates normal flow. 3.2.2 Plasminogen Activators. These throm-
in morbidity and mor- bolytic drugs have exploited or been patterned
are associated with restoration of TIM1 after the plasmin-based fibrinolytic system
e 3. In the GUSTO I study, the mortality that normally dissolves small intravascular
IMI grade 3 flow 90 min clots in vivo (154). This fibrinolytic system is
thrombolysis was less highly regulated such that unwanted fibrin
an one-half that in patients with TIM1 thrombi are removed while fibrin in wounds is
ades 0 or 1 flow (152). unaffected. Fibrinolysis is normally initiated
1 Thrombolysis has been found to benefit when t-PA is released from endothelial cells in
- ose patients treated within 12 h of the onset response to such signals as the stasis produced
3 chest pain, but improvements in survival by a vascular occlusion. This endothelial cell-
- are greatest when treatment is begun within derived t-PA is rapidly cleared from blood or
Myocardial Infarction Agents

inhibited by a circulating inhibitor, plasmino- sive bleeding and hemorrhagic stroke can en-
gen activator inhibitor-1, and thus exerts little sue. Yet in large clinical trials, the benefits of
effect on circulating plasminogen. By con- timely thrombolysis have vastly outweighed
trast, the t-PA that binds to fibrin converts the drawbacks (2, 153).
fibrin-bound plasminogen to plasmin. Plas-
minogen and plasmin both bind to fibrin at 3.2.3 Anticoagulants. The benefits of
sites near their lysine-rich amino termini, thrombolytic therapy are improved signifi-
sites that are also required for binding of plas- cantly with the concomitant use of the anti-
min to a,-antiplasmin, a 452 amino acid glyco- platelet agent, aspirin, which is an irrevers-
protein that instantaneously inactivates plas- ible inactivator of platelet cyclooxygenase,
min. Therefore, fibrin-bound plasmin is highly and the classical anticoagulant, heparin,
effective because it is shielded from inhibition, which is a thrombin inhibitor (156). Anti-
whereas plasmin that escapes into the circula- platelet agents and thrombin inhibitors help
tion is rapidly inhibited (154). maintain vessel patency by preventing reoc-
t-PA (alteplase) is a first generation tissue clusion. These anticoagulants have proved
plasminogen activator produced commercially beneficial even in the absence of thromboly-
by recombinant DNA technology. It is synthe- sis, presumably because of the inhibition of
sized using human cDNA for natural human new thrombotic events (153, 156). It is cur-
tissue type plasminogen activator and is ex- rently recommended that all aspirin-toler-
pressed in and harvested from cultured Chi- ant individuals suffering from an apparent
nese hamster ovary cells. t-PA is a 527 amino acute mvocardial
" infarction be immediatelv
acid serine protease with minimal activity in given chewable aspirin, and daily low dose
the absence of fibrin, but when bound to the aspirin is also recommended for most post-
fibrin in a thrombus it converts occluded plas- myocardial infarction patients (157). The
minogen to plasmin, initiating local thrombol- thienopyridines, clopidogrel or ticlopidine,
ysis (154). Accelerated t-PA administration which inhibit platelet function by inhibiting
achieves TIM1 grade 3 more rapidly than the binding of fibrinogen to activated plate-
streptokinase and has been associated with lets, should be used in patients who are al-
more favorable clinical outcomes (155). lergic to aspirin or who suffer from gastro-
Alteplase must be continuously infused be- intestinal bleeding (156). In conjunction
cause of its short half-life. Variants of t-PA with thrombolysis for an acute myocardial
with deletion of specific domains or specific infarction, adequate heparinization is par-
amino acid substitutions have shown reduced ticularly important in patients receiving
plasma clearance and are suitable for bolus t-PA because this drug has far less of a sys-
injections (155). Reteplase (rPA) is a single temic anticoagulant effect than streptoki-
peptide chain molecule consisting of 355 nase or APSAC (154, 156, 158). t-PA plus
amino acids, starting with serinel and ending heparin has been found to be slightly but
with proline527 of the original t-PA sequence. significantly more efficacious a t improving
It also lacks amino acids valine4 through glu- survival of patients with acute myocardial
tamatel75. Tenecteplase (TNK-tPA)contains infarction, despite a slightly increased risk
a threonine to asparagine substitution at po- of stroke (153, 156).
sition 103 of t-PA, an asparagine to glycine
substitution at position 117, and the replace- 3.2.4 Clycoprotein Ilblllla Receptor Block.
ment of lysine296, histidine297, arginine298, ers. When platelets are activated, the glycop-
and arginine299 by alanines. Reteplase and rotein IIbDIIa (GP IIbDIIa) receptor under.
tenecteplase have reduced plasma clearance goes a change in configuration that increases
and can be administered as a single bolus its affmity for binding to fibrinogen and other
(TNK-tPA) or as a double bolus 30 min apart ligands (159). Fibrinogen binding to receptors
(rPA), offering the possibility for pre-hospital on different platelets results in platelet aggre
initiation of thrombolysis (155). gation. GP IWIIIa receptor antagonists pre
When exogenous thrombolytic agents are vent fibrinogen binding and therefore prevent
applied at their recommended dosages, exces- platelet aggregation (159). The available GP
Treatment for Myocardial Infarction

1Ia antagonists have different pharmaco- 3.3 Treatment of Arrhythmias Induced by


odynamic properties. Ab- Myocardial Ischemia
ab is a Fab fragment of a humanized mu- Interventions that alter ion flux across the
antibody that has a short plasma half-life sarcolemma should also alter the potential for
strong aMinity for the receptor. Some re- arrhythmias induced by myocardial ischemia.
or occupancy can persist for weeks, and In particular, one would predict that either
elet aggregation gradually returns to nor- ATP-sensitive potassium channel antagonists
24-48 h after the drug is discontinued. or calcium channel antagonists would protect
Eptifibatide is a cyclic heptapeptide that against the formation of malignant arrhyth-
cine-aspartate sequence. mias under these conditions. The following
tide mimetic of the argi- section will briefly review the experimental
sequence of fibrinogen. and clinical experience with these ion channel
se two synthetic antagonists bind less antagonists. We shall first begin with a brief
tly than abciximab, with receptor occu- description of the major types of anti-arrhyth-
cy in equilibrium with plasma levels. They mic drugs.
e a half-life of 2-3 h and are more specific 3.3.1 Classification of Anti-Arrhythmic
the GP IIb/IIIa receptor than abciximab, Drugs. Anti-arrhythmic drugs are widely clas-
ch can also bind to the vitronectin receptor sified by their effects on cardiac electrical
endothelial cells and the MAC-1 receptor properties using the system devised by
Vaughn Williams (161, 162). In this schema,
GP IIb/IIIa receptor antagonists have drugs are assigned to one of four classes. The
ficial in patients with class I drugs block sodium channels and are
table angina or non-S-T segment elevation subclassified based on their effects on action
These patients are not potential conduction. The class IA drugs pro-
but may undergo duce a moderate prolongation of conduction
s. The GP IIb/IIIa and repolarization. Representative examples
duce the incidence of of class IA drugs include quinidine, procain-
rcutaneous interven- arnide, and disopyramide. The class IB drugs,
which include lidocaine and tocainide, exert
he GUSTO V trial compared the effects of little or no effect on conduction and repolar-
(rPA) with half-dose ization. In contrast, the class IC drugs (fle-
abciximab (160). Sev- cainide, encainide) exhibit a marked prolonga-
ggested that the com- tion of conduction and repolarization. The
on of a low dose plasminogen activator class IC drugs have been shown to increase
a IIb/IIIa receptor antagonist improved cardiac mortality in patients recovering from
durability, and completeness of myo- myocardial infarction most likely as a result of
nary data from the an increased incidence of sudden death (a pro-
that at 30 days there arrhythmic effect fibrillation), and as such,
fewer nonfatal reinfarctions in the com- should no longer be used to suppress arrhyth-
up and there was less need for ur- mias in this patient population (163). Class I1
revascularization (rescue angioplasty). anti-arrhythmic drugs block p-adrenergic re-
e reteplase group, 5.9%of patients had ceptors and are the only agents widely ac-
at 30 days compared with 5.6% in the cepted to reduce the incidence of sudden car-
dose reteplase plus abciximab group (p = diac death in post-infarction patients (see
lications were more below). Class III drugs prolong repolarization
the combination therapy group, and effective refractory period, most likely
there was no increase in intracranial hem- through modulation of potassium channels.
). Data for 1-year mortality are Examples include amiodarone, dofetilide,
e will establish ibutilide, bretylium, and d-sotalol. Pro-ar-
mplete reperfusion rhythmic properties have also been reported
for some (d-sotalol, dofedilide), but not all,
Myocardial Infarction Agents

(amiodarone) class I11 anti-arrhythmic drugs with an increased expression of functional T-


(164, 165). These drugs have been shown to type calcium channels. Vascular smooth mus-
prolong QT interval, promote life-threatening cle contains both L- and T-type calcium chan-
tachyarrhythmia, torsades de pointes, and in- nels that each contribute to the regulation of
crease cardiac mortality in some patient pop- smooth muscle calcium, and thereby, vascular
ulations. Amiodarone is, in fact, the only class tone (172, 173). Given the widespread distri-
I11 agent that has been shown to reduce car- bution of calcium channels within the car&
diac mortality in post-infarction patients (166, vascular system, it is not surprising that cal-
167).Because this drug blocks a number of ion cium channel antagonists have received
channels (e.g., L-type calcium channels and a considerable therapeutic interest. Indeed,
number of potassium channels including the since their discovery in the 1960s (177-179),
ATP-sensitive potassium channel) (168), it calcium antagonists have become increasingly
has been difficult to ascertain the mechanism more important in the treatment of various
responsible for this cardioprotection (169). Fi- cardiovascular diseases, most notably hyper
nally, class IV drugs inhibit calcium channels tension and angina pectoris (180). The use
(see below). calcium channel antagonists has
As described above, myocardial ischemia restricted to the treatment of suprave
elicits profound changes in the regulation of lar arrhythmias. In contrast, the
intracellular calcium and potassium. In the potential in the management of life-th
following section, we shall focus on agents that ing ventricular arrhythmias, particularly
may be particularly useful in the modulation ing myocardial ischemia, has received less
of calcium and the ATP-sensitive potassium tention. The following sections of this chap
currents during myocardial ischemia and will summarize experimental and
could thereby protect against malignant ar- studies that illustrate the anti-arrhyth
rhythmias. The section will close with a brief tential of individual calcium channel ant
discussion of the well-established effects of nists.
P-adrenergic receptor antagonists on cardiac
mortality after myocardial infarction. I t 3.3.3 Verapamil. Veraparnil, a b
should be emphasized that the list of agents acetonitrite, is structurally similar to pap
described in the following sections represents ine and was first synthesized in 1962 (
only a few of the many agents used to treat Verapamil blocks calcium entry equally
cardiac arrhythmias. For more detailed pre- both cardiac and smooth muscle through
sentation of specific agents, particularly class I tions on both L- and T-type calcium chan
and class I11 agents, please refer to refs. 170 (30, 182). Verapamil, like most calciu
and 171. nel antagonists, is not completely selecti
calcium channels and has been shown to
3.3.2 Calcium Channel Antagonists. Cal- avariety of potassium channels (301, i
cium antagonists represent a large, structur- the ATP-sensitive potassium channel,
ally diverse group of chemicals that share the sient outward current, and the rapi
ability to inhibit calcium entry into muscle ing component of the delayed-rectifier cu
cells through actions on the calcium-selective (I&). Of particular note, verapamil has
ion channels. Cardiac tissue contains two shown to block human ether-a-go-go-re
types of calcium channels: channels with a gene (HERG)-encoded channels (
long activation (L-type) and channels that ac- fected into various cell lines at clinically r
tivate transiently (T-type). The L-type cal- vant concentrations (183). Howev
cium channels are located in both atrial and or not the verapamil-mediated inhibitio
ventricular tissue, whereas the T-type chan- these potassium currents contributes to
nels are more predominate in atrial (i.e., pace- clinical effects of this drug has not yet b
maker) cells (172,173). However, pathological determined. The blockade of calci
conditions including ventricular hypertrophy smooth muscle elicits vasodilation with c
(1741, dilated cardiomyopathy (1751, or myo- sponding reductions in arterial pressure
cardial infarction (176) have been associated increases in coronary blood flow. In car
3Treatment for Myocardial Infarction

muscle, verapamil tends to depress both con- major cardiac events (cardiac death or second
tractile force and impulse conduction. The sec- myocardial infarction). Sudden death was re-
ond action is particularly obvious at the A-V duced by 20-26% in patients treated with verap-
arnil-results comparable with those obtained
In 1968, Kaumann and Aramendia (184) with P-adrenergic antagonists (199, 200). This
were the first to demonstrate that verapamil protection, however, was noted only in patients
can protect against ventricular fibrillation in- without evidence of heart failure. Verapamil had
duced by ligation of a coronary artery. These no beneficial effects on major cardiac events in
findings have been confirmed by many other patients with heart failure, and in fact, may have
animal studies (76, 184-187). In intact ani- increased cardiac mortality (198).The reduction
mals, verapamil prevents ventricular arrhyth- in sudden death may reflect inhibition or reduc-
mias induced by myocardial ischemia (76,187) tion of calcium loading in ventricular cells, but
and attenuates the reduction in ventricular no direct data yet support this suggestion.
fibrillation threshold that accompanies coro-
nary artery occlusion (185, 188, 189). Billman 3.3.4 Diltiazem. Diltiazem, a benzodiaz-
further demonstrated that verapamil com- epine derivative, is structurally quite distinct
pletely suppresses ventricular fibrillation in- from verapamil and was first identified as a
duced by either cocaine (190) or the combina- calcium antagonist in 1971 (201). Diltiazem
tion of exercise and acute myocardial ischemia has pronounced effects on both vascular
(76). In a similar manner, verapamil, in com- smooth and cardiac muscle. This drug elicits
bination with the angiotensin-converting relaxation of the vascular smooth muscle, in-
enzyme inhibitor trandolapril, prevented ven- creasing coronary blood flow and reducing ar-
tricular tachyarrhythmias induced by myocar- terial pressure. Diltiazem depresses A-V con-
dial ischemia followed by reperfusion in anes- duction, but has a lesser negative inotropic
thetized pigs (191). In contrast, verapamil effect than verapamil. Diltiazem can also in-
failed to prevent ordered re-entrant arrhyth- hibit the delayed rectifier potassium current
mias induced by programmed electrical stim- but not at clinically useful concentrations (30,
ulation (80, 84, 191, 192). 183). Less consistent antiarrhythmic actions
Clinical experience with verapamil to date have been noted for diltiazem than for vera-
has been somewhat inconsistent. For exam- pamil, perhaps because of its less potent car-
ple, verapamil is generally ineffective in the diac actions. Diltiazem reduced regional dif-
treatment of either stable re-entrant arrhyth- ferences in impulse conduction in ischemic
mias or arrhythmias induced by programmed tissue (60, 202), prevented slow-response ac-
electrical stimulation (80,84,191, 1921, which tion potentials (33), and abolished calcium
also probably result from re-entry. In con- transient or electrical alternans (36,921. In a
trast, some reports indicate that arrhythmias similar manner, diltiazem decreased the am-
associated with acute myocardial ischemia or plitude of T wave alternans and the incidence
exercise-induced ventricular tachycardia re- of ventricular tachyarrhythmias in chloralose-
spond favorably to verapamil(191, 193). Vera- anesthetized dogs (203). Diltiazem also pro-
pamil also significantly decreased the frequency tected against ventricular fibrillation in anes-
and severity of ventricular arrhythmias in pa- thetized animals (92, 204). In unanesthetized
tients with left ventricular hypertrophy (194). canine preparations, diltiazem delayed the
Verapamd has been shown to be effective in the time to onset of malignant arrhythmias, but it
treatment of idiopathic left ventricular tachy- failed to prevent ventricular fibrillation in-
wdia (a relatively rare but distinct entity in duced by irreversible coronary artery occlu-
young, mostly male, patients) and to a lesser ex- sion (205). Billman further demonstrated that
tent in the treatment of right ventricular out- diltiazem prevented ventricular fibrillation in-
flow ventricular tachycardia (which is more duced by cocaine (190) or acute myocardial
common in female subjects) (195-197). Finally, ischemia (76). In contrast, diltiazem failed to
in a large Danish multicenter study (198) of pa- prevent ischemic changes in the ventricular
tients recovering from myocardial infarction, fibrillation threshold (206),as well as arrhyth-
verapamil significantly reduced the frequency of mias associated with reperfusion (203, 207).
Similar mixed results have been reported very high concentrations (76). Nifedipine also
in clinical studies of the antiarrhythmic poten- failed to reduce mortality in patients recover-
tial of diltiazem. Diltiazem was found to re- ing from myocardial infarction (216). A reflex
duce the reinfarction rate by approximately tachycardia induced by nifedipine's reduction
51% in patients with myocardial infarction, of arterial blood pressure may, in fact, worsen
but in contrast to verapamil, did not affect the condition of such patients by placing a
overall mortality rates (208). Furthermore, higher metabolic demand on the damaged
diltiazem failed to reduce stable ventricular heart. Indeed, much of the recent controversy
arrhythmias during a 12-h recording period (217) surrounding its use may be related to
(209). However, in a study involving over 2400 this reflex action. The increased metabolic de-
patients with myocardial infarction, diltiazem mand placed on the heart may counteract the
produced a significant decrease in mortality in beneficial actions of reduced arterial blood
a subgroup of patients without radiographic pressure (211).
evidence of pulmonary congestion (i.e., heart
failure) (210).It is unclear whether the reduc- 3.3.6 Flunarizine. Flunarizine is a difluor-
tion in mortality arises from blockade of car- onated piperazine derivative that, under phys-
diac calcium channels and the resultant pre- iologic conditions, interacts weakly with both
vention of cellular calcium overload. The sodium and calcium (L- and T-type) channels
subgroup analysis further demonstrated that (218,219). However, during hypoxia, flunariz-
diltiazem provoked a dramatic increase in ine inhibits increases in cytosolic calcium and
mortality in patients with myocardial infarc- prevents tissue damage, particularly in neural
tion complicated by pulmonary congestion tissue (218). It seems reasonable that flunariz-
(210). Calcium antagonists, therefore, may ine may act as a calcium overload antagonist
have deleterious effects in patients with com- and thereby prevent malignant arrhythmias
promised cardiac function (211). during myocardial ischemia. Flunarizine pro-
tected against isoproterenol-induced cardiac
3.3.5 Nifedipine. Nifedipine, a dihydro- lesions and ischemic injury of the rat heart
pyridine, was first synthesized in 1971 (212). (218). It reduced ventricular fibrillation by
In contrast to either diltiazem or verapamil, at 100% and ectopic beats during occlusion and
therapeutic concentrations, nifedipine acts reperfusion of the left anterior descending cor-
primarily on vascular smooth muscle. Nifedi- onary artery (218); however, a much higher
pine acts as a more potent vasodilator than dose was required for protection equivalent to
either diltiazem or verapamil; cardiac actions that noted with verapamil (218). Flunarizine
are noted only at much higher concentrations. prevented ventricular tachycardia due to de-
Therefore, it is not surprising that nifedipine layed afterdepolarizations induced by ouabain
exhibits few antiarrhythmic properties in in- toxicity but not arrhythmias due to activation
tact animals or patients. Nifedipine, with a of re-entrant circuits (220, 221). Based on
few exceptions (see below), is generally inef- these findings, Vos et al. (220) proposed that
fective in the treatment of experimentally in- flunarizine may be used to differentiate be-
ducedischemic arrhythmias (76,213,214).Ni- tween arrhythmias arising from calcium over-
fedipine failed to alter the reduction in load (triggered activity) and arrhythmias due
ventricular fibrillation threshold induced by to re-entry. Flunarizine has also been shown
coronary artery occlusion (214) and to prevent to prevent torsades de pointes (a severe poly-
reperfusion arrhythmias (207). However, it morphic ventricular tachycardia associated
has been reported that nifedipine prevents with early afterdepolarizations) induced by
ventricular fibrillation in the ischemic rat the selective IKr blocker almokalant in dogs
heart. This protection is attributed to im- with chronic A-V block (222) or anesthetized
proved coronary perfusion (i.e., less myocar- rabbits (almokalant combined with p-adren-
dial ischemia) rather than to direct cardiac ac- ergic receptor stimulation with methoxamine)
tions of the drug (215). Nifedipine protected (223). In the latter study, neither the intracel-
against malignant arrhythmias induced by ex- lular calcium chelator BAPTA-AM nor the
ercise plus myocardial ischemia, but only at sarcoplasmic reticulum calcium release dis-
Treatment for Myocardial Infarction

revent the arrhyth- with frequent ventricular arrhythmias. In


. The authors, therefore, concluded that contrast, intravenous magnesium signifi-
through the T-type cantly reduced the incidence of arrhythmias
crucial role in the during acute myocardial infarction (233-235).
ntes (223). Finally, The most consistent results from magnesium
that flunarizine therapy have been reported for the treatment
icular fibrillation of torsades de pointes. Tzivonis and co-work-
or exercise plus ers (236,237) reported that intravenous mag-
ocardial ischemia; however, large reduc- nesium sulfate completely abolished torsades
were noted. The de pointes within 1-5 min after either the first
or second bolus injection of 2 g (8 mM) magne-
e use of flunarizine in the management of sium sulfate. However, the same regimen
ntricular arrhythmias. However, given the failed to prevent recurrent polymorphic ven-
tential for severe cardiac mechanical de- tricular tachycardia in patients with ischemic
heart disease (236).Given these often conflict-
deleterious effects, particularly in pa- ing results, the therapeutic value of magne-
h compromised cardiac function. sium in the management of ventricular ar-
e long-term administration of flunarizine rhythmias has been questioned (231).

ce a high incidence of neuromotor symp- 3.3.8 Mibefradil. Experimental and clini-


cal evidence described above suggests that cal-
cium antagonists have the potential to protect
gnesium. It is well established that against malignant arrhythmias induced by
is an important regulator of cyto- myocardial ischemia. Calcium antagonists
cium and has, in fact, been called "na- that exert demonstrable direct cardiac actions
ologic calcium channel blocker" in therapeutic concentrations (verapamil, flu-
his inorganic calcium channel antago- narizine) are, in general, more effective anti-
been reported to correct a variety of arrhythmic agents in myocardial ischemia
disorders caused by calcium overload than antagonists that are more selective for
-229). For example, magnesium tenni- vascular smooth muscle (nifedipine). However,
digitalis-induced arrhythmias (227), pre- the more effective calcium channel antagonists
afterdepolarizationselicited by also depress cardiac mechanical function and of-
chloride (228), and suppressed reperfu- ten produce deleterious effects in patients with
compromised cardiac function (198, 210)-the
er showed that the intrave- very patients at greatest risk for malignant ar-
injection of magnesium sulfate prevented rhythmias (238). The challenge, therefore, is to
ventricular fibrillation induced by acute develop calcium antagonists that modulate myo-
al ischemia in conscious dogs with cardial calcium, particularly during ischemia,
ed myocardial infarctions. In contrast, Eu- without compromising cardiac function.
(230) reported that magnesium infusion Mibefradil (Ro 40-5967), a benzimidazolyl-
substituted tetraline derivative, represents a
ation induced during isch- new class of calcium channel antagonists with
or reperfusion in anesthetized dogs. these clinically important characteristics
Similar conflicting results have been re- (239,240).Mibefradil is relatively selective for
d in humans (231).Oral magnesium sup- the T-type calcium channel but also blocks the
s have been advocated as a nonphar- L-type channel (241) and the delayed rectifier
gical means of controlling frequent potassium current (242). Mibefradil has also
ias for a number of years been reported to inhibit sarcolemmal ATP-po-
elded only modest re- tassium channels in adrenal zona fasciulata
, For example, Zehender et al. (232) found cells (243) but to activate mitochondrial ATP-
daily magnesium supplements reduced sensitive potassium channels, thereby facili-
only 17% in patients tating ischemic pre-conditioning and limiting
Myocardial Infarction Agents

myocardial infarction size in isolated rat patients with ischemic heart disease (267,
hearts (244, 245). With regards to calcium 268). Given the high bioavailability ( ~ 9 0 % )
channels, mibefradil was found to have a 10- and long plasma half-life (17-25 h) (173,240),
to 15-fold greater affinity in uitro for the T- this drug was a strong candidate for once-a-
type calcium channel than for the L-type cal- day dosing. However, mibefradil did not alter
cium channel (246, 247). At therapeutic con- cardiovascular mortality in patients with con-
centrations in uiuo, the cardiovascular actions gestive heart failure (MACH I study) (269). In
of mibefradil seem to be mediated primarily fact, patients co-medicated with antiarrhyth-
through actions on the T-type calcium chan- mic drugs including amiodarone exhibited a
nel ( 4 0 % ) with lesser actions on the L-type significantly increased rate of death (269). It
calcium channel ( ~ 2 0 % )Mibefradil
. was also was also discovered that mibefradil inhibits
found to be highly selective for vascular, as cytochrome P450, the major enzyme involved
opposed to cardiac, tissue (173,239,240,248). in the metabolism of many cardiovascular
Sarsero et al. (248) investigated the vascular- drugs (270). As a result, a number of adverse
to-cardiac selectivity of a variety of calcium interactions (including pronounced bradycar-
channels antagonists using human small ar- dia) between mibefradil and other medica-
teries (aortic vaso vasorum) and right atrial tions were reported, which prompted the with-
trabeculae. They reported the followingvascu- drawal of this drug from the market in 1998
lar-to-cardiac selectivity ratios: mibefradil, 41; (271-273).
felodipine, 12; nifedipine, 7; amlodipine, 5; These adverse reactions not withstanding,
and verapamil, 0.2. It is therefore not surpris- it should be noted that mibefradil has also
ing that mibefradil has been reported to in- been reported to have favorable actions on car-
crease coronarv " blood flow and reduce arterial diac rhythm. For example, mibefradil reduced
blood pressure with little or no negative ino- regional differences in action potential dura-
tropic effects (240, 249, 250). Indeed, mibe- tion induced by myocardial ischemia (274)and
fradil binds at or near the same membrane significantly reduced the incidence of ventric-
sites as verapamil but exerts few negative ino- ular fibrillation brought about by the combi-
tropic effects on the isolated heart (240, 251- nation of exercise and acute ischemia (78). In
254). Mibefradil has the same affinity for the contrast to either verapamil or diltiazem, this
[3H]desmethoxyverapamil binding site as ve- protection was afforded without adversely af-
rapamil, but it elicits an approximately 10-fold fecting A-V conduction or inotropic state (78).
less reduction in myocardial force (239). In ad- Mibefradil prevented ischemically induced re
dition, mibefradil improves myocardial func- ductions in ventricular fibrillation threshold
tion during experimental myocardial isch- without depressing the maximal rate of leR
emia, whereas in the same study, verapamil ventricular pressure development. However,
not only failed to protect against ischemia but only higher doses of mibefradil, which de
also produced severe cardiac depression (240). pressed ventricular contractile function, could
In marked contrast to either diltiazem or ve- prevent arrhythmias induced by reperfusion
rapamil, mibefradil does not seem to alter con- in the anesthetized pig (189). These authora,
tractile function in animal models of heart therefore, concluded that both the L- and T.
failure or myocardial infarction (78,240,250- type calcium currents contribute to arrhyth-
256). Initial studies indicated that mibefradil mia formation during ischemia and reperfu-
was well tolerated by patients (257),reducing sion. Mibefradil was also found to be less
arterial pressure without adverse effects on effective than verapamil in the prevention o
cardiac contractile function, even in patients ventricular fibrillation induced by reperfusio
with heart failure (258). A number of clinical in the isolated rat heart (275). In cont
studies demonstrated that this drug was effec- to ventricular arrhythmias, mibefradil
tive in treating patients with hypertension found to be particularly effective in the tre
(259-262) and angina pectoris (263-268). In- ment of atrial fibrillation (276, 277). The
deed, mibefradil was also found to be particu- type calcium channel number does not ch
larly effective in suppressing both exercise-in- as a consequence of chronic atrial fibrillati
duced and "silent" ischemic episodes in whereas the L-type calcium channel dens
3 Treatment for Myocardial Infarction

decreases (2781, and as such, the T-type cal- bated the nonischemic arrhythmias induced
cium current may contribute to maintenance by programmed electrical stimulation (284).
of this arrhythmia. Indeed, mibefradil reduced For example, mibefradil (as well as verapamil
regional differences in atrial refractory period and diltiazem) increased both duration and
(i.e., decreased heterogeneity in repolariza- rate of ventricular tachycardia in approxi-
tion), terminated atrial fibrillation, and pre- mately 50% of the animals tested (284). Fi-
vented the ion channel remodeling associated nally as noted above, mibefradil inhibits pref-
with chronic atrial fibrillation (276, 277). T- erentially calcium entry through T-type
type selective calcium channel antagonists calcium channels (283,285). The expression of
may, therefore, represent a novel approach for the T-type calcium channels has been shown
the management of this clinically important to increase as a consequence of a variety of
and often intractable atrial arrhythmia. cardiac diseases including myocardial infarc-
It is still unclear how mibefradil, in con- tion (174-176). Therefore, it is possible that
trast to verapamil or diltiazem, can protect an increased activation of T-type calcium
against ischemic arrhythmias with minimal channels after myocardial infarction also may
adverse actions on contractile function. It is contribute significantly to the induction of
possible that a unique electropharmacologic ventricular fibrillation, particularly 'during
property of mibefradil may be responsible for myocardial ischemia.
this cardioprotection. At physiologic cell mem-
brane resting potentials, verapamil is 20 times 3.3.9 Sodium/Calcium Exchanger Antago-
more potent than mibefradil in the inhibition nists. As previously noted, the electrogenic so-
of calcium entry into cardiac myocytes (279). dium/calcium exchanger plays a major role in
However, in depolarized cells, verapamil and the regulation of intracellular calcium in both
mibefradil are equipotent in the inhibition of normal and diseased hearts (286, 287). Be-
calcium entry (279). More recent studies have cause this protein can move ions (and charge)
confirmed these findings (247,280-283). Bez- in either direction, mode-selective antagonists
prozvanny and Tsien (282)demonstrated that may have important therapeutic applications.
mibefradil is a more effective inhibitor of the For example, during myocardial ischemia, the
L-type calcium current at reduced (depolar- sodium pump can no longer function properly
ized) membrane potentials. They concluded (because of reductions in ATP levels),and as a
that this voltage dependency resulted from a result, cellular sodium levels increase. This el-
preferential binding to the open and inacti- evation in intracellular sodium reverses the
vated state of the L-type calcium channel. As direction of the sodium/calcium exchanger so
noted above, significant numbers of ventricu- that sodium is extruded and calcium is taken
lar cells depolarize during myocardial isch- up by the myocytes. As we have seen, this in-
emia (59). Therefore, mibefradil may be par- creased calcium entry exacerbates calcium
ticularly selective for ischemic tissue-the overload and may lead to afterdepolarizations
tissue most vulnerable to calcium overload that trigger arrhythmias. The up-regulation
and arrhythmia formation. As such, mibe- of this sodium/calcium exchanger that occurs
fradil would exert little negative effect on nor- as a consequence of heart failure has been
mal tissue, but during ischemia, would be- linked to systolic dysfunction and arrhyth-
come an effective mvocardial
" calcium channel mias in these patients (288, 289). Therefore,
antagonist. Perhaps because of its membrane one might speculate that drugs that suppress
potential sensitivity, mibefradil may act as an sodium/calcium exchanger activity may be
ischemia-selective calcium channel antago- beneficial, reducing calcium overload and
nist. Indeed, mibefradil completely suppresses thereby improving the electrical stability of
ventricular tachycardia induced by pro- the heart.
grammed electrical stimulation during myo- Recently, KB-R7943, an amphiphilic mole-
cardial ischemia but fails to prevent electri- cule that contains a positively charged isothio-
cally induced arrhythmias under nonischemic urea group, has been reported to be a potent
conditions (284). In fact, mibefradil, as well as and selective sodium/calcium exchanger an-
other calcium channel antagonists, exacer- tagonist. KB-R7943 dose-dependently inhib-
Myocardial Infarction Agents

ited the whole cell Nat/Ca2+ exchanger cur- myocytes (303). Furthermore, KB-R7943 pre-
rent recorded in rat (290), guinea pig vented the cell-to-cell propagation of calcium
ventricular myocytes (291,292), smooth mus- overload mediated hypercontraction in 16 of
cle cells (290), and Na+/Ca2+ exchanger- 20 rat myocyte cell pairs (304). KB-R7943 also
transfected fibroblasts (290) in low (IC,, = reduced both the incidence and the duration of
0.3-2.4 a) concentrations. Importantly, KB- arrhythmias provoked by hypoxidreoxygen-
R7943 exerted a more potent inhibition of the ation in guinea pig papillary muscle prepara-
reverse mode than on the forward mode of the tions (299).Finally, KB-R7943 (5 mgtkg iv bo-
Nat/Ca2+ exchanger (290, 292-294). For ex- lus) significantly prolonged atrial effective
ample, in guinea pig myocytes, lower concen- refractory period in a time-dependent fashion
trations of KB-R7943 were required to block but did not alter either ventricular refractory
the outward Na+/Ca2+ exchanger current period or A-V nodal conduction (AH and HV
(IC,, approximately 0.32 a) than that re- interval) nor did it affect sinus cycle length,
quired for the inward current (IC,, = 17 p&¶). QT interval, or mean arterial pressure in
Similar results were obtained in cultured neo- anesthetized dogs (305). This dose of KB-
natal rat cardiomyocytes (290) and with the R7943 also prevented tachycardia-induced
canine Na+/Ca2+exchanger expressed in Xe- shortening of atrial refractory period in the
nopus oocytes (293). anesthetized dogs (306).Because both calcium
KB-R7943 has also been reported to be a overload and reductions of atrial refractory
particularly effective Na+/Ca2+exchanger an- period have been implicated in the mainte-
tagonist under conditions that would favor nance of atrial fibrillation (276), the authors
myocardial calcium overload. This drug proposed that KB-R7943 might be potentially
blocked calcium entry into rat ventricular useful in the treatment of this atrial
myocytes induced by either removing extra- arrhythmia.
cellular sodium or by the cardiac glycoside It should be noted, however, that KB-
strophanthidin, thereby reducing diastolic R7943 could also inhibit a number of other ion
calcium and abolishing spontaneous calcium channels in cardiac tissue obtained from a
oscillation (291). Interestingly, this reduction variety of species. At concentrations up to
in calcium overload did not alter the positive 30 p&¶, KB-R7943 had little effect on the
inotropic response to the glycoside (291). KB- Na+/Ht exchanger, sarcolemmal Ca2+ ATPase,
R7943 suppressed ouabain-induced arrhyth- or Na+/Kt ATPase (290). However, at 30 pA4
mias in isolated guinea pig atria and signifi- but not 10 a, KB-R7943 could block voltage-
cantly increased the dose of ouabain required dependent Nat and Caf cpannels (290). In
to provoke arrhythmias in anesthetized guinea pig ventricular myocytes, KB-R7943
guinea pigs (295). Inhibition of the Na+/Ca2+ inhibited the Na+ current, Ca2+ current, and
exchanger with KB-R7943 reduced calcium the inward rectifier K+ current with an IC,, of
overload and cell death associated with hypox- approximately 14, 8, and 7 a, respectively
idreoxygenation of isolated cardiac cells or (292). KB-R7943 may also inhibit the calcium-
ischemia/reperfusion in a variety of different activated chloride current (307). In isolated
preparations including rat cardiomyocytes blood perfused canine right atrium and left
(290, 296-298), guinea pig papillary muscle ventricle, KB-R7943 (0.03-3 p k f ) elicited a
(299, 300), and isolated perfused rat heart negative inotropic response as well as a nega-
(296-298, 301) or rabbit hearts (302). KB- tive followed by positive chronotropic effect
R7943 also reduced the infarction size in iso- (308). These responses may have resulted
lated perfused rabbit hearts (30 min of global from the nonspecific inhibition of ion channels
ischemia followed by 2 h of reperfusion) (302) in addition to actions on the Na+/Ca2+ ex-
or in intact pigs subjected to 48 min of isch- changer current. In contrast, KB-R7943
emia followed by 10 min of reperfusion com- (5a)
2
did not alter rat ventricular myocyte
pared with placebo-treated animals (a 34% re- Ca +, sarcoplasmic reticular calcium levels, or
duction in infarct size) (296). KB-R7943 also steady-state twitch amplitude (291). Further-
protected against the calcium overload in- more, the resting potential was not altered by
duced by lysophatidyl choline in isolated rat KB-R7943 treatment but the action potential
3 Treatment for Myocardial Infarction

plateau duration increased, an observation tional conduction block and thereby remove
that is consistent with inhibition of Kt cur- the substrate for re-entry in much the same
rents (291). KB-R7943 has been reported to manner as sodium channel (class I anitar-
inhibit nicotinic and glutamate [N-methyl-D- rhythmic drugs) antagonists. Thus, calcium
aspartate (NMDA)] receptor-mediated cal- channel agonists could be antiarrhythmic in
cium entry in neural tissue (309,310). Finally, certain settings. Cabo et al. (312), in fact, re-
the reverse mode selectivity of KB-R7943 has ported that the calcium channel agonist Bay Y
recently been questioned. The preferential in- 5959 prevented the initiation of electrically in-
hibition of the reverse mode of the Naf /Ca2+ duced ventricular tachycardia that resulted
exchanger by KB-R7943 has been shown to from re-entry in nearly one-half of the animals
disappear under certain conditions (301,311).
tested (anesthetized dogs with a healing,
For example, KB-R7943 equipotently blocked
4-day-old infarction). The effects of this drug
both direction of the exchanger current under
ionic conditions that favored bidirectional on ischemically induced arrhythmias were not
conduction (301). However, the selectivity for investigated, nor was this drug completely
the outward current was maintained for the successful even under conditions that favored
canine sodium/calcium exchanger expressed re-entry. In contrast, the calcium channel ag-
in frog oocytes even during conditions that fa- onist Bay K 8644 has been shown to induce
vored bidirectional transport (293). afterdepolarizations (313), and ventricular
In summary, the preliminary studies de- fibrillation during myocardial ischemia in an-
scribed above suggest that KB-R7943 may act imals previously shown to be resistant to ma-
selectively to block the reverse mode of the lignant arrhythmias (76).Furthermore, eleva-
sodium calcium exchanger. As such, KB- tions in intracellular calcium would both
R7943 could be particularly effective against directly (increased heart rate and inotropic
alterations in cardiac function induced by state) and indirectly (increased arterial pres-
ischemic calcium overload. Indeed, this drug sure, afterload) place a greater metabolic de-
was shown to reduce arrhythmias, protect mand on the heart, and could thereby exacer-
against cell death, and reduce the myocardial bate ischemia in diseased hearts. Although it
infarction size induced by ischemialreperfu- is possible that calcium agonists may termi-
sion. It should be noted that side effects of this nate re-entrant arrhythmias in certain set-
h g caused by nonspecific actions on a num- tings (electrically induced arrhythmias), this
ber of other ion channels and neurotransmit- limited protection is achieved at considerable
ter receptors could limit its application in the risk. The resulting calcium overload would not
clinic. However, it seems reasonable that se-
only increase the risk for afterdepolarizations
lective sodium/calcium exchanger (particular-
and triggered automaticity (see above) but
ly the reverse mode) antagonists offer an ex-
would also place a severe metabolic burden on
citing and novel approach for the management
of malignant arrhythmias. what may be an already compromised heart.
As such, these agents would be clearly coun-
3.3.10 Calcium Channel Agonists. As pre- terindicated in the vast majority of patients
viously noted, elevations in intracellular with existing cardiac disease-the very pa-
calcium can uncouple cardiac cells delaying tients at the greatest risk for lethal ventricu-
electrical conduction (see above), thereby con- lar arrhythmias (314). It is, therefore, reason-
tributing to the formation of re-entrant cir- able to conclude that the risks associated with
cuits. It has recently been proposed that an calcium channel agonists greatly exceed the
enhancement of the L-type calcium current putative benefits for this therapy. Unless cal-
could lead to further decreases in the gap junc- cium channel agonists can be developed that
tion conduction, increasing the electrical re- selectively target regions of the heart where
sistance between cells and thereby eliminat- conduction is compromised without affecting
ingre-entrant pathways (312). In other words, normal cardiac or vascular muscle, it is un-
calcium channel agonists could convert a uni- likely that these agents will gain widespread
directional conduction block into a bidirec- clinical acceptance.
Myocardial Infarction Agen

3.3.1 1 ATP-Sensitive Potassium Channel the inhibition of ATP-sensitive potassi


Antagonists. It is now generally accepted that channels located on the coronary vascu
the activation of the ATP-sensitive potassium smooth muscle.
channel during myocardial ischemia provokes There are a few clinical studies that illu
a potassium efflux and reductions in action trate the anti-arrhythmic potential of ATP
potential duration that lead to dispersion of sensitive potassium channels (330-332, 397
repolarization. Because heterogeneity of repo- Cacciapuoti et al. (397) found that gliben-
larization plays a crucial role in the induction clamide significantly reduced the frequency
of ventricular fibrillation, drugs that prevent and severity of ventricular arrhythmias r e
ATP-sensitive potassium channel activation corded during transient ischemia in non-insu-
lin-dependent diabetic patients with coronary
should be particularly effective in the suppres-
artery disease. This drug, however, did not af-
sion of malignant arrhythmias induced by
fect non-ischemic arrhythmias nor did it
ischemia. change the number or length of the ischemic
The sulfonylurea drug glibenclamide has episodes.
been shown to prevent hypoxia induced reduc- Glibenclamide significantly reduced the in-
tions in action potential duration in single cidence of ventricular fibrillation in non-insu-
cells, isolated hearts, and intact animals (110, lin-dependent patients with acute myocardial
112, 113, 115-120, 122, 315-320). This drug infarction (332). Glibenclamide also reduced
can attenuate the ischemically induced the S-T segment elevation and prevented ven-
changes in the S-T segment in intact anesthe- tricular fibrillation induced by chest impact (a
tized or unanesthetized animals (134, 135). baseball delivered at 30 mph to the chest of
Glibenclamide also prevented arrhythmias in- anesthetized swine) (333). The authors con-
duced by ischemia in isolated hearts (118, cluded that "selective ATP-sensitive p o t s
321-324). Furthermore, both glibenclamide sium channel activation may be a pivotal
and glimepride reduced blood glucose and de- mechanism in sudden death resulting from
creased the incidence of irreversible ventricu- low energy chest wall trauma" (i.e., Commotio
lar fibrillation induced by reperfbsion (after a Cordis) "in young people during sporting ac-
6-min coronary occlusion) in anesthetized rats tivities" (333).
(325).Glibenclamide has also been reported to Glibenclarnide is not selective for cardiac
reduce the incidence of life-threatening ar- sue. As noted above, this drug can profoun
rhythmias induced by coronary artery ligation reduce coronary blood flow through actions
in the conscious rat (326)and the anesthetized vascular smooth muscle and can also pro
rabbit (3271, as well as improve survival in the insulin release, thereby provoking hypo
anesthetized rat during ischemia and reperfu- rnia (146). These non-cardiac actions
sion (328). Billman et al. (146, 329) further limit the anti-arrhythmic potential of
demonstrated that glibenclamide prevented clamide in the clinic. Cardioselectivecomp
ventricular fibrillation induced by the combi- should have fewer side effects and woul
nation of acute ischemia during submaximal fore provide a better therapeutic option t
exercise in animals previously shown to be nonselective ATP-sensitive potassium
susceptible to sudden death. It should be antagonist glibenclamide.
noted, however, that in these animals gliben- Several different ATP-sensitive potassi
clamide significantly reduced both the exer- channel subtypes have been identified.
cise- and reactive hyperemia-induced in- ATP-sensitive potassium channel consis
creases in coronary blood flow as well as pore-forming subunit coupled to a sul
depressed ventricular function (large reduc- urea receptor (334-336). The functional c
tions in left ventricular dP/dt maximum) (146, nel forms as a hetero-octomer composed
329). In the isolated working rabbit heart, tetramer of the pore and four sulfonyl recept
glibenclamide provoked an immediate de- subunits. At present, two different pore-
crease in coronary blood flow reducing for- ing subunits have been identified, bot
ward flow to zero (140). Thus, glibenclamide which produce an inward rectifier pot
has potent vasoconstrictor effects as a result of current (Kir 6.1 and Kir 6.2) (138,337).
3 Treatment for Myocardial Infarction

rent sulfonylurea receptor subtypes have ischemic preconditioning (21, 345). Recently,
n isolated: SURl (on pancreatic islet cells), Liu et al. (337) showed that the mitochondrial
UR2A (on cardiac tissue), and SUR2B (on ATP-sensitive potassium channel most closely
cular smooth muscle) (335, 336, 338, 339). resembles the KirG.l/SURl subtype. Thus,
Thus, six different potassium channel pore HMR 1883 could inhibit cardiac membrane
and sulfonylurea receptor combinations are ATP-sensitive potassium channels with mini-
ssible. Suzuki et al. (137) recently showed mal effects on mitochondrial channels.
at Kir 6.2 and Kir 6.1 were required for car- HMR 1883 also prevented ischemically in-
and vascular smooth muscle ATP-sensi- duced changes in the S-T segment in anesthe-
e potassium channel activity, respectively. tized mice (138), anesthetized swine (346),
hus, Kir 6.2lSUR2A most likely forms the and conscious dogs (135). In the conscious
cardiac cell membrane ATP-sensitive potas- dogs with healed myocardial infarctions, both
sium channel, whereas Kir 6.1/SUR2B is lo- HMR 1883 and glibenclamide prevented isch-
cated on vascular smooth muscle. It should emically induced reductions in effective re-
therefore be possible to develop compounds fractory period (146). HMR 1883 significantly
that selectively inhibit (or activate) a particu- reduced monophasic action potential shorten-
lar ATP-sensitive potassium channel subtype. ing induced by coronary artery occlusion in
A drug that selectively blocks the Kir 6.21 anesthetized pigs (347). HMR 1883 also re-
SUR2A subtype should prevent ischemically duced cardiac mortality in anesthetized pigs
induced changes in cardiac electrical properties (346, 348) and prevented ventricular fibrilla-
(e.g., reductions in action potential duration) tion induced by myocardial ischemia and
and thereby prevent arrhythmias without the reperfusion in rats (349). Finally, both gliben-
side effects noted for the nonselective ATP-sen- clamide and HMR 1883 significantly reduced
sitive channel antagonist glibenclamide. the incidence of ventricular fibrillation in-
The sulfonylthiourea drug HMR 1883 and duced by myocardial ischemia in conscious
its sodium salt HMR 1098 were recently devel- dogs with healed anterior wall myocardial in-
I oped to block the cardiac ATP-sensitive potas- farctions (146). In contrast to glibenclamide,
sium channel (340). HMR 1883 inhibited the HMR 1883 did not alter plasma insulin or
sarcolemmal cardiac ATP-sensitive potassium blood glucose levels in these animals (146).
channel activated by the channel opener ril- Furthermore, glibenclamide, but not HMR
T makalin at a much lower concentration 1883, significantly reduced exercise-induced
1 (guineapigmyocytesIC,, = 0.6-2.2 a) than increase in mean coronary blood flow and pro-
2 was required to promote insulin release (9- to voked large reductions in left ventricular
- 50-fold higher concentration was required to dP/dt maximum (both at rest and during exer-
3 block pancreatic RIN m5F cells) (341). Fur- cise) (146). Thus, HMR 1883 seems to act se-
- thermore, and in contrast to glibenclamide, lectively on the cardiac cell membrane ATP-
s HMR 1883 did not alter hypoxia-induced in- sensitive potassium channel and thereby
I- creases in coronary blood flow in Langendorff- prevents ischemically induced arrhythmias
e perfused guinea pig hearts (341). Importantly, with minimal effects on either pancreatic or
?1 HMR 1883 did not alter flavoprotein autoflo- smooth muscle channels.
rescence, an index of mitochondrial redox In contrast to ATP-sensitive potassium
n state (342). In contrast, 5-hydroxydecanoic channel antagonists, channel agonists have
.e acid, a selective blocker of mitochondrial been reported to induce arrhythmias during
a channels, completely inhibited the flavopro- ischemia in both isolated hearts and intact an-
1- tein florescence (22). In agreement with these imals (322, 350, 351). The channel agonist
1- in vitro findings, HMR 1883 did not prevent pinacidil facilitated ventricular fibrillation
a the cardioprotective effects induced by isch- during myocardial ischemia in isolated rat
)r emic preconditioning in either rat (343) or (322) or rabbit (350) hearts with reduced po-
n- rabbit (344). Accumulating evidence suggests tassium levels. Di Diego and Antzelevitch
of that the activation of mitochondrial ATP-sen- (147) found that the activation of the ATP-
m sitive potassium channels plays a crucial role sensitive potassium channel with pinacidil
3e in the mechanical protection that results from caused marked inhomogeneities of refractory
period, which provoked extrasystoles. These brane potential that occur during phase 3 of
extrasystoles were blocked by glibenclamide the cardiac action potential) are dependent on
in strips of isolated canine myocardium. They delayed repolarizations (59) and may be pre-
concluded that this dispersion of repolariza- vented by ATP-sensitive channel activators
tion and refractory period formed a substrate (359-363). For example, the ATP-sensitive
for reentry. Indeed, the ATP-sensitive potas- potassium channel agonists pinacidil
sium channel oDener cromakalim reduced ef- orandil can abolish early afterdepolarizatio
fective refractory period and increased vulner- induced by cesium chloride (363) or BAY
ability for re-entry (139). Furthermore, this 8644 (362). These agents have also been
drug was also shown to increase interventric- ported to suppress delayed afterdepolariza
ular dispersion of refractory period and in- tions (oscillations that occur after repolariza
duced ventricular fibrillation in 5 of 12 iso- tion of the preceding action potential
lated rabbit hearts under normoxic conditions produced by cardiac glycosides (361) in
(140). Chi et al. (351) further demonstrated lated tissue. It is also important to note
that pinacidil increased the frequency of ven- some ATP-sensitive potassium channel a
tricular fibrillation during myocardial isch- nists have been reported to promote ische
emia in unanesthetized dogs. This drug had no preconditioning and thereby reduce the m
effect on arrhythmias induced by electrical chanical dysfunction induced by ischemia. I
stimulation in normally perfused tissue. Cro- deed, the ATP-sensitive potassium ch
makalim also failed to reduce arrhythmias in- opener diazoxide may act somewhat se
duced by ischemia in either isolated rabbit tively on mitochondrial ATP-sensitive pot
hearts (140) or intact dog (352). It should be sium channels (22,23).
noted that the pro-arrhythmic effects of these In summary, activation of cardiac c
agents have been reported only at high doses, membrane ATP-sensitive potassium chan
doses that often produce hypotension and cor- during myocardial ischemia promotes po
responding reflex tachycardia (351). More re- sium efflux, reduction in action potential
cently, pinacidil failed to alter the dispersion ration, and inhomogenieties in repolarizatio
of refractory period or promote arrhythmia thereby creating a substrate for re-en
formation in animals with healed myocardial Drugs that block this channel should be
infarctions (353).Clinical trials with ATP-sen- ticularly effective anti-arrhythmic agents
sitive potassium channel agonists as antihy- deed, it is interesting to note that m
pertensive agents have been reported not to rently available anti-arrhythmic
induce arrhythmias (354, 355). However, including verapamil, mibefradil, quinidine
Goldberg (121) reported that 30% of patients docaine, and amiodarone have also been
taking pinacidil as an antihypertensive medi- ported to inhibit ATP-sensitive potassi
cation showed abnormal ECG (T wave) channels at therapeutic concentrations (1
changes suggestive of alterations of repolar- 356,364,365). Therefore, the inhibition of
ization. It is possible that in the presence of ATP-sensitive potassium channel may be
ischemia these cardiac actions of channel ago- quired for anti-arrhythmic actions d
nists become more pronounced, increasing the myocardial ischemia. HMR 1883 (or i
propensity for life-threatening arrhythmias. dium salt HMR 1098) selectively blocks
It must be emphasized, however, that un- diac sarcolemmal ATP-sensitive potas
der certain conditions, potassium channel channels. As such, this drug attenuates
activation may also have anti-arrhythmic emically induced changes in cardiac elec
properties, particularly against arrhythmias properties, thereby preventing malignan
resulting from abnormalities in repolarization rhythmias without the untoward effecta
(356-358). ATP-sensitive potassium channel other drugs. Because, as noted above,
agonists have been reported to show anti-ar- ATP-sensitive potassium channel only
rhythmic properties in some nonischemic and comes active as ATP levels fall, HMR 1
a few ischemic experimental models (326,328, the added advantage that this drug wou
359-363). Arrhythmias that result from early exert actions on ischemic tissue with 11
afterdepolarizations (oscillations of mem- no effect noted on normal tissue. Thus, se
Treatment for Myocardial Infarction

antagonists of the cardiac cell surface farction patients are not placed on these drugs
-sensitive potassium channel may repre- (371-373). For example, Viskin et al. (373)
ly ischemia selective anti-ar- found that only 58% of infarct survivors with
hmic medications and should be free of no contraindication to P-adrenergic blockers
e pro-arrhythmic effects that have plagued actually received these drugs at the time of
ently available anti-arrhyth- hospital discharge. More importantly, only
hat HMR 1883 11% of the patients received doses clinically
diac mortality even in high proven to reduce cardiac mortality; the major-
advanced ischemic heart ity of patients received substantially lower
doses! It has been conservatively estimated
that if cardiologists would adhere to the guide-
3.3.12 P-adrenergic Receptor Antagonists. lines for the use of p-adrenergic blockers as
noted above, myocardial ischemia and in- suggested by the American College of Cardiol-
ction can provoke pronounced changes in ogy, nearly 1900 deaths would be averted an-
lation of the heart that, in nually nationwide (373).
, have been implicated in the formation of In contrast to the long-term benefits of
ntricular fibrillation. As a rule, cardiac elec- p-adrenergic receptor antagonists, the influ-
ced by activation of the ence of these agents during acute phase myo-
pathetic nervous system, whereas in- cardial infarction is less definitive. There have
tic activity may protect been at least 32 trials involving approximately
ent of malignant ar- 29,000 patients in which P-adrenergic recep-
. Both clinical (4) and tor blockers have been initiated within the
ies have shown that first few hours of either confirmed or sus-
ia elicits increases in pected myocardial infarction (200, 369, 370).
pathetic tone coupled with reductions in The pooled data from these studies suggest
ympathetic activity. Furthermore, the that early treatment with p-adrenergic recep-
est changes in auto- tor blockers results in a 20-30% reduction of
ed the greatest pro- 'infarction size, and a 15% decrease in cardiac
death (39). Myocar- mortality, probably as the result of a corre-
on has also been reported to elicit spondingreduction in the incidence of ventric-
s in the autonomic control of ular fibrillation (369). However, a careful
-368). In fact, the relative mor- analysis reveals that results, particularly with
calculated to be more than regards to ventricular fibrillation, vary de-
pending on the agent used. p-Adrenergic re-
hetic tone (mea- ceptor antagonists with intrinsic sympatho-
by R-R interval variability), compared mimetic activity failed to alter the incidence of
those patients with more normal auto- ventricular fibrillation (200, 374, 375). The
majority of the studies using p-adrenergic re-
rising that interventions that reduce car- ceptor antagonists failed to report significant
effects of sympathetic activation, particu- reductions in the incidence of ventricular fi-
/3-adrenergic receptor antagonists, have brillation during acute myocardial infarction
the subject of considerable investigation. (376380). For example, metoprolol did not
ast 25 random- reduce the number of patients that experi-
lling over 23,000 patients) of enced ventricular fibrillation (377, 378). Al-
energic blockade on the secondary pre- though atenolol reduced overall mortality
ion of cardiac events have been completed 15%,this drug (380) did not affect the number
Refs. 200, 369, 370). The pooled data re- of patients that died as the result of malignant
that this treatment resulted in a 23% re- arrhythmias. In contrast, propranolol almost
on in mortality with a 32% reduction in completely eliminated death due to ventricu-
en deaths. Recent studies show that de- lar fibrillation during acute myocardial infarc-
s benefits of long-term p-ad- tion (381, 382). These data suggest a better
ergic therapy, a large number of post-in- anti-arrhythmic protection can be achieved
Myocardial Infarction Agents

with complete (i.e., PI- and P2-) rather than hearts of animals resistant to these malign
selective (i.., - P-adrenergic receptor arrhythmias (50). Thus, activation of P2-
blockade. These data further indicate that the renergic receptors during myocardial i
activation of myocardial P-adrenergic recep- emia could culminate in ventricular fib
tors may play a particularly important role in tion. In this regard, it is interesting to note
the induction of malignant arrhythmias and that there are isolated clinical reports in
acute myocardial infarction. which p,-adrenergic agonists have precipi-
There is now a growing body of evidence tated sudden death as a consequence of car-
demonstrating that in addition to PI-adrener- diac actions in asthmatic patients (370,383).
gic receptors, ventricular myocytes also con- In summary, the data described above
tain functional &-adrenergic receptors (48). clearly demonstrate that P-adrenergic recep
Furthermore, these receptors may become tor antagonists reduce cardiac mortality in
particularly important in the regulation of post-infarction patients. This reduction
contractile function in certain disease states mortality largely results from a reduction h
(28,48,49). For example, as a consequence of the incidence of arrhythmogenic deaths. FUF
chronic heart failure, there is a substantial thermore, the data indicate that nonselecti
loss of PI-adrenergic receptors with little or no P-adrenergic receptor antagonists offer
loss in P2-adrenergic receptors (51). The fail- more complete protection than the so-call
ing heart therefore may become more depen- cardioselective P- (Dl-) adrenergic receptor
dent upon the activation of P2-adrenergic tagonists. It would seem that the acti
receptors for inotropic support during sympa- ventricular &adrenergic receptors co
thetic stimulation. Indeed, recent studies voke life-threatening arrhythmias, p
demonstrated that the P2-adrenergicagonist larly during myocardial ischemia or in
zinterol elicited significantly larger calcium tients with compromised cardiac function.
transients (increases in cytosolic Ca2+ in re-
sponse to electrical field stimulation) in ven- 3.4 Prevention of Remodeling
tricular myocytes prepared from failing hearts
as compared to normal tissue (49). As noted 3.4.1 Ace Inhibitors. Large multice
above, cytosolic calcium plays a critical role in trials have shown conclusively that angio
the induction of ventricular fibrillation. P2- sin-converting enzyme (ACE) inhibitors
Adrenergic receptor-mediated changes in nificantly improve post-infarction su
Ca2+influx could lead to the formation of ar- (13,148, 158,383-386). Unlike the case
rhythmias and sudden death in myocardial in- the calcium channel antagonists, this e
farction patients. It is interesting to note that seems to be generic and unrelated to the use
heart failure patients and patients with poor any particular drug. It is currently reco
left ventricular contractile function are at a mended that ACE inhibitor therapy be
substantially greater risk of sudden death ated 1-2 days post-infarct (13, 148, 158,
compared with patients with a more normal 386). ACE inhibitors are especially impo
pump function (314). One might speculate in patients with a reduced ejection fract
that changes in cytosolic Ca2+ mediated by a measure of the efficiency of cardiac pum
greater activation of myocardial P2-adrenergic who are most prone to develop sympt
receptors may provoke lethal cardiac arrhyth- congestive heart failure after the infar
mias in these patients. Recently, the p2-adren- healed (13, 148, 158, 383-3861. In pati
ergic receptor antagonist ICI 118,552 was with more normal cardiac function, it is
shown to prevent canine ventricular fibrilla- rently believed that ACE inhibitors can be
tion induced by myocardial ischemia during continued after 6 months, at which time
the last minute of submaximal exercise (50). jury-related remodeling of the heart sh
Furthermore, the j3,-adrenergic receptor ago- have ceased (383). However, it has been
nist zinterol elicited much larger Ca2+ tran- gued that the anti-thrombotic effects of
sients in ventricular myocytes prepared from inhibitors may contribute significantly
the hearts of animals susceptible to ventricu- their salutary effects on post-infarction m
lar fibrillation compared with cells from tality, in which case more prolonged t
mmary and Conclusions

ent may prove beneficial (383). Studies risk for developing congestive heart failure
re ACE inhibitors were given very early in (148, 158, 383, 385,389, 390).
treatment of acute myocardial infarction
ed to show any addi- 3.4.2 Clucose/lnsulin/Potassium. Because
benefit, and there may have been a ischemic myocardium is profoundly deener-
increase in mortality most likely as a gized, there has long been the view that met-
of hypotension in some patients (149). abolic support should help to preserve viable
ere is debate as to whether early ACE inhib- tissue. In experimental animal models of isch-
e infarct expansion emia and reperfusion (391) the infusion of
reduce long-term mortality if restricted to clinically relevant concentrations of glucose
ne low blood pres- plus insulin plus potassium led to greater re-
covery of mechanical function and reduced
he mechanisms responsible for the pro- cells necrosis as monitored by enzyme release
cts of ACE inhibitors in the post- (391). A pilot trial of glucose/insulin/potas-
d myocardium are not fully understood. sium demonstrated reduced mortality in pa-
e name implies, converts inactive tients with acute myocardial infarctions un-
I to active angiotensin 11; it also dergoing reperfusion therapy (392) and
radykinin. Thus, ACE inhibitors Apstein and Taegtmeyer have written an edi-
synthesis of angiotensin I1 and torial urging a large prospective clinical trial
degradation of bradykinin. De- of this therapy (393). However, the costs of
s in angiotensin I1 decrease aldosterone such a trials would be prohibitive, especially
d reduce blood pressure. This is because thrombolytic therapy in GUSTO V re-
beneficial in the setting of hyperten- duced 30-day mortality to less than 6% (160).
ading of the heart may account Nevertheless, salvage of viable myocardium
some of the effects of ACE inhibition post- may have longer-term effects to prevent car-
dion. There are also direct cardiac effects diac dilatation and failure. In this context, it is
the ACE inhibitors that cannot be repli- noteworthy that insulin administered at
with equivalent reductions in blood reperfusion inhibited apoptosis in cultured
ssure using other means (148). Angioten- heart cells (394). Insulin activates the serine-
I1 binds to specific receptors on cardiac threonine kinase Akt, and Akt activation has
es and fibroblasts (387, 388) and in- been shown experimentally to prevent isch-
hypertrophy (heart muscle cell emic (395) and reperfusion-induced (396) car-
last hyperplasia (3871, and re- diomyocyte injury.
eling of the extracellular connective tis-
). Myocyte hypertrophy, an in-
e in the size of individual heart muscle 4 SUMMARY A N D CONCLUSIONS
ppropriate response when the
dium must replace the muscle mass Great advances have been made in the treat-
to an infarct in order to perform its nor- ment of acute myocardial infarction. Defibril-
contractile work. But for reasons that are lation, p-adrenergic antagonists, and throm-
yet adequately understood, hypertrophy bolysis have significantly reduced in-hospital
lead to eventual heart failure. Extracellu- mortality. Long-term treatment with nonspe-
remodeling realigns individual myocytes, cific p-blockers, ACE inhibitors, and aspirin
mal heart into a more spher- benefit large numbers of post-infarct patients
(eccentric hypertrophy) by preventing arrhythmias, ventricular re-
). This process is poorly understood, but modeling, and reocclusion of the infarct-re-
is a very poor prognos- lated vessel. Nevertheless, sudden cardiac
gn.By blocking myocyte hypertrophy and death continues to be a leading cause of mor-
acellular remodeling, ACE inhibitors tality. The possibility that p,-adrenergic re-
e treatment of post- ceptors are involved in ventricular fibrillation
cially those with rela- suggests that p,-selective antagonists may not
ly large infarcts who are at the greatest be the drugs of choice for patients at risk for
. Myocardial Infarction Agents

lethal arrhythmias. Additional work is needed S. L. Hazen, D. A. Ford, and R. W. Gross,


to develop appropriate therapies for the pre- J. Biol. Chem., 266,56295633 (1991).
vention of sudden cardiac death in patients S. C. Armstrong and C. E. Ganote, Am. J.
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Myocardial infarction continues to be a K. Y. Hostetler and E. J. Jellison, Mol. Cell.
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soa Peptides
JAMESL.STANTON
LWDY L. WEBB
detabolic and Cardiovascular Diseases
Tovartis Institute for Biomedical Research
h m i t , New Jersey

Contents
1 Introduction, 194
2 Angiotensin 11,195
2.1 Biosynthesis and Metabolism, 195
2.2 Structural Features, 195
2.3 Receptors, 196
2.3.1 AT, Receptors, 196
2.3.2 AT, Receptors, 197
2.4 Biological Actions, 197
2.5 Inhibitors and Antagonists of the Renin-
Angiotensin System, 197
2.5.1 Renin Inhibitors, 197
2.5.2 ACE Inhibitors, 198
2.5.3 AT, Receptor Antagonists, 198
3 Arginine Vasopressin, 199
3.1 Biosynthesis, Metabolism,
and Structural Features, 199
3.2 Receptors, 200
3.3 Biological Actions, 200
3.4 Antagonists, 201
4 Endothelin Peptide Family, 201
4.1 Structural Features, 201
4.2 Biosynthesis and Metabolism, 202
4.3 Receptors, 203
4.4 Biological Actions, 203
4.4.1 Cardiovascular, 203
4.4.2 Endocrine Actions, 205
4.4.3 Renal Actions, 205
4.5 Pathological Roles of Endothelin, 205
4.6 Antagonists and Inhibitors, 206
4.6.1 Antagonists, 206
4.6.2 Endothelin-Converting Enzyme
Inhibitors, 208
5 Neuropeptide Y, 208
%urger's Medicinal Chemistry and Drug Discovery 5.1 Structural Features, 208
b h ~dition,Volume 3: Cardiovascular Agents and 5.2 Biosynthesis and Metabolism, 208
iocrines 5.3 Receptors, 209
by Donald J. Abraham 5.3.1 Y, Receptors, 209
0-471-37029-0 O 2003 John Wiley & Sons, Inc. 5.3.2 Y, Receptor, 210
Endogenous Vasoactive Peptides

5.3.3 Y, Receptor, 210 8.1.1 CGRP, 218


5.3.4 Y, Receptor, 210 8.1.2 ADM, 220
5.3.5 Y, Receptor, 210 8.2 Receptors, 220
5.3.6 y, Receptor, 210 8.3 Biological Actions, 221
5.4 Biological Actions, 210 8.3.1 CGRP, 221
5.4.1 Cardiovascular, 210 8.3.2 ADM, 221
5.4.2 Renal, 211 8.4 Therapeutic Potential and Antagonists, 221
5.4.3 Other Effects, 212 8.4.1 CGRP, 221
5.5 Antagonists, 212 8.4.2 ADM, 221
6 Urotensin-11, 212 9 Tachykinins, 221
6.1 Biosynthesis, Metabolism, 9.1 Biosynthesis, Structure, and Metabolism,
and Structural Features, 212 221
6.2 Receptor, 213 9.2 Receptors, 222
6.3 Biological Actions, 213 9.3 Biological Actions, 222
7 Natriuretic Peptide Family, 214 9.4 Antagonists, 224
7.1 Biosynthesis, Metabolism, 10 Bradykinin, 224
and Structural Features, 214 10.1 Biosynthesis, Structure, and Metabolism,
7.1.1 ANP, 214 225
7.1.2 BNP, 216 10.2 Receptors, 225
7.1.3 CNP, 216 10.3 Biological Actions, 225
7.2 Receptors, 217 10.4 Antagonists, 226
7.3 Biological Actions, 217 11 Vasoactive Intestinal Peptide and Related
7.3.1 ANP, 217 Peptides, 226
7.3.2 BNP, 217 12 Other Peptides, 228
7.3.3 CNP, 218 12.1 Somatostatin, 228
7.4 Therapeutic Potential, 218 12.2 Gastrin-Releasing Peptide, 229
8 Calcitonin Gene-Related Peptide Family, 218 12.3 Neurotensin, 230
8.1 Structure, Biosynthesis, and Metabolism, 218 12.4 Relaxin, 230

1 INTRODUCTION improved techniques in immunohistochem-


istry, molecular biology, chromatography,
The control of vasomotor tone regulates the electrophysiology, and pharmacology have
overall cardiovascular system and its re- led to a wealth of discoveries that have dra-
sponses to physiological and pathophysio- matically reshaped our understanding of the
logical stress. It provides differential blood autonomic nervous system. With the isola-
flow to regional beds at times of normal tion and characterization of neuropeptides
stress, such as exercise, and during condi- such as substance P and vasoactive intesti-
tions of pathological stress, such as conges- nal peptide in the 1970s and 1980s, there
tive heart failure. Changes in vascular tone came revitalization in attempts to identify
permit rapid and sometimes large shifts of nonadrenergic, noncholinergic transmit-
blood volume to meet the needs of the body, ters. Indeed, many newly discovered pep-
for example, during hemorrhage. Although tides were found to be widespread in auto-
the vascular system regulates many of its nomic and sensory neurons innervating the
functions at the site of the blood vessel itself, vasculature, and they had potent vasoactive
other factors that may control vascular tone effects on many blood vessels (1-3). As more
include the autonomic nervous system, cir- neuropeptides were isolated throughout the
culating hormones functioning in an endo- 1980s, the number of peptides found to occur
crine role, and reflex metabolic control. For in peripheral autonomic or sensory neurons
many years, studies on neurohormonal con- continued to increase. The existence of more
trol of the vasculature have been dominated than one transmitter substance in some
by the role of catecholamines and acetylcho- nerves, cotransmission, is now also widely
line. Over the past two decades, new and recognized (4, 5).
The major role of neuropeptides in the vas- were discovered more than 100 years ago by
culature appears to be mainly modulatory, Tigerstedt and Bergmann, who described a
such that they may produce short- or long- pressor system originating in the kidney (10).
term influences on the release or action of
other neurotransmitters, either directly or in- 2.1 Biosynthesis and Metabolism
e is increasing evi-
The synthesis and metabolism of Ang I1 are
s, forming the in-
outlined in Fig. 4.1 (11-13). The process is ini-
nermost layer of blood vessels, play a crucial
tiated when the enzyme renin acts on circulat-
role in the vasodilatory response of the vessel
ing angiotensinogen to release the decapep-
to acetylcholine and to a growing number of
tide angiotensin I (AngI). Ang I, which has no
other substances (6-9).Considerable research
intrinsic activity, is rapidly converted to the
into vascular control mechanisms has led to
octapeptide Ang I1 through the action of an-
the concept of dual regulation of blood vessel
giotensin-converting enzyme (ACE). Ang I1 is
tone, whereby both nerves and the endothe-
subsequently metabolized by aminopepti-
m are involved. There is also considerable
dases to yield [angiotensin,-,I (Ang 111) and
volvement of en-
[angiotensin,-,I (Ang IV). Ang I can also be
tides in a variety of
cleaved to produce [angiotensin,-,I. Ang 111,
gical activities, including pain
Ang IV, and [angiotensin,,] show some bio-
ansmission, behavioral actions, and meta-
logical activity, but their relative importance
olic processes. Vasoactive peptides have been
is still under investigation (13-18).
an important role in many
Various stimuli, such as hyponatremia, hy-
uations, for example, asthma,
povolemia, and hypotension, cause renin to be
ritis, vascular headaches, hypertension,
released within the kidney and into the circu-
iovascular diseases. In this
lation. Renin release is mediated by means of
dence for the differ-
intrarenal vascular and tubular mechanisms,
ous vasoactive peptides,
as well as the sympathetic nervous system.
ir interaction with other systems, the pos-
Ang I1 itself exerts a negative feedback inhibi-
in diseases, and the
tion on renin release and also synthesis (19).
rapeutic use of vasoac-
Extrarenal tissues, such as the brain, heart,
ve peptide agonists and antagonists. Atten-
adrenals, and blood vessels, have been shown
on will be given largely to those peptides
to contain renin or renin-like enzyme or
r vascular effects as va-
mRNA for renin, but at much lower levels
constrictors, including angiotensin, argi-
than those in the kidney (20-22). The kidney
thelins, neuropeptide
is the major source of plasma renin. Renin spe-
asodilators, includ-
cifically hydrolyzes the amide bond between
s, calcitonin gene-re-
the amino acids in positions 10 and 11 of the
ins, bradykinin, and
N-terminus of angiotensinogen to form Ang I.
de. Other endoge-
Angiotensinogen, the only known physiologi-
sess vascular activity,
cal substrate for renin, is an a-2-globulin that
e include somatosta-
is synthesized in the liver and released into
, gastrin-releasing peptide, neurotensin, the circulation. Ang I is converted into the bi-
ologically active octapeptide, Ang 11, by angio-
tensin-converting enzyme (ACE, also called
kininase 11), a zinc-containing enzyme found
ANCIOTENSIN I I
in many tissues. ACE, unlike renin, has a
broad substrate specificity and cleaves several
octapeptide angiotensin I1 (Ang 11)is the
other peptides (e.g., bradykinin, substance P).
ncipal effector of the renin-angiotensin
stem (RAS) and plays an important role in
2.2 Structural Features
e control of blood pressure and in the patho-
nesis of a variety of cardiovascular diseases, Many analogs of Ang I1have been synthesized,
cluding hypertension. Aspects of the RAS and considerable information on the struc-
Endogenous Vasoactive Peptides

Angiotensinogen

NH,- 0Asp Arg Val Tyr Ile His Pro Phe His Leu Val Ile His Ser 0- R

A
renin

Angiotensin I (decapeptide)
1 5 10

A
Angiotensin converting enzyme (ACE)

Angiotensin I I (octapeptide)

A
Arninopeptidase
I
Angiotensin I l l (Heptapeptide)
2 8

I
arninopeptidases carboxypeptidases

Figure 4.1. Formation and degradation of ANG I, ANG 11, and ANG 111. The presumed sites of
action of the specific peptidase enzymes are indicated by arrowheads.

ture-activity relationship (SARI is available 2.3 Receptors


(23,241. As is evident from the activity of Ang
111, most of the essential information resides Ang I1 exerts its wide variety of physiological
in the C-terminal heptapeptide. Phenylala- actions by binding to specific receptors located
nine in position 8 is critical. Its removal from in the plasma membranes of various tissues
any of the angiotensins abolishes agonist ac- (25). In humans, two receptor subtypes have
tivity. The other aromatic residues in posi- been identified, designated AT, and AT,.
tions 4 and 6, the guanido group in position 2,
and the C-terminal carboxyl are involved 2.3.1 AT, Receptors. The AT, receptor is a
mainly in binding to the receptor site. Position member of the seven transmembrane G-pro-
1 is not critical, but replacement of aspartic tein-coupled receptor family. Binding of Ang
acid in position 1 with sarcosine (N-methyl- I1 to the AT, receptor initiates a series of
glycine) enhances binding and slows hydroly- events, including stimulation of phospho-
sis by aminopeptidases. Substitution at either lipase C and subsequent activation of protein
position 4 or position 8 within the Ang I1 se- kinase C and release of intracellular calcium
quence has produced analogs with antagonis- (26, 27). Specific AT, receptors have been
tic properties. identified in multiple tissues, including vascu-
lar smooth muscle, adrenal cortex and me- renal tubules. The effects of Ang I1on the pe-
dulla, kidney, uterus, brain, liver, and heart ripheral autonomic nervous system include
(25,26).The AT, receptor has been cloned (28, sympathetic stimulation, facilitation of adren-
29). Mice lacking the AT, gene (30) show re- ergic transmission, and adrenal medullary re-
duced blood pressure, increased water uptake, lease of noradrenaline. These multiple neu-
and increased urine excretion. Mice overex- roendocrine actions of Ang I1 contribute to
pressing the AT, gene (31, 32) show myocyte arterial vasoconstriction and blood pressure
hyperplasia and lethal heart block. Most of the regulation. In the central nervous system Ang
physiological responses of Ang I1are mediated I1 mediates a direct pressor response by in-
through the AT, receptor (25, 33, 34). In ro- creasing efferent sympathetic activity and
dents, but not humans, two forms of AT, stimulating the release of vasopressin (48,491.
(AT,, and AT,,) have been identified and are Ang I1 may also influence blood pressure by
pharmacologically indistinguishable (35). contributing to both vascular and cardiac hy-
pertrophy (50). In addition, Ang I1has a direct
2.3.2 AT, Receptors. The AT, receptor positive inotropic action on the heart. It also
subtype was identified with radioligand bind- has been linked to cardiac fibrosis (51, 521,
ingstudies (36,371. The AT, receptor is also a oxidative stress (53), and atherosclerosis (54,
member of the seven transmembrane G pro- 55). All of these effects are mediated through
tein-coupled receptor family (38). It is coupled the AT, receptor. The cardiovascular func-
to various tyrosine and serinetthreonine phos- tions produced by the AT, receptor include
phatases and mediates protein dephosphory- suppression of cell proliferation (561, apopto-
lation (39). The AT, and AT, receptors have sis (57, 58), and stimulation of renal NO pro-
only 34% sequence homology (38). The AT, duction (59).
receptor is expressed during fetal develop-
ment but not during normal adult life (40,411, Inhibitors and Antagonists of the
2.5
and may play a role in fetal growth and differ- Renin-Angiotensin System
entiation. The receptor has been identified at
low density in adults in adrenal glands, brain, Angiotensin-converting enzyme (ACE) inhibi-
uterus, kidney, and heart (42, 43). Under tors were the first commercially available
pathological conditions, such as vascular in- blockers of the RAS. More recently, AT, recep-
jury, heart failure, and myocardial infarction, tor antagonists have been marketed and renin
AT, receptors are reexpressed in adults, and inhibitors have been studied clinically. These
may oppose the pathologic effects induced by agents have been used primarily for the treat-
the AT, receptors. AT,-deleted mice have ment of hypertension and congestive heart
n generated and show increased blood failure.
salt retention (44,45).
2.5.1 Renin Inhibitors. The enzyme renin,
an aspartyl endoprotease, is highly specific for
pressure by acting at the hydrolysis of the Leu1'-Val1' bond of an-
ous sites, including vascular smooth mus- giotensinogen, so inhibition of renin produces
d the central and pe- selective blockade of the RAS (60). A number
s (11, 12). It is a very of orally active, nonpeptidic renin inhibitors
particularly of the re- have been described (61) and several have
vasculature, and, therefore, can directly been tested in humans. Ro42-5892 (1) and
ease vascular resistance. The effects of CGP 38560A (2) (Fig. 4.2) have been shown to
g I1 on the kidney include modulation of lower blood pressure in primates (62,63) and
a1blood flow, glomerular filtration rate, tu- humans (63-65). To date, no renin inhibitors
ar sodium and water reabsorption, and in- have reached the market, primarily because of
ition of renin release (46,47). In the adre- rapid elimination in vivo and cost of synthesis.
cortex Ang I1 acts directly on the zona However, recent reports indicate that these
e aldosterone, a hor- issues may be surmountable (66). See Fig. 4.2,
one with potent sodium-retaining effects on structures (3)and (4).
Endogenous Vasoactive Peptides

OMe
(4)

d X
N
I
COOH

Figure 4.2. Chemical structures of Ro42-5892 (11, CGP 38560A (2), compound 10 (31, compound
rac-30 (4),captopril (S), enalapril(6), losartan (71, and valsartan (8).

2.5.2 ACE Inhibitors. The enzyme ACE is a (Fig. 4.2). Since then, many additional ACE
zinc-containing metalloprotease. ACE inhibi- inhibitors have become available (69).
tors block the conversion of Ang I to Ang 11.
The first commercially available ACE inhibi- 2.5.3 AT, Receptor Antagonists. Saralasin,
tors were captopril (67) and enalapril (68) a peptide analog of Ang 11, was the first re-
199

Arginine Vasopressin

Figure 4.3. Schematic illustration of bovine prepro-vasopressin structure and the primary struc-
ture of arginine vasopressin.

saralasin is not used clinically. In 1982 AVP is synthesized as a larger prohormone in


wa and coworkers discovered some the hypothalamus and is carried by axonal
transport to the posterior pituitary, where it is
c Ang I1 antagonists (71). These com- stored for later release. The vasopressin gene
unds served as leads to develop losartan is divided into three exons by two short inter-

onist, and valsartan (73) (Fig. 4.2). Since leads to a long pre-mRNA, which is then

ared (73-75). These agents are as effective translatable mRNA of about 750-800 bases.
ACE inhibitors, but avoid some of their The mRNA is then translated to give the ini-
hanism-related side effects, such as cough, tial precursor or "preprohormone." This pre-
h, and angioedema. prohormone consists of the following peptide
sequences beginning at the N-terminus: a sig-
nal peptide required for further processing;
3 ARGlNlNE VASOPRESSIN vasopressin; a tripeptide-bridging structure;
neurophysin; and a single arginine residue
ginine vasopressin (AVP) was originally joining the neurophysin sequence to a 39
dentified more than 100 years ago as a pitu- amino acid residue that will become a glyco-
gland extract that was shown to be a po- peptide (Fig. 4.3) (81). This precursor is pack-

pressin as a result of the development of active nonapeptide vasopressin. Vasopressin


Endogenous Vasoactive Peptides

which was originally thought to result from an yield terlipressin, glypressin, and ornipressin,
inability to synthesize vasopressin, more spe- novel therapeutics to treat acute bleeding ep-
cifically occurs because of gene mutations of isodes, variceal bleeding associated with por-
the signal peptide and in neurophysin, thus tal hypertension, and to act as a local vasocon-
indicating the importance of these peptide strictor during surgeqy (98-100). Recently,
fragments in the appropriate expression and the first known vasopressin analogs to possess
secretion of a physiologically active vasopres- selective vasodepressorjhypotensive activity
sin (83). Recently, it has become apparent that have been described (101). These compounds
vasopressin also may act in a paracrine or au- may prove useful in treating hypertension.
tocrine manner. Vasopressin and its mRNA
have been identified in various peripheral tis- 3.2 Receptors
sues, including the adrenal gland, testis,
Three distinct vasopressin receptors have been
ovary, thyroid gland, spleen, pancreas, heart,
cloned, sequenced, and expressed and are desig-
and intestine (84, 85).
nated V,,, Vlbr and V, (102-104). Vasopressin
Once released, AVP is subject to proteolysis
receptors belong to the seven transmembrane G
catalyzed by peptidases. In the periphery,
protein-coupled receptor family (105). Binding
AVP is converted into inactive metabolites by
of vasopressin to its receptor results in receptor
peptidases of kidney and liver attacking its C-
internalization and subsequent desensitization.
terminal portion or by an N-terminal cleaving
Receptor recycling occurs in the case of the V,
enzyme in plasma (86,871. In the brain N- and
receptor but not with the V, receptor (105). The
C-terminal cleaving activities appear to occur
vasopressin V, receptor is expressed mainly in
in different cellular compartments. C-termi-
the medullary renal tubules, coupled to a chol-
nal cleavage of AVP (Arg-Gly-NH,) was seen
era toxin-sensitive G protein Gs and activates
in soluble fractions of brain tissue (88), and
CAMP (106). Activation of the V, receptor in-
N-terminal cleavage (release of Tyr2) predom-
duces insertion of water channels (aquaporins)
inated in membrane preparations (89). Simi-
into the lurninal surface of renal collecting tu-
lar to AVP's proteolytic susceptibility in the
bules with a resultant increase in water reab-
periphery, N-terminal cleavage in the brain is
sorption and is responsible for the well-known
mediated by aminopeptidase activity. Trypsin
antidiuretic effects. The V,, receptor, known as
and carboxamido-peptidase are candidates for
the vascular receptor, is expressed on vascular
cleavage of the C-terminal Arg-Gly-NH, bond
smooth muscle, spleen, adipocytes, brain, testis,
(90, 91); the postproline cleaving enzyme can
liver, reproductive organs, and adrenal cortex
split the Pro7-Arg bond (92). In vivo studies
(106). Vasopressin binding to the V,, receptor
have shown that the putative vasopressin
activates phospholipases &,C, and D, generates
fragments are relatively most abundant in ex-
inositol phosphates and diacylglycerol, stimu-
trahypothalamic brain regions and [vasopres-
lates protein kinase C, and mobilizes intracellu-
sin,-,] and [vasopressin,,l amino acid frag-
lar calcium and Nat-H+ exchanger (106). The
ments of vasopressin can account for up to
classical vasopressor actions of vasopressin are
30% of the AVP content (93). The products of
mediated by the V,, receptor. The pituatary V,,
AVP processing have potent and selective cen- ~ ~

receptor, also known as the V, receptor, poten-


tral activities (94, 95).
tiates the release of ACTH through activation of
The structure of AVP can be modified in a
phospholipase C and protein kinase C (106). Fi-
variety of ways to give analogs exhibiting in-
nally, evidence has recently been put forward
creased antidiuretic potency, different antidi-
describing a novel dual angiotensin IUvasopres-
uretic/pressor selectivity, and increased dura-
sin receptor possessing equal affinity for vaso-
tion of action. Examples of analogs exhibiting
pressin and angiotensin 11and is expressed on
these different characteristics are reviewed
medullary tubules (107).
elsewhere (96). One such modified peptide,
desmopressin (dDAVP), has become the cor-
3.3 Biological Actions
nerstone for the treatment of bleeding disor-
ders such as hemophilia A and von Willebrand It is generally recognized that AVP plays arole
disease (97). Other structural modifications in normal blood pressure regulation as well as
in the pathogenesis of certain forms of exper- The role of AVP in long-term regulation of
imental hypertension, especially low renin blood pressure and in the pathogenesis of hy-
models such as the deoxycorticosterone ace- pertension is still controversial. Although
tate-salt model (108). On a molar basis AVP is plasma levels of AVP have been reported to be
a more powerful vasoconstrictor, through its increased in many models of hypertension, it
action on endothelial vascular smooth muscle has not been possible to demonstrate that
cells, than angiotensin I1 or catecholamines chronic administration of AVP, at levels simi-
(109, 110). Pharmacological doses are re- lar to those seen in hypertension, can sustain
quired to achieve a significant increase in ar- an elevated arterial pressure (121). Further-
more, it has not been possible to consistently
terial blood pressure. It appears that the rise
demonstrate that administration of pressor
in blood pressure in response to physiological
antagonists of AVP causes more than a tran-
concentrations of AVP is blunted by an in- sient fall in blood pressure in any model of
crease in total peripheral resistance and a fall experimental hypertension (121). AVP acting
in cardiac output (111-113). Baroreflex mech- on the V, receptor and/or alterations in med-
anisms appear to contribute to lowering of car- ullary blood flow could account for an escape
diac output in conscious dogs (112). AVP could from chronic V,, blockade (115,122).
also have negative inotropic actions that con-
tribute to a fall in cardiac output (114). Acti- 3.4 Antagonists
vation of the V, receptor effectively opposes There have been substantial improvements in
V,,mediated vasoconstriction and could ac- the design of specific antagonists for AVP act-
count, in part, for the lack of sensitivity of ing throughv, and V, receptors. Early success
blood pressure in response to increasing doses with the design of subtype-specific antagonists
of AVP. Specifically, the integrated control of was achieved through the synthesis of peptidic
the renal medullary circulation appears to be analogs of AVP (101, 123). The design and
intimately involved in the overall systemic hy- synthesis of nonpeptidic AVP antagonists, ei-
pertensive response to AVP (115). There are ther nonselective or subtype selective, have
regional differences in blood flow responses to been only a recent innovation and initially
exogenous AVP. AVP infusion in dogs pro- were hampered by the high species differences
duced a decrease in blood flow to the skin, skel- observed in ligand selectivity (124). The first
etal muscle, pancreas, thyroid gland, and fat, reported orally active, nonpeptidic antagonist
and to a lesser extent to myocardium, gastro- was the V,, antagonist, OPC-21268 (125).
intestinal tract, and brain. Some organs did Subsequent to this disclosure, additional non-
not show any fall in blood flow at all, particu- peptidic antagonists have been reported with
larly kidney and liver (116). Long-term expo- V,,, V,, and dual V,,/V, antagonistic activity
sure to vasopressin has growth-promoting ef- (Fig. 4.4) (126, 127). Notably, SR 121463A, a
fects on the vasculature as well (117). high affmity antagonist for the V, receptor (Ki
In addition to its peripheral vasoconstric- = 0.26 + 0.04 nM), is highly selective and
tor and renal effects, AVP has been shown to orally active (127). To date, none of these non-
produce cardiovascular effects that are medi- peptidic antagonists has reached the market,
ated by the central nervous system (118).Va- although numerous agents are in advanced
sopressin-containing cells and AVP receptors stages of clinical testing. These antagonists al-
are located in areas known to be involved in ready have been shown to be effective in ani-
cardiovascular regulation (119). Experimen- mal models of heart failure and hyponatremic
tal evidence thus supports a central role for states, and are powerful aquaretic agents
AVP in hypertension through its ability to in- (128-131).
crease sympathetic outflow and suggests an
involvement in the circadian rhythm of blood 4 ENDOTHELIN PEPTIDE FAMILY
pressure (119). Furthermore, a significant in-
4.1 Structural Features
teraction between vasopressin and angioten-
sin I1 has been demonstrated in the central Endothelin (ET), identified several years ago
control of blood pressure (120). as a potent vasoconstrictor, constitutes a fam-
Endogenous Vasoactive Peptides

ily of structurally very closely related peptides


(132-133), originally isolated from rodent
sources (ET-3) as well as human and porcine
sources (ET-1). To date, four distinct mem-
bers of the family, endothelin-1 (ET-I), endo-
thelin-2 (ET-2), endothelin-3 (ET-31, and va-
soactive intestinal constrictor (VIC), have
been identified in mammalian systems (Fig.
4.5) (134). Endothelins have a common struc-
ture of 21 amino acids with two cysteine-cys-
teine disulfide bonds between positions 1-15
and 3-11 and a hydrophobic C-terminus (res-
idues 16-21) (Fig. 4.5). These peptides share
sequence homology with sarafotoxins (Fig.
4.5) isolated from the venom of the burrowing
asp, Atractaspis engaddensis (135,1361, which
also shares most of their biological character-
istics. The main differences between the endo-
thelin isopeptides is in their amino N-terminal
segments. The biochemistry and biological ac-
tivity of the ETs can be found in several recent
reviews (137-140).

4.2 Biosynthesis and Metabolism


Endothelin is encoded by a well-characterized
gene located on human chromosome 6 (141).
In endothelial cells, endothelin is synthesized
as a 203 amino acid precursor protein called
prepro-ET-1, which is cleaved by a dibasic
Figure 4.4. Chemical structures of OPC-21268 pair-specific endopeptidase to form first pro-
and SR 121463. ET-1 or "big ET-1" a 38 (human) or 39 (por-

Endothelin-1 Cys-Ser-Cys-Ser-Ser-Leu-Met-Asp-Lys-Glu-Cys-Val-Tyr-Phe-Cys-His-Leu-Asp-Ile-lle-Trp
(porcinelhumanlratlcanine)

Endothelin-2 Cys-Ser-Cys-Ser-Ser-Trp-Leu-Asp-Lys-Glu-Cys-Val-Tyr-Phe-Cys-His-Leu-Asp-Ile-Ile-Trp
(human)

Vasoactive Intestinal Constrictor (VIC)


(mouse)
Cys-Ser-Cys-Asp-Ser-Trp-Leu-Asp-Lys-Glu-Cys-Val-Tyr-Phe-Cys-His-Leu-Asp-lle-lle-Trp

Sarafotoxin-6c (representativeof the sarafotoxin family of peptides; S6-a,b,c,d)

Figure 4.5. Amino acid sequences of endothelin-1, endothelin-2, endothelin-3, vasoactive intestinal
constrictor, and sarafotoxin8c, one of the sarafotoxin family of peptides. The endothelin peptides all
consist of 21 amino acids, with considerable homology between other members of this family of
peptides, in which each peptide contains two essential intramolecular disulfide bonds. Amino acids
differing from endothelin-1are designated in bold.
4 Endothelinyeptide Family

cine) amino acid peptide (132) (Fig. 4.6). Big and ET-3 were found to be rapidly removed
ET-1 is then converted to active ET-1 (21 from the circulation with a half-life of less
amino acid residues) by a neutral metallopro- than 2 min. No ET catabolites were found in
tease termed endothelin-converting enzyme the blood, and the radioactivity was localized
(ECE-1) (142). ECE has been cloned and sev- primarily in lungs, kidneys, and liver, suggest-
eral ECE-1 isoforms have been identified, des- ing that these organs are the principal sites of
ignated ECE-la, ECE-lb, and ECE-lc, and ap- metabolism of ET peptides (159, 160). In vitro
parently a single gene encodes the various experiments have demonstrated that ET pep-
isoforms (135-138). Whereas ECE-la is pri- tides can be degraded by a purified neutral
marily located on the plasma membrane, endopeptidase 24.11. Circulating ET-1 can
ECE-lb is within the intracellular compart- also be cleared by receptor-mediated uptake
ment and ECE-lc is expressed in the Golgi (161).
(146). ECE-1 is a highly glycosylated protein,
is contained within the zinc metalloendopep- 4.3 Receptors
tidase family that includes NEP 24.11, and
may also be involved in the degradation of bra- Endothelin's actions are mediated by at least
dykinin (147). two subtypes of ET receptor. Two distinct ETA
The lack of secretory storage granules in and ETB receptors have been cloned and ex-
endothelial cells suggests that ET-1 is made hibit 60% homology (162, 163). The predicted
upon demand (148). In most cases, induction structure of both receptors is similar to seven
of ET-1 secretion above basal levels requires transmembrane G-protein-coupled receptors.
2-5 h and most results from enhanced prepro- For the ETA receptor, the rank order of po-
ET-1 gene expression (149, 150). Endothelin tency is ET-l > ET-2 %- ET-3 and the affinity
mRNA in cultured endothelial cells is induced of binding to ET-1 is about 100 times greater
by several vasoactive substances including than that of ET-3 and subserves vasoconstric-
thrombin, angiotensin 11, vasopressin, and tion (164, 165). ETB receptors show a similar
epinephrine. The interaction between the en- affinity for all three ETs and for the sarafotox-
dothelin and renin-angiotensin systems ap- ins and subserve vasodilatation as well as va-
pears to be particularly crucial in the regula- soconstriction (164, 166,167). The distinction
tion of cardio-renal function (151). In between ETAand ETBreceptors has been con-
addition, ET-1 release is induced by hemody- firmed by the recent development of selective
namic stimuli such as shear stress, as well as agonists and antagonists. Radioligand binding
by ET-1 itself (for reviews, see Refs. 140, 152- studies and in situ hybridization studies with
154).In human coronary arteries, a dual-pro- cDNA receptor probes have demonstrated
cessing pathway has been postulated. In addi- that ET receptors are widely distributed, in
tion to the well-characterized constitutive keeping with the multiple actions of these pep-
pathway, a regulated secretory pathway may tides (132,168,169). Recently, the ETBrecep-
exist where endothelin is contained within tor has been shown to act as a clearance recep-
Weibel-Palade bodies (155). Various factors tor for removing endothelin from the
activate phospholipase C, which results in the circulation (170). Subsequent to endothlein
activation of protein kinase C through the for- receptor stimulation, a complex cascade of sec-
mation of 1,2-diacylglycerol,and in mobiliza- ond-messenger pathways becomes activated
tion of calcium ions through the production of and contributes to the diversity of endothelin
inositol-1,4,5-trisphosphate.The production actions (171).
of ET-1 is also regulated by a negative path-
way involving a guanosine 3',5'-cyclic mono- 4.4 Biological Actions
phosphate (cGMP) mechanism. It was found
that agents such as atrial natriuretic peptide, 4.4.1 Cardiovascular. After the discovery
nitric oxide, and nitrovasodilators, which in- of ET-1 it became apparent that the endothe-
crease cGMP levels, reduce the secretion of lins were the most potent vasoconstrictor sub-
ET-1 (156-158). Following intravenous ad- stances yet identified in vascular preparations
ministration to animals, radiolabeled ET-1 from humans and experimental animals (132).
Endogenous Vasoactive Peptides
4 Endothelin Peptide Family

The vascular effects of endothelin are medi- 4.4.3 Renal Actions. Endothelins can cause
ated by ETA and ET, receptors located pri- a profound increase in renal vascular resis-
marily on vascular smooth muscle and endo- tance, decrease in renal blood flow, and de-
thelial cells, respectively (172-174). After crease in glomerular filtration in association
intravenous administration, ET-1 elicits a with a decrease in sodium excretion and an
characteristic initial, short-lived depressor re- increase in plasma renin activity (179, NO),
sponse followed by a secondary and prolonged effects that resemble those seen in acute renal
pressor response (132, 174). Prostacyclin and failure. Infusion of antiendothelin antibodies
nitric oxide are predominantly responsible for has been found to exert a renal-protective ef-
the vasodilation, whereas the vasocontriction fect in ischemia (185, 186). The renal vascula-
following ETA activation is mediated by in- ture is several times more sensitive to the con-
strictor effects of ET than are other vascular
creases in intracellular calcium flux (175,
regions (187). Interestingly, in addition to
176). In addition to the well-characterized ef-
ETA-mediated vasoconstriction, activation of
fects on vascular tone, additional vascular ef- the ETBreceptor also has been shown to elicit
fects of endothelin include thrombogenesis, renal vasoconstriction (188). A more detailed
neutrophil adhesion, mitogenic effects, and al- review of the renal actions of endothelin has
terations of vascular permeability (177, 178). been published (189).
4.4.2 Endocrine Actions. Endothelins ap- 4.5 Pathological Roles of Endothelin
pear in low concentrations in the blood circu- The powerful vasoconstrictive and mitogenic
lation and act in a paracrine manner. Locally properties of endothelins suggest that they
synthesized endothelins are secreted ablumi- may have an important role in the develop-
nally into the interstitial space, where they ment of numerous cardiovascular diseases.
may act on nearby cells. For example, the va- Plasma endothelin concentrations are ele-
soconstrictor actions of ET-1 in vivo are op- vated in many pathological conditions includ-
posed by the concomitant release of circulat- ing cardiac hypertrophy and failure, myocar-
ing atrial natriuretic peptide (ANP) (179, dial infarction, and in various acute and
1801, and locally produced prostacyclin and chronic renal pathologies (171, 190-192). A
endothelium-derived relaxing factor (181), all growing body of evidence supports a contribu-
of which are potent vasodilators and may play tory role for endothelin in types I and I1 dia-
a role in modulating the pressor response to betes mellitus and its associated vascular com-
ETs. Endothelin gene expression is regulated plications (193,194). The endothelin system is
by several vasoactive agents such as thrombin activated in septic and cardiogenic shock
and adrenaline, which are known to stimulate (195). The plasma ET-1 level associated with
phosphoinositide hydrolysis in endothelial essential hypertension remains controversial.
cells (132). Angiotensin, vasopressin, calcium Higher plasma ET-1 levels have been mea-
ionophore A23187, endotoxin, and hypoxia are sured in patients with essential hypertension
also known to stimulate gene expression and than in normal individuals (196). However,
release of endothelin (181). ET, in turn, can endothelin acts primarily in an autocrine and
increase plasma levels of various vasoactive paracrine manner where the levels deter-
hormones including aldosterone and cat- mined in plasma may represent only spillover
echolamines (179, 180). Several studies have from tissues and, consequently, may serve as
reported the inhibitory effect of ET on the re- an unreliable diagnostic marker. Tissue levels
lease of renin in vitro (182, 183). In vivo stud- would appear to provide a more accurate as-
ies, however, have shown a significant in- sessment. Increased endothelin production
crease in plasma renin activity after has been shown in endothelium of small arter-
intravenous infusions of pharmacological ies from hypertensive patients (197).Circulat-
doses of ET (179, 180). There appears to be a ing and tissue levels of endothelin are elevated
complex interaction between the renin-angio- in patients with atherosclerosis and correlate
tensin and endothelin systems under normal with the severity of disease (198). Further-
and pathological conditions (184). more, endothelial cells, macrophages, and
206 Endogenous Vasoactive Peptides

Table 4.1 Endothelin Receptor Antagonists


Rank Order of
Antagonist Reference Receptor Affinity for ETs References
BQ-123 ETA
BQ-153 ETA
BQ-485 ETA
BQ-610 ETA
A-127722 ETA
A-216546 ETA
BE-18257A ETA
BE-18257B ETA
BMS 182874 ETA
EMD 122946 ETA
FR 139317 ETA
50-235 ETA
TAK-044 ETA
TTA-386 ETA
LU135252 Darusentan ETA
ZD 1611 ETA
TBC-11251 Sitaxsentan ETA
RPR 111844 ETA
IRL 1038 ETB
BQ-788 ETB
RES 701-1 ETB
A192621 ETB
A308165 ETB
IRL 3630 Nonspecific ETA/ETB
L-749,329 Nonspecific ETA/ETB
PD 145065 Nonspecific ETA/ETB
PD 142893 Nonspecific ETA/ETB
Ro-46-2005 Nonspecific ETA/ETB
Ro-47-0203 Bosentan Nonspecific ETALET,
Ro-61-0612 Tezosentan Nonspecific ETA/ET,
SB 209670 Nonspecific ETA/ET,
SB 217242 Enrasentan Nonspecific ETA/ETB

smooth muscle cells, all of which are cellular indirectly through stimulation of vascular en-
components of atherosclerotic lesions, can ex- dothelial growth factor (204).
press endothelin (199, 200). A primary role
4.6 Antagonists and Inhibitors
has been suggested for endothelin in the
pathogenesis of pulmonary hypertension. Considering the potential importance of ETs
Plasma levels of endothelin are elevated in pri- in various physiologic and pathophysiologic
mary pulmonary hypertension (201)and ET-1 conditions, a major effort has been made to
expression is increased in small muscular pul- develop specific receptor antagonists to mod-
monary arteries from patients with pulmo- ify the actions of ET. More recently, the char-
nary hypertension (190,202).The observation acterization of the enzymes responsible for
that endothelin levels are increased in various generating ET has led to the design of specific
forms of cancer and the demonstration of a enzyme inhibitors.
potent mitogenic effect of endothelin has led I
to speculation of ET-1 involvement in angio- 4.6.1 Antagonists. Various compounds have
genesis (203). In addition to direct effects on been reported in recent years as ETA- or
cell growth, endothelin may alter mitogenesis ETB-selectiveand as mixed ETNBreceptor an-
4 Endothelin Peptide Family

ET receptor antagonists

Bu-t
0
OMe HO
Bosentan A-216546

ECE inhibitors

H O \ p ~ N / N
HO' 11 H 0
0

Compound 47 SM-19712
Figure 4.7. Structures of key ET receptor antagonists and ECE inhibitors.

tagonists (Table 4.1 and Fig. 4.7). Several evaluated clinically for the treatment of hy-
comprehensive reviews that define the struc- pertension, congestive heart failure, and sub-
turd requirements necessary for receptor arachnoid hemorrhage (208). Evidence for a
subtype specificity and their overall activity role of endothelin in various cardio-renal dis-
profiles have been published (205-207). The eases, including hypertension, heart failure,
most clinically advanced compound, bosentan, diabetes, and renal failure, has been strength-
is a mixed antagonist and is being developed ened by results from extensive preclinical and
for heart failure and pulmonary hypertension clinical testing of endothelin antagonists
(208).Bosentan competitively antagonizes the (209-211).
binding of radiolabeled endothelin to ETAand There also are other classes of drugs that
ET, receptors with K,values of 4.7 and 95 nM, interfere with endothelin release or modify its
respectively. Bosentan is well absorbed (50%) action, such as angiotensin-converting en-
and peak plasma levels are achieved in hu- zyme inhibitors, calcium-channel blockers,
mans between 2 and 3 h after oral administra- and angiotensin I1 receptor antagonists (140).
tion (208). Bosentan reduced systemic vascu- Endothelin antagonists have great potential
resistance in hypertensive and heart for use in the treatment of patients with car-
ure patients and had a significant benefit in diovascular pathology, including systemic hy-
e pulmonary vascular bed. Bosentan has re- pertension, myocardial ischemia, restenosis
ntly been approved for the treatment of pul- after angioplasty, and congestive heart fail-
monary hypertension. Bosentan is also being ure. Results of various ongoing clinical trials
Endogenous Vasoactive Peptides

are expected to further clarify our under- stored with different transmitters such as nor-
standing of the role of endothelin in disease adrenaline (254). Two structurally related
processes. peptides in mammals, peptide YY (PYY) and
pancreatic polypeptide (PP) (Fig. 4.8), are also
4.6.2 Endothelin-ConvertingEnzyme Inhibi- members of the PP-family. PYY was originally
tors. Although a specific enzyme processing purified from pig intestine (255). PYY is pro-
step was hypothesized at the time of the initial duced by endocrine cells of the intestine and
description of endothelin (132), the isolation the pancreas of all vertebrates investigated
and characterization of ECE occurred some (see Ref. 256 and references therein). Pancre-
years later. ECE was described as a mem- atic polypeptide (PP) was originally discov-
brane-associated, glycosylated metallopro- ered in chicken pancreas (257) and has subse-
tease of greater than 100 kDa (240,241). ECE quently been isolated or cloned in several
has been purified from bovine endothelial tetrapods. PP appears to be expressed exclu-
cells, denoted as ECE-1, and is expressed in- sively in endocrine cells of the pancreas (258).
tra- and extracellularly (242). ECE is highly Each member of the PP-family consists of two
related to another metalloprotease, neutral antiparallel helices: a polyproline-like helix
endopeptidase 24.11 (NEP) (243). Conse- (residues 1-8) that through hydrophobic resi-
quently, the development of selective endothe- dues is connected with an amphiphilic a-helix
lin-converting enzyme (ECE) inhibitors has (residues 15-30), creating a hairpinlike loop.
proven to be a significant challenge. The first The three-dimensional structures are sup-
enzyme inhibitors to be produced were pep- ported by X-ray crystallographic data of avian
tidic and were isolated from fermentation PP (259) as well as NMR, circular dichroism,
broths, FR901533 (2441, B-90063 (2451, and and molecular modeling (260, 261).
TMC-66 (246). Initial efforts with nonpeptidic NPY and PYY are only about 50% homolo-
ECE inhibitors demonstrated nanomolar po- gous with PP in the primary structure; never-
tency; however, significant inhibitory activity theless, all three peptides retain the key amino
against NEP 24.11 was retained (247). More acids necessary to hold the distinct tertiary
recently, a nonpeptidic ECE inhibitor, structure (260). The structure of the PP-fam-
SM-19712, has been described (248) with high ily is stable in aqueous solution (260,262).The
potency against ECE (IC,, = 42 nM)and high folded structure results in close association of
selectivity (inactive against ACE and NEP at the carboxy- and amino-terminal parts of the
100 m. See Figure 4.7. Early results from in molecule, which is important for receptor rec-
vitro and in vivo test systems confirm the ex- ognition (263). The amino acid composition of
pected biological efficacy of ECE inhibition the individual peptides is well conserved, both
(249-25 1). during evolution and among species (264,
265). In the case of NPY, the only identified
substitution between species is that the me-
5 NEUROPEPTIDE Y
thionine at position 17 in human, rats, guinea
pigs, and rabbits is replaced by leucine in pigs
5.1 Structural Features
(265). Shortly after their discovery, it was ob-
Neuropeptide Y (NPY), a 36 amino acid pep- served that the entire NPY and PYY molecules
tide isolated in 1982 (2521,is a potent vasocon- were necessary to evoke vasoconstriction,
strictor as well as having significant effects on whereas N-terminally truncated forms of NPY
feeding, memory, intestinal secretions, and and PYY were quite effective in suppressing
adrenocortical function. It is a member of the sympathetic nerve activity.
pancreatic polypeptide family (PP-family). It
5.2 Biosynthesis and Metabolism
derives its name from having amino- and car-
boxy-terminal tyrosine residues (single letter The DNA that codes for NPY is located on
code Y) (Fig. 4.8). Neuropeptide Y is widely chromosome 7 in humans (266). The corre-
distributed in the central and peripheral ner- sponding mRNA contains a single open read-
vous system of numerous mammalian species ing frame for a prepro-NPY sequence consist-
including humans (253) and the peptide is co- ing of 97 amino acids (267). Cleavage of the
NH2 NH2
I I
Neuropeptide Y Peptide W (PYY)

Pancreatic polypeptide (PP)

Figure 4.8. Amino acid sequences of human neuropeptide Y, peptide YY, and pancreatic polypep-
tide. Amino acids differing from neuropeptide Y are indicated by fdled circles.

signal peptide yields pro-NPY consisting of a tion to a N-terminal truncated NPY3-,, by


69 amino acid sequence. This prohormone is dipeptidyl peptidase IV.This cleavage of NPY
further processed by a convertase, which splits and PYY results in inactivation of these pep-
off the 30 amino acid sequence containing the tides with respect to biological effects medi-
CPON (C-flankingpeptide of NPY). Two addi- ated by the Y, receptor subtype, but not by Y,
ional enzymatic modificationslead to the ma- receptor subtype (269).
ture NPY, which is C-terminally amidated
(Fig. 4.9).
5.3 Receptors
Like other peptide hormones, NPY is syn-
thesized in the endoplasmic
. reticulum of the NPY elicits its physiological effects by activat-
neuronal Golgi apparatus. NPY migrates by ing at least six receptor subtypes, Y,-Y, and y6
way of specific vesicles along the axon and is (270). These receptors belong to the family of
.es released into the synaptic cleft as a result of seven transmembrane G-protein-coupled re-
In, nerve impulses. Peripherally, it is released ceptors and their activation leads to inhibition
?Y from predominantly postganglionic, sympa- of adenylate cyclase (271). Receptors Y,, Y,,
ng thetic neurons. NPY is localized in larger ves- Y,, Y,, and y6 have been cloned (270).
ides; high frequency stimulation leads to the
migration of these vesicles within the presyn- 5.3.1 Y, Receptors. The NPY Y, receptor
aptic membranes and the release of NPY is located in distinct regions of the brain and in
on :(268). This release is regulated by presynaptic vascular smooth muscle cells of most blood
re- 1 NPY autoreceptors, which can also inhibit the vessels. [Pro3,] NPY has high affinity for Y,,
ad- i release of noradrenaline, and by presynaptic whereas P P and N-terminally truncated NPY
ist- [ a,-adrenergic receptors. Released NPY and derivatives bind with low affinity (270-274).
the j PYY may also be exposed to further degrada- Y, elicits pressor and contractile effects on
Endogenous Vasoactive Peptide!

1 30 65 68 97 diates presynaptic actions involving the sup


pression of neurotransmitter release (287).
Signal peptide N PY CPON
5.3.3 Y, Receptor. The Y3 receptor recog
nizes NPY but not PYY (288). Central admin.
I Signal peptidase istration of NPY and related peptides into thc
nucleus of the tractus solitarius of rat brair
evoked changes in cardiovascular responseE
associated with activation of Y3 receptori
(289,290). Y3 receptors have been identified in
the bovine adrenal medulla (291), in rat car
diac membranes (292), heart, adrenal gland
brain stem, and hippocampus (269,271,272).

5.3.4 Y, Receptor. The Y, receptor has a


high affinity for PP and low affinity for NPY!
and is probably not an NPY receptor (271,

30
1Carboxypeptidase 272).

5.3.5 Y, Receptor. The Y5 receptor has a


F~ - ElY-OH
high aMinity for NPY, PP, and N-terminally
truncated forms of NPY (269, 270, 293). The
Y, receptor is localized in the brain as well m

30
Jalpha amidating enzyme

65
in testis and pancreas (293,294). With Y,, the
Y5 receptor appears to mediate NPY effects on
food intake (277,293,295).
- NH2 5.3.6 y6 Receptor. This receptor is written
in lowercase to indicate that it is a nonfunc-
Figure 4.9. Biosynthesis of NPY. CPON, C-flank- tional pseudogene in rats and primates be-
ing peptide of NPY. cause of a single base deletion in the sixth
transmembrane region leading to a truncated
receptor (296,297).
vascular smooth muscle (275,276), as well as
mediating (with the Y5 receptor) NPY effects 5.4 Biological Actions
on feeding (277).
5.4.1 Cardiovascular. NPY can regulate
5.3.2 Y, Receptor. The Y, receptor is pre- cardiovascular function through the central
dominantly localized on the presynaptic mem- nervous system by a specific action on various
branes of postganglionic, sympathetically in- nuclei (298). In the periphery NPY can modu-
nervated neurons of the peripheral nervous late cardiovascular function by acting on the
system (278,279). Y, receptors have also been vasculature, heart, and kidney. In general,
identified in the proximal tubule of kidney NPY can act at a pre- or postsynaptic level.
(2801, in parasympathetic neurons (2811, and Presynaptic NPY receptors exist in the vascu-
in red blood cells (282). Centrally, Y, receptors lature, heart, and kidney, where they mediate
seem to be the dominant NPY receptor, which inhibition of noradrenaline release as well as
are particularly numerous in the hippocam- their own release from sympathetic nerve end-
pus (283,284). The Y, receptor has high affm- ings (299, 300). Presynaptic NPY receptors
ity for PYY and N-terminally truncated forms may also exist on parasympathetic nerve end-
of NPY (285). As for the Y, receptor, C-termi- ings (e.g., in the heart), where they inhibit the
nal amidation of NPY is essential for activity release of acetylcholine (301, 302). The
(286). Among its actions, the Y, receptor me- postsynaptic effects of NPY at the neurovas-
ar junction can be further divided into di- First, NPY can cause powerful coronary vaso-
and indirect (i.e., potentiating effects) constriction. Second, NPY may act presynap-
03). Thus, NPY can cause direct vasocon- tically to inhibit the release of cardiac auto-
riction of some vessels, most notably the cor- nomic neurotransmitters. Finally, NPY may
mesenteric, and renal vascu- act directly on cardiac muscle to alter contrac-
beds, but can also potentiate the tion, chronotropy, and electrical conduction
effects of agents such as nor- acutely, and to affect protein synthesis and
enaline, angiotensin, or serotonin in some hypertrophy chronically (312). It is well docu-
ssels, including those in which NPY does not mented that systemic administration of NPY
se direct vasoconstriction. Direct vasocon- reduces cardiac output, and all of the above
of NPY have been docu- cardiac and extracardiac effects of NPY may
mented in isolated vessel preparations, iso- potentially participate in this reduction (304).
lated tissues as well as in uivo in conscious, NPY binding at the Y, receptor has been im-
esthetized, and pithed animals, and in situ plicated in blood pressure control (3131, and
the human forearm (289, 304-306). Direct appears to exert its hypertensive effects under
effects of NPY have been conditions of high sympathetic nervous sys-
s as well as veins, although tem activity, particularly in response to stress
may be a more effective vasoconstrictor (314). Stress, which is generally thought to
arteries than of veins (307). The direct va- contribute to the development of primary hy-
onstricting effects of NPY are predomi- pertension, raises the plasma levels of NPY
by Y, receptors and fre- and noradrenaline in humans (315). Sympa-
the influx of extracellular thetic nerve activity is often increased in hy-
cium through voltage-operated channels. pertensive patients and plasma levels of NPY
view of its powerful vasopressor effect in and noradrenaline are elevated compared
vo, NPY is a surprisingly poor vasoconstric- with those of normotensive controls (316).
ge blood vessels usually re- These observations, suggesting differences in
PY (3081, but it is possible central and peripheral NPY system between
ness to NPY may increase normotensive and hypertensive individuals,
sel diameter (309). Alterna- indicate that NPY may play an important role
ely, a vasoconstrictor tone may be required in primary hypertension.
n of a-adrenoceptors) to en-
able isolated blood vessels to respond to NPY 5.4.2 Renal. Modulation of renal function
(289, 310); this is compatible with the obser- by NPY can also occur at multiple levels,
vation of reciprocal facilitation of NPY and which include indirect effects through sys-
noradrenaline (310). The effectiveness of the temic hemodynamics, the central nervous sys-
two agents may be enhanced at the sympa- tem, or modulation of release of hormones af-
c neuroeffector junction. NPY is also ca- fecting renal function, and direct effects on
of inhibiting the vasorelaxing effects of renal blood flow and tubular function (317).
s such as acetylcholine (289,311). Thus, NPY can reduce renin release and plasma re-
may be involved in the regulation of vas- nin activity (318-322). Systemic NPY admin-
ral autonomic neuroeffector istration has been reported to elevate plasma
ctions where the peptide seems to act in levels of atrial natriuretic peptide (323). NPY
ncert with other transmitters. reduces renal blood flow in a variety of species
The cardiac effects of NPY are complex and experimental settings (321, 324, 3251, an
! (304). NPY can affect cardiac function indi- effect that, at least in rats, occurs by way of a
b rectly through central effects, by alterations Y,-like receptor (322,326,327). In contrast to
in afterload secondary to its direct and poten- the antidiuretic effects of other renal vasocon-
1 tiating vasoconstricting effects, and by alter- stricting neurotransmitters and hormones,
ations in preload, which may be enhanced be- NPY enhances diuresis and natriuresis in uivo
? cause of venous vasoconstriction or reduced in anesthetized rats (325, 328).
? secondary to histamine release. At least three Together, these data demonstrate that
possible sites of action exist within the heart. NPY can modulate cardiovascular function in
Endogenous Vasoactive Peptide

multiple ways by acting on specific brain areas


as well as on peripheral tissues. The net effect
of these actions depends on the spatial and
temporal release pattern of NPY, and on the
absence or presence of other neurotransmit-
ters and hormones that may be potentiated in
their cardiovascular effects or the release of
which may be inhibited by NPY. Moreover,
NPY not only affects acute cardiovascular
events, but might also be involved in chronic
processes such as the development of hyper-
trophy, hyperplasia, or both.

5.4.3 Other Effects. NPY elicits other sig-


nificant biological effects, showing antiepilep-
tic (329), anxiolytic (3301, and angiogenic
(331) activity. It regulates a variety of pitu-
itary hormones (332) and adrenocorticoid
function (333). In particular, NPY stimulates
feeding and appears to play a key role in the
regulation of eating behavior (334,335). CGP 71683A

5.5 Antagonists Figure 4.10. NPY Y, and Y, antagonists.


Primarily because of their potential antiobe-
sity properties, industrial and academic labo- of posttranslational enzymatic cleavages a1
ratories have exerted considerable efforts to basic processing sites (Fig. 4.11) (342). U-I1 it
discover nonpeptidic NPY antagonists, with located at the C-terminal portion of prepro-U. ge
11. Expression of prepro-U-I1 mRNA has been ha
selectivity for the Yl or Y5 receptor. The
first reported Yl-selective agent, BIBP3226, detected in human brain (medulla oblongata) PU
spinal cord, adrenal glands, and kidney (343- tu
blocked NPY-induced vasoconstriction and
345). U-11, but not its mRNA, has been found su
pressor response (336) (see Fig. 4.10). CGP
Wf
71683A, a selective Y5 antagonist, blocked in vascular and cardiac tissue (344). Rat,
mouse, porcine, and human U-I1 have been na
NPY-induced feeding by 50% in rats (337).
Since then, many more Y, and Y5 antagonists cloned (346). No reports describing the metab.
olism and inactivation of U-I1 have appeared. tic
have been reported (334, 338), and these
However, pretreatment of fish U-I1 with car. bit
should generate valuable data on the role of
boxypeptidase A destroys bioactivity in vitro, su
NPY in cardiovascular and metabolic diseases.
presumably by cleavage of the C-terminal de
6 UROTENSIN-II amino acid (Val) (347). PIC
U-I1 consists of 12 amino acids in various cic
Urotensin-I1 (U-11),one of the most potent va- fish species and 11 amino acids in the human in1
soconstrictors yet identified, was first charac- isoform (Fig. 4.12). All isoforms of U-I1 con- an
terized over 30 years ago in certain species of tain a hexapeptide loop formed by a disulfide is
fish, where it was isolated from spinal cord bridge spanning two cysteines and the amino co:
tissue (339). At first U-I1was thought to reside acids within the loop (positions 5-10 in human (31
only in fish, but a human isoform was identi- U-11) are conserved in all species (348).In con- ac:
fied in 1998 (340). trast, the N-terminal region of U-I1 has con-
6.:
siderable variation; for example, rat and hu-
6.1 Biosynthesis, Metabolism,
and Structural Features
man U-I1 have 48% sequence identity (Fig. u-
4.12). U-I1 has some structural similarity to f0l
Human U-I1 is synthesized from prepro-U-11, somatostatin-14, but these two peptides are mt
a 124 amino acid peptide (340,341),by a series probably not derived from a common ancestral tif
Signal peptide U-ll

21 114
I Signal peptidase

124

U-ll

1 Pro-hormone convertases

Figure 4.11. Biosynthesis of U-11.


st
is
J-
: gene (349).The solution conformation of U-I1 75% homology with GPR14 (344). U-I1 binds
has been studied by NMR (350) and a com- selectively to its receptor, and although soma-
?n puter model of the three-dimensional struc- tostatin-14 has some structural similarities to
d, ture has been developed (351). These studies U-11, it does not bind (344). The human U-II
3-
suggest that the hexapeptide loop region has a receptor has been detected in heart and pe-
ld ripheral vascular tissue as well as brain, skel-
well-defined structure, whereas the N-termi-
~t, nal portion is disordered. etal muscle, and spinal cord (344, 355, 356).
?n Experiments have shown that the hexapep- On binding to its receptor, U-I1 stimulates
b- tide C-terminal region of U-I1 is required for phospholipase C, which produces an increase
d.
biological activity (348). This conclusion is in inositol phosphate (357), and results in an
LT-
supported by NMR studies suggesting a well- influx of extracellular calcium and an intracel-
'0,
defined loop structure as well as by the com- lular release of calcium (344, 348,358).
lal
plete conservation of this region across all spe-
cies. SAR studies have indicated that the 6.3 Biological Actions
us
intact hexapeptide ring plus its flanking Human U-I1 is the most potent vasoconstrictor
an
amino acids, each containing an acidic group,
In- yet identified, and is more than 10 times more
is the minimum core structure that exhibits
.de potent than endothelin-I (356). Its vasoconstric-
contractile potency similar to that of U-I1
no tor properties are seen in a variety of mamma-
(352, 353). Removal of the C-terminal amino
an lian vascular tissues, including airway and pul-
acid results in complete loss of activity (347).
In- monary tissues (359361). Administration of
In- U-I1 to anesthetized monkeys led to myocardial
6.2 Receptor
1u- depression and circulatory collapse (344).Thus,
pig. U-I1 was identified as the endogenous ligand U-I1 may influence cardiovascular homeostasis
to for the orphan receptor GPR14, a seven trans- and be involved in the pathology of ischemic
are membrane G-protein-coupled receptor iden- heart disease and congestive heart failure. U-I1
;ral tified in the rat (354). The human receptor has and CGRP are the only two neuropeptidesfound
Endogenous Vasoactive Peptides

Human

Rat
Figure 4.12. Structures of
urotensin-I1 from goby (fish),
human, and rat. Shaded amino
acids indicate the hexapeptide
sequence that is conserved in
all species.
HN
,
00000
- GI" His Thr Ala

in motoneurons of the spinal cord, suggesting closed by a disulfide bond between two cysteine
that U-I1 might also have a modulating role at residues (363). This disulfide crosslink between
neuromuscular junctions (362). cysteines 7 and 23 is essential for all known bio-
logical activity ofANP (364).ANP is synthesized
7 NATRlURETlC PEPTIDE FAMILY predominantly in the cardiac atria. ANP is en-
coded by a single gene, which in humans is lo-
The natriuretic peptide family currently con- cated on chromosome 1, band p36, and is com-
sists of three structurally similar but geneti- posed of three exons and two introns (365).
cally distinct endogenous peptides: atrial na- Human ANP preprohormone is initially trans-
triuretic peptide (ANP), brain natriuretic lated from rnRNA within the atrial muscle and
peptide (BNP), and C-type natriuretic peptide in a variety of other tissues (366, 367) as a 151
(CNP). They are involved in the regulation of amino acid peptide (Fig. 4.14). This preprohor-
cardiovascular function causing natriuresis, mone is processed within the endoplasmic retic-
diuresis, and vasorelaxation. ulum by a serine protease to form a prohormone
(pro-ANP, also known as [ANPI-,,,] or y-AN?)
7.1 Biosynthesis, Metabolism, consisting of 126 amino acids after removal of
and Structural Features the 25 amino acid signal peptide from its N-ter-
The three natriuretic peptides ANP, BNP, and mind. This peptide, pro-ANP, is the major form
CNP each have a 17 amino acid ring structure, of ANP stored in the atrial granules of rats and
in which some of the amino acid residues are other species (368, 369). Upon stimulation,
conserved (Fig. 4.13). these granules move to the cell surface, releas-
ing the stored pro-ANP. In most other regulated
7.1.1 ANP. The circulating, biologically ac- hormone systems, the mature hormone is pro-
tive form of human ANP is composed of a 28 cessed from the precursor before storage in
amino acid peptide with a 17 amino acid ring granules.
7 Natriuretic Peptide Family

ANP BNP

H2N

CNP

Figure 4.13. Structures of the mature form of the human natriuretic peptide family. The shaded
circles indicate identical amino acids in the ring of the three peptides.

ANP is therefore distinct from most other tissue, especially in subjects with heart failure
hormones in being packaged in the prohor- (373). The release of ANP from the heart is
mone form, and therefore requires a process- enhanced by an increase in central volume,
ingmechanism that acts during or after secre- which stretches the atrium of the heart (370,
tion. Pro-ANP is cleaved into the biologically 372), and by an increase in heart rate (374).
active hormone ([ANP99-126 1, also known as Accordingly, increased circulating levels of
a-ANP) which is released together with the ANP are seen in congestive heart failure (3751,
N-terminal [ANP,,,] (370-372). Humans chronic renal failure (3761, and in severe hy-
differ from other species in having P-ANP, an pertension (377). Circulating ANP is cleared
antiparallel dimer of ANP, present in atrial rapidly, by both an enzymatic hydrolysis and a
Endogenous Vasoactive Peptide

ANP BNP CNP


I
signal peptide 4

1
Processed products IN-Terminal peptidel

Figure 4.14. Processing pathways and major molecular forms of the three natriuretic peptides in
humans.

receptor mechanism (clearance receptor). amino acids in human) precursor called pre-
Plasma half-life is approximately 3 min (378). pro-BNP and a 106 amino acid residue peptide
Endopeptidase 24.11 localized to the brush called pro-BNP (in humans it has 108 amino
border of the proximal tubule of the kidney, acids) (Fig. 4.14). Both prepro-BNP and pro-
lung, and vascular endothelium (379-381) BNP have BNP at their C-terminal ends. Pro-
cleaves ANP between residues of Cys (105) BNP is probably generated from prepro-BNP
and Phe (106) and between Ser (123) and Phe by proteolytic cleavage of a 26 amino acid res-
(124) disrupting the 17-membered ring, yield- idue signal peptide from its N-terminus. Pro-
ing biologically inactive peptides (382, 383). BNP, like ANP, is the major tissue form in
The cleaved product has been identified in hu- porcine cardiocytes. Thereafter, pro-BNP is
man coronary sinus plasma (384), indicating cleaved to produce biologically active BNP. In
that endopeptidase 24.11 most likely acts on humans, BNP is produced primarily in ven-
ANP in vivo. ANP is also removed from the tricular myocytes in response to increases in
circulation by binding to the natriuretic pep- ventricular pressure and volume and is re-
tide receptor-C (NPR-C) in vascular endothe- leased constitutively into the blood (393).Also
lium (385). BNP is secreted in response to physical exer-
cise (394).
7.1.2 BNP. Brain natriuretic peptide was In contrast to ANP, human BNP is the ma-
originally isolated in 1988 from porcine brain jor storage form in the heart, which indicates
(386). Although it was called brain natriuretic that posttranslational processing of BNP pre-
peptide, subsequent studies in pigs (387,388) cursor occurs in the heart and not during se
and in humans (389-391) showed that BNP cretion as in the case of ANP (395). BNP has a
levels in the heart are much higher than levels plasma half-life of 22 min (396-398) and is
in the brain. The human form consists of a 32 removed from circulation by cleavage with en-
amino acid peptide and, like ANP, has a 17 dopeptidase 24.11 and by uptake by the recep
residue ring structure closed by a disulfide tor NPR-C.
bridge (Fig. 4.13). BNP shows significant vari-
ation in structure across species. Separate 7.1.3 CNP. Structurally, CNP is the most
genes encode BNP. This peptide is also syn- conserved member of the natriuretic peptide
thesized as a prohormone, in which the biolog- family. It is considered to be the more primi-
ically active species is contained at the C-ter- tive natriuretic peptide, from which ANP and
minus (392). In porcine cardiac atria, there BNP evolved through gene duplication (399).
appear to be two major storage forms of BNP, The homology between rat and pig precursors
consisting of a 131 amino acid residue (134 is 97% and between human and pig prepm-
7 Natriuretic Peptide Family

CNP is 96% (400-402). A biologically active tions (415). All three peptides are bound by
peptide has two mature forms, one containing the NPR-C receptor (385, 416, 417). NPR-C
22 amino acids and the other 53 amino acids, internalizes the bound natriuretic peptides
both of which have been identified in porcine and delivers them to lysosomes for degrada-
brain (403). The porcine CNP precursor mol- tion (409, 410, 418). Thus, this receptor has
ecule consists of 126 amino acid residues and been characterized as a "clearance receptor."
is termed prepro-CNP (Fig. 4.14). Cleavage of
prepro-CNP yields a 103-residue peptide pro- 7.3 Biological Actions
CNP. Thereafter pro-CNP is cleaved to pro-
duce active CNP-22. Processing after this res- 7.3.1 ANP. ANP has diverse actions affect-
idue generates another endogenous CNP ing cardiovascular, renal and, endocrine ho-
(CNP-53).The C-terminus of CNP-53 is iden- meostasis. The main effects occurring during
tical to CNP-22, and CNP-53 is often referred infusion of ANP to healthy humans are a de-
to as the N-terminal extended form of CNP crease in blood pressure, by direct arteriolar
(401,403,404).CNP differs from other family vasodilation (419,420) and from a decrease in
members ANP and BNP in a number of re- cardiac output (421,422);an increase in natri-
spects, most notably that the C-terminus of uresis and diuresis attributed to changes in
CNP ends at the second cysteine residue and renal hemodynamics, especially an increase in
lacks any further C-terminal extension from glomerular filtration rate (423);a reduction in
the ring structure (Fig. 4.13). The stimuli for intravascular volume both by inducing diure-
synthesis and release of CNP appear to be cy- sis and by shifting intravascular fluid to the
tokines, growth factors, and mechanical interstitial space (424); inhibition of plasma
stretch (405, 406). CNP is released mainly renin activity and aldosterone secretion from
from vascular endothelial cells, but has also the adrenal cortex (425-427);and antagonism
been found in the kidney, brain, and intestine, of antidiuretic hormone (428). Genetic mouse
and does not circulate in plasma. The degra- models presenting alterations in expression of
dation of CNP, like ANP and BNP, occurs by genes for ANP or its receptors indicate that
way of endopeptidase 24.11 and the NPR-C genetic defects in ANP or natriuretic receptor
receptor (407). activity may play a role in salt-sensitive hyper-
tension as well as other sodium-retaining dis-
7.2 Receptors
eases such as congestive heart failure (CHF)
Autoradiographic studies have shown a wide- and cirrhosis (429). ANP infusion also influ-
spread distribution of ANP-binding sites in ences the sympathetic nervous system, proba-
many tissues, including endothelium, vascu- bly by inhibiting the baroreceptor-mediated
lar smooth muscle, adrenal glands, lung, and activation of renal sympathetic nerves as well
brain (408). Three natriuretic peptide recep- as increasing noradrenaline release (421,430,
tors have been identified. Two of these, natri- 431). ANP causes antimitogenic and antipro-
uretic peptide receptor A and B (NPR-A and liferative effects in glomerular mesangial
NPR-B), have guanylate cyclase activity and cells, in vascular smooth muscle cells, and in
mediate the biological activity of the natri- the heart (432) and arterial wall (433). ANP
uretic peptides (409, 410). A natural product inhibits the synthesis of the potent vasocon-
polysaccharide, HS-142-1, was shown to bind strictor endothelin I (434)and induces apopto-
competively at the ANP receptor, blocking sis in neonatal rat cardiomyocytes (436). ANP
ANP binding (411). NPR-A and NPR-B each also appears to be a local mediator of intestinal
share about 30% sequence homology with ty- function (435).
mine kinases, but possess no kinase activity
(412). Extensive studies have shown that ANP 7.3.2 BNP. The natriuretic, endocrine,
and BNP act through the NPR-A receptor and and hemodynamic effects of BNP are compa-
CNP acts through the NPR-B receptor (385, rable to those of ANP (437-439). Infusion of
413,414).The third receptor, NPR-C, does not BNP into healthy humans at physiological
contain the guanylyl cyclase and protein ki- doses produces effects that are similar to those
nase domains and does not mediate cGMP ac- seen with ANP. BNP had no effect on heart
Endogenous Vasoactive Peptides

rate and blood pressure, but potently inhib- endopeptidase 24.11 inhibitors showed effi-
ited the renin-angiotensin axis and had a cacy in treating mild to moderate heart fail-
large natriuretic effect. Transgenic mice over- ure. The inhibitor candoxatril is marketed for
expressing BNP have lower blood pressure this indication (471). Dual inhibitors of neu-
(440). Antimitogenic effects of BNP appear to tral endopeptidase 24.11 and angiotensin-con-
be similar to those of ANP (441). Like ANP, verting enzyme could be more efficacious for
BNP inhibits vascular smooth muscle cell pro- hypertension and CHF, and clinical studies
liferation by blocking the proliferative actions have been carried out (472,473). In addition,
of angiotensin I1 and endothelin I in the heart measurement of plasma levels of natriuretic
and other tissues (442-444). Thus overall peptides or the N-terminal ANP prohormone,
ANP and BNP appear to function as an inte- [ANP,,-,,I, has been suggested as a diagnos-
grated cardiac natriuretic peptide system and tic and prognostic tool to determine the sever-
may play a significant role in vascular remod- ity of CHF (459, 474) and acute myocardial
eling and resetting vascular compliance. infarction (475, 476).

7.3.3 CNP. Unlike ANP and BNP, CNP


has little effect on natriuresis and diuresis 8 CALCITONIN GENE-RELATED
(445) but acts in a local paracrine manner on PEPTIDE FAMILY
vascular endothelial cells as a vasorelaxant The calcitonin gene-related peptide family
and inhibitor of cell proliferation (446-448). consists of calcitonin (CT), calcitonin gene-
CNP may also act as a local neuroendocrine related peptide (CGRP), adrenomedullin
regulator because it is found in high concen- (ADM),and amylin. Human CGRP shows 19%
trations in the hypothalamus (449,450) and in homology with calcitonin, 35% homology with
the anterior pituitary (4021, as well as other adrenomedullin, and 51% homology with amy-
endocrine glands (451).In addition CNP inter- lin. CT maintains skeletal muscle mass during
acts with other endothelial modulators, the ni- calcium stress and plays a central role in mod-
tric oxide system (452), and endothelin (453, ulating calcium homeostasis. Amylin stimu-
454). When these observations are taken to- lates the breakdown of glycogen in skeletal
gether, CNP appears to serve as a local para- muscles and thus helps to regulate blood glu-
crine and autocrine factor in the regulation of cose levels. Amylin does not produce signifi-
vascular wall biology. cant vasoactive effects and will not be dis-
cussed further. CGRP and ADM are potent
7.4 Therapeutic Potential vasodilators, and are described below.
Because they influence renal, hemodynamic,
and endocrine function, are vasorelaxant, and 8.1 Structure, Biosynthesis, and Metabolism
inhibit cardiovascular cell growth, natriuretic
peptides have been suggested as playing an 8.1.1 CGRP. The existence of this peptide
important role in hypertension (455, 456), was predicted on the basis of the alternative
congestive heart failure (457-4671, renal fail- processing of the calcitonin gene (477, 478).
ure (462-4641, restenosis (444,465), and ath- Calcitonin gene-related peptide, a 37 amino
erosclerosis (466, 467). Given their peptidic acid neuropeptide (Fig. 4.15), first identified in
nature and rapid degradation, the natriuretic 1982 (478, 479) as an extremely potent vaso-
peptides are not themselves suitable as thera- dilator, is approximately 1000 times more po-
peutic agents. ANP gene therapy has been ex- tent than acetylcholine or substance P (480,
plored in a rat model (468). Inhibition of neu- 481). This peptide is distributed throughout
tral endopeptidase 24.11, which should the central and peripheral nervous systems
prolong the duration of action of the natri- (482-484) and is located in areas involved in
uretic peptides, has been studied both pre- cardiovascular function (485- 487).
clinically and clinically for hypertension and Calcitonin and both of the very similar
congestive heart failure. In clinical studies, al- forms of CGRP (alpha and beta) are encoded
though they did not lower blood pressure in on chromosome 11 (488). Alternate splicing of
most hypertensive patients (469,470), neutral the primary RNA transcript of the calcitonin
8 Calcitonin Gene-Related Peptide Family

Adrenomedullin

Figure 4.15. Amino acid sequences of alpha- and beta- forms of human calcitonin gene-related

1E peptide (hCGRP)and adrenomedullin. Amino acids differing from alpha-CGRP are indicated by filled
circles in beta-CGRP.

gene results in the production of two biologi- 75% identity between salmon and human
d y active peptides, calcitonin and a 37 amino forms. Whereas calcitonin is largely confined
:id peptide, now designated aCGRP (Fig. to thyroid cells, CGRP is widely distributed
4.16). A second gene on chromosome 11, unre- throughout the central and peripheral ner-
ted to calcitonin, produces PCGRP (Fig. vous systems (481,482). Only aCGRP mRNA
15) (489). Human aCGRP and PCGRP differ is found in the heart (490) and the cardiovas-
P
by only three amino acids. The structure of cular effects of aCGRP are generally more po-
CGRP is well conserved among species, with tent than those of the p-form (491). The CGRP
Endogenous Vasoactive Peptide!

1 8183 120125 128 transmitters in sensory neurons, particularlj


alpha substance P (502). In the heart, the amount oj
CGRP CGRP is higher in the atria than in the ventri
prepro-alpha CGRP cles (503). Release of CGRP is stimulated bj
Endopeptidases electrical stimulation as well as by prostaglan.
dins, potassium, bradykinin, capsaicin, lactic
lalphal- Gly-OH acid, low pH, and ischemia (504). The degra.
dation of CGRP has not been fully elucidated:
but a tryptase enzyme and chymotrypsin may
be involved (505, 506).

8.1.2 ADM. Adrenomedullin, a 52 amina


1 22 42 45 97 185 acid peptide discovered in 1993 (507), is ex-
Signal pressed in a wide range of tissues and has a
peptide PAMP ADM
wide range of action, including vasodilation,
natriuresis, and regulation of cell growth.
Endopeptidases ADM has 24% homology with CGRP. It con-
tains a disulfide bridge between cysteines 16
1- -Gly-OH and 21 and a C-terminal amidated tyrosine,

1alpha amidating enzyme both features of which are necessary for bio-
logical activity. The gene for ADM is located
on chromosome 11 (508) and produces a 185
amino acid precursor, prepro-adrenomedullin,
Figure 4.16. Biosynthesis of alpha-CGRP and which is cleaved by proteases and amidated,
ADM. like CGRP, by the enzyme peptidylglycine C-
amidating monoxygenase to generate active
ADM (Fig. 4.16) (509,510). ADM is expressed
structure contains a Cys2-Cys7 disulfide in the adrenal medulla, heart, lung, kidney,
bridge and C-terminal carboxamide, both es- and pituitary gland (511, 512), as well as in
sential for activity. A three-dimensional com- plasma and endothelial cells (513). The
puter model of aCGRP has been constructed plasma half-life of ADM in humans is about 22
(492). Cleavage or derivatization of the disul- min (514) and ADM appears to be degraded by
fide bond destroys agonist activity (493). The neutral endopeptidase 24.11 and amino pepti-
region of the peptide between Val-8 and dases (515, 516).
Arg-18 has the potential to form an amphi-
pathic a-helix, and this is evident by NMR
8.2 Receptors
studies (494,495). The fragment [CGRP,,,]
acts as an antagonist (496). The N-terminal CGRP receptors have been studied in humans
amino acids are essential for agonist activity and in the rat and are widely distributed in the
(493). The removal of the C-terminal amino nervous system (517, 519), including discrete
acid results in a significant loss of activity brain areas, and in the cardiovascular system
(497). CGRP mRNA produces prepro-CGRP (520). CGRP receptors in general match pep-
consisting of 128 amino acids, which is subse- tide distribution (518). Two cell surface recep-
quently cleaved at paired dibasic amino acids tors, CGRP, and CGRP,, have been postu-
(498) and finally converted enzymatically to lated (489, 521, 522). CGRP receptors belong
the active 37 amino acid carboxamide (499). to the family of seven transmembrane G-pro-
CGRP is abundant in sensory neurons and tein-coupled receptors, and binding leads to
present at lower levels in motor neurons, colo- activation of adenylate cyclase, which in-
calized with acetylcholine (500). CGRP is also creases cyclic adenosine monophosphate pro-
found in many endocrine cells and organs, duction (523-526).ADM shows potent binding
where it is stored in secretory vesicles (501). at CGRP receptors (527) but also has a distinct
CGRP is often colocalized with other neuro- receptor (528-530). It is also a member of the
ein-coupled receptor family and, like 8.4 Therapeutic Potential and Antagonists
, stimulates adenylate cyclase.
8.4.1 CGRP. CGRP elicited various benefi-
cial effects in congestive heart failure patients
3 Biological Actions
(547,548) and showed improvements in renal
disease (549,550) as well as in Raynaud's syn-
8.3.1 CCRP. Intravenous administration
drome (551, 552). It was active in a rat model
CGRP produces strong, dose-dependent hy- of pregnancy-induced hypertension (553).
nsive and tachycardia responses both in CGRP might also contribute to inflammation
rat (531-533) and in human subjects (554), migraine (5551, and gastrointestinal
nochemical studies have shown disorders (556); for these indications a CGRP
CGRP is distributed broadly within the antagonist would be needed. The fragment
diovascular system as a dense peripheral [CGRP,-,,I has antagonistic properties (492),
rk that innervates the arteries, and several nonpeptidic antagonists have
s, heart, and viscera (531, 535-538). been reported (Fig. 4.17) (557-559).
RP has significant and selective regional
effects and has been shown to 8.4.2 ADM. ADM shows potential in pul-
r decrease vascular monary hypertension (560, 561), congestive
he coronary, common carotid, heart failure (5621, and renal failure (563).
al, mesenteric, and hindquarter vascular
(481, 539), in addition to causing an in-
e in the rate and force of contraction in
heart (540). The coronary vasculature has
ed to be a particularly sensi- Tachykinins consist of three related peptides:
target (480, 481). Systemic administra- substance P, neurokinin A (substance K; neu-
creases blood pressure in a romedin L; NKA), and neurokinin B (Table
ependent manner in both normotensive 4.2) (564,565). Substance Pis the most potent
sand humans and in spontaneously hy- of the tachykinins in inducing vasodilatation
sive rats (482,539),whereas in patients and microvascular permeability, two charac-
failure, the decreases in teristic features of substance P's action.
lmonary wedge pressure
iac output and contractil-
9.1 Biosynthesis, Structure, and Metabolism
were more pronounced (541). The primary
for this blood pressure reduction is pe- Substance P is a peptide containing 11 amino
al arterial dilation (481, 539). On the acids (Table 4.2) derived from two distinct
t vasodilator effects and genes encoding for prepro-tachykinin A and B
perivascular localization of CGRP, it has (Fig. 4.18) (566, 567). Prepro-tachykinin A,
postulated that this peptide plays a role depending on alternative RNA splicing, will
e regulation of blood pressure and re- yield either prepro-tachykinin a, P, or y (568,
both under normal 569). The a- and y-prepro-tachykinin precur-
ns and in the pathophys- sors possess a single substance P product. The
of hypertension (482,483). P-prepro-tachykinin isoform additionally pos-
sesses a 10 amino acid neurokinin A peptide
3.2 ADM. ADM has a biological profile sequence at positions 98-107 (568) (Fig. 4.18).
to that of CGRP, showing potent vaso- The tachykinins are degraded by several pro-
g (542) and natriuretic activity (543) teolytic enzymes that may vary regionally.
regulates local and systemic vascular tone The main degradative enzymes are angioten-
otensive effects with- sin-converting enzyme (ACE), neutral endo-
chycardia (545) as well as renal activity peptidase 24.11 (NEP),and dipeptidyl (amino)
actions ADM appears peptidase (DPPIV) (554). Although numerous
antagonist to endo- enzymes contribute to the metabolism of the
tachykinins, both ACE and NEP are mem-
Endogenous Vasoactive Peptides

NH2
I

Figure 4.17. Chemical struc-


tures of BIBN4096BS (1) and
SB-273779 (2).

brane-bound peptidases optimally located to substance P), whereas neurokinin B preferen-


effectively subserve a metabolic role. tially stimulates NK, receptors (NK, potency:
neurokinin B > neurokinin A > substance P)
9.2 Receptors (571, 572). NK, receptors are located both in
the central nervous system and in peripheral
The three tachykinin receptor types are desig-
tissues, whereas NK, receptors are predomi-
nated NK, (substance P receptor), NK, (neu-
nantly found in the periphery and NK, recep-
rokinin A receptor), and NK, (neurokinin B
tors are mainly restricted to the brain (564,
receptor) and belong to the seven transmem-
573).
brane G-protein-coupled receptor family
(569, 570-572). Individual tachykinin pep-
9.3 Biological Actions
tides preferentially activate different tachyki-
nin receptor subtypes. For example, substance Substance P is thought to act principally on
P is more potent at NK, receptors than at NK, endothelial cells (574). By stimulating NK, re-
or NK, sites, and displays the following rank ceptors, substance P affects G-proteins and
order of potency: substance P > neurokinin A phospholipase C (564) to induce several down-
> neurokinin B. Alternatively, neurokinin A stream signaling events that include mobiliza-
is more potent at activating NK, receptors tion of intracellular calcium and nitric oxide
(N% potency: neurokinin A > neurokinin B > (NO) (575, 576). Activation of NK, receptors

Table 4.2 Tachykinin Receptors and Peptides


Endogenous
Receptor Peptide Structure
NKI Substance P Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-N
N& Neurokinin A His-Lys-Thr-Asp-Ser-Phe-Val-Gly-Leu-Met-NH2
NK3 Neurokinin B Asp-Met-His-Asp-Phe-Phe-Val-Gly-Leu-Met-NH2
Preprotachykinin (PPT) B

Alternative splicing
3 distinct mRNAs

Neurokinin B
Substance P

Substance P Figure 4.18. Schematic illus-


Neurokinin A tration of substance P and re-
Neuropeptide K lated tachykinin formation.

in the periphery may also be brought about permeability within the respiratory system
indirectly by release of substance P from var- and thereby is important in respiratory func-
icosities of afferent neurons (577). tion. In the airways, as in other regions, selec-
Importantly, tachykinins contribute to the tive NK, agonists increase the leakage of vas-
localized tissue response to injury. Injury may cular proteins across the endothelium and
be induced by physical, chemical, or thermal mucus hypersecretion (585). This results in
stimuli to evoke the release of substance P edema within the interstitial tissues, as well
from sensory neurons (578). This phenome- as extravasation of exudate into the airway
non, referred to as neurogenic inflammation, lumen. Moreover, substance P's effects are
although occurring in many vascular beds, not mimicked by NK, or NK, agonists, sug-
and splanchnic gesting that NK, and NK, receptor types do
of NK, receptor not affect vascular permeability in the respi-
otein extravasa- ratory system as in other systems (587).
is a hallmark feature of neurogenic in- Substance P also indirectly regulates car-
mation and occurs primarily in cutaneous diovascular function through its actions on
d splanchnic vessels (580,581).Substance P other physiological systems. For example, sub-
been shown to be a potent vasodilator stance P is coreleased with calcitonin gene-
ich are contin- related peptide and together, they cause vaso-
gent on neuronal release, appear to be highly dilatation in guinea pig submucosal arterioles
species dependent and inconsistent (579). and coronary vasculature of numerous species
Thus, evidence to support a role for substance (588, 589), whereas vasoactive intestinal pep-
Pin neurogenic vasodilatation is less compel- tide may potentiate the effects of substance P
ling than that for demonstrating its effects to on the microvasculature in some systems
increase vascular permeability (579). The (590). Importantly, substance P opposes the
attenuated by actions of the potent vasoactive peptide endo-
ive NK, antagonists, such as SR140333 thelin-1 (591). At least some of substance P's
, but is minimally blocked by selective actions result from its ability to suppress the
antagonists and not blocked by selective synthesis and release of catecholamines from
antagonists, further suggesting a role for the adrenal medulla (592), as well as alter-
e NK, receptor type (583-585). ations in ACTH-corticosterone production
In addition to vascular leakiness and local (593).It has recently been shown that the final
modulate warnidation step necessary for full activity of
ntral and pe- substance P can occur directly within endo-
586). Impor- thelial cells and can be released by shear stress
es vascular (594,595). This endothelial-derived substance
Endogenous Vasoactive Peptides

Figure 4.19. Chemical structures of CP-


96,345 (11, L-733,060 (21, and SR 142801
(3).

P can then evoke vasorelaxation through a ni- was shown to inhibit plasma extravasation
tric oxide dependent mechanism (594). Inter- without affecting blood pressure and heart
estingly, the immediate precursor before the rate in rats (601). The cardiovascular (rise in
a-amidation, substance P-Gly,also possesses a blood pressure and heart rate) and behavioral
vasorelaxant potency similar to that of the ma- reactions that occurred in response to a nox-
ture peptide (596). ious stimulus were attenuated by central ad-
ministration of NK, antagonists (602). SR
9.4 Antagonists 142801, a novel nonpeptidic NK, antagonist,
As with the other vasoactive peptide systems, devoid of agonist properties, has proved useful
initial synthetic attempts to design receptor in defining the functional role of tachykinin
antagonists focused on modifications of the receptors in the periphery. The pressor effects
endogenous peptide. Spantide, [D-Argl, evoked by systemic administration of NK,
' , 1']-substance P, is a competitive
~ - T r p ~ ,Leu agonists and neurokinin B were inhibited by
NK, peptide antagonist created by a general SR 142801 (603). These findings underscore
strategy of replacing the N- or C-terminal the important role subserved by subtype-spe-
amino acids of substance P (597). Spantide, cific antagonists in defining the constellation
although a potent substance P antagonist, in- of tachykinin-induced effects.
duces histamine release and is neurotoxic at
high doses (597). Additional peptide analogs
that act as antagonists at NK,, NK,, and NK,
receptors are described elsewhere (564, 597,
598). Bradykinin was first identified in 1949 as an
The first nonpeptidic NK, receptor antago- extract from ox blood (604). It was more than
nist reported was CP-96,345 (599, 600). Sub- 10 years later that the complete peptide se-
sequently, nonpeptidic antagonists of NK, quence was unequivacally described (605,
and NK, receptors were reported (Fig. 4.19) 606). Since that time, major strides have been
(571, 600). The NK, antagonist, L-733,060, made in our understanding of this peptide
weight or low molecular weight kininogen
(HMWK or LMWK)

Kallikrein

Lys-bradyknin

1/ Bradykinin
Aminopeptidase

J
/ \
Kininase I
kininase I, II, NEP,
carboxypeptidase M
%
'
Figure 4.20. Formation and degradation of
[des~rg~]
bradykinin Inactive products bradykinin.

through the use of molecular techniques and partments can alter significantly the overall
the discovery of selective receptor antagonists. degradation pattern of BK.

10.1 Biosynthesis, Structure, and Metabolism 10.2 ~~~~~t~~~


Bradykinin (BK) is a nonapeptide with the fol- The biological actions of BK in mammals are
lowing structure: Arg-Pro-Pro-Gly-Phe-Ser- brought about by the activation of two distinct
Pro-Phe-Arg [BK,-,I. Bradykinin is formed in receptors. Originally, the two-receptor classi-
the plasma as a component of the inflamma- fication of B, and B, was based on an opposing
tory response at sites of tissue damage (607) pharmacological pattern of responses (612).
(Fig. 4.20). The initiating event is the activa- The rank order of potency of a series of ligands
tion of Factor XI1 in the blood, which occurs at for the B, receptor is as follows: [desArggl-BK
sites of tissue injury. Activated Factor XI1 > [TJT(M~)~]-BK > BK, whereas at the B, re-
(XIIa)converts prekallikrein to the active pro- ceptor: [Tyr(Me)']-BK > BK > [desArg91-BK.
tease kallikrein. Kallikrein can then enzymat- This two-receptor distinction later was con-
ically digest high molecular weight kininogen firmed with the cloning and expression of the
to yield bradykinin. Alternative splicing of the B, and B, genes (613-615). The kinin B, and
primary transcript of the kininogen gene re- B, receptors belong to the seven transmem-
sults in the synthesis of low molecular weight brane G-protein-coupled receptor family
kininogen. In humans, this protein is a sub- (616). The B, receptor is responsible for the
strate for tissue kallikrein and yields lysyl-BK most notable physiologic effects of BK in
([LysO]-BK),also known as kallidin (607,608). mammals and this receptor is considered to be
Tissue kallikrein and plasma kallikrein are constitutively expressed (617). In contrast,
structurally unrelated enzymes. Once formed, the B, receptor is inducible and its expression
the kinins are short-lived with a half-life of is upregulated at the site and time of tissue
less than 30 s (609). Although numerous en- injury (617,618).
zymes are capable of degrading the kinins,
ACE and NEP 24.11 are the predominant pro- 10.3 Biological Actions
teases responsible for BK7sshort half-life in
the circulation and in tissues (610, 611). ACE The B, and the B, receptors are members of
preferentially cleaves BK between the Pro7- the seven transmembrane G-protein-coupled
Phe8 and Phe5-Sere bonds, whereas NEP di- receptor family. The B, receptor is coupled to
gests BK between Gly4-Phe5 and Pro7-Phes Gai and Gaq, whereas the B, receptor couples
bonds (611).The relative distribution and pro- to Gaq/ll and Gai,,, (619, 620). Kinin recep-
portion of these enzymes within tissue com- tor activation, through the actions of the G
Endogenous Vasoactive Peptides

proteins, can stimulate various intracellular Recently, nonpeptidic antagonists have been
pathways including phospholipases &,C, and described (Fig. 4.21) (633). These novel antag-
D, leading to protein phosphorylation. Selec- onists will no doubt further delineate BK re-
tive protein phosphorylation then results in ceptor subtypes and aid in clarifying the role of
the generation of intracellular calcium and BK in various pathophysiological conditions.
prostaglandin and nitric oxide release (621).
BK increases vascular permeability at the site
of tissue injury and also possesses potent va- 11 VASOACTIVE INTESTINAL PEPTIDE
sodilator activity, two critical components of A N D RELATED PEPTIDES
the local inflammatory response (622). In ad-
dition to nitric oxide-mediated BK vasodilata- Vasoactive intestinal peptide (VIP) was first
tion, an endothelium-derived hyperpolarizing isolated from porcine intestine (634). The pep-
factor, resistant to inhibitors of the nitric ox- tide derives its name from the profound and
ide system, has been shown to contribute to long-lasting vasodilatory action seen upon sys-
BK-induced vascular relaxation (623). A role temic administration (635). VIP is a highly ba-
for the mitogen-activated protein kinase fam- sic, single-chain linear polypeptide, contain-
ily has also recently been demonstrated as a ing 28 amino acid residues in its sequence with
mediator of BK activity (624). a C-terminal asparagine amide (636).The pri-
Bradykinin has been implicated as an im- mary sequence of VIP is identical in most
portant factor in mediating numerous physio- mammals, with the guinea pig being the one
logical and pathophysiological processes, espe- notable exception (637).
cially those within the cardiovascular and VIP is derived from a 170 amino acid pre-
renal systems (625). Genetic manipulations of cursor, prepro-VIP (638). The prepro-VIP
the BK receptor system (knockouts, trans- peptide contains another biologically active
genic animals) suggest that BK may be impor- peptide, referred to as PHI (peptide with N-
tant in the development of the blood pressure terminal histidine and a C-terminal isoleucine
phenotype (626). A role for kinins in blood amide). The human equivalent of PHI has a
pressure regulation, cardiac ischemia, myo- C-terminal methionine and is referred to as
cardial infarction and remodeling, and renal PHM. PHIPHM is structurally related to
disease has been shown (625-627). The clini- VIP, and shares many of its biological actions,
cal effects of the ACE inhibitors in cardiovas- although it is generally less potent than VIP
cular disease, in part, are attributed to BK (639, 640). VIP appears to be coreleased with
(628, 629). PHIPHM (641);VIP also can be released with
acetylcholine and together they act synergisti-
10.4 Antagonists
cally on peripheral vascular targets (642).
Efforts to identify selective and specific antag- VIP is a member of a family of regulatory
onists for BK receptors have been pursued for peptides that also includes pituitary adenylate
more than 20 years. Numerous chemical and cyclase-activating peptide (PACAP). PACAP
amino acid substitutions have been made in is a basic 38 amino acid a-amidated peptide
an attempt to increase potency, selectivity, structurally related to VIP (643). The recep-
and duration of action (610, 630, 631). The tors for these peptides are members of the
carboxy terminal arginine appears to be par- seven transmembrane G-protein-coupled su-
ticularly important, in that its presence or ab- perfamily that also includes glucagon, gluca-
sence exerts a dramatic impact on agonistlan- gon-like peptide, secretin, and growth hor-
tagonist receptor selectivity (630). One mone-releasing factor (644, 645). PACAP
notable B, antagonist is HOE-140, a peptidic binds with high affinity to three distinct recep-
antagonist with high potency and long dura- tors, whereas VIP interacts specifically with
tion of action [&g-Arg-Pro-Hyp-Gly-Thi- only two of these receptors (646). The recep-
Ser-,Tic-Oic-Arg] (631). Through the use of a tors for these peptides have been designated
series of antagonists for B, and B, receptors, by different names based on the binding char-
evidence for species differences and for novel acteristics of various ligands; however, the
non-B,/B, receptors has been put forth (632). recommended nomenclature is PAC,, VPAC,,
11 Vasoadive Intestinal Peptide and Related Peptides

LF16-0687

Figure 4.21. Chemical structures of FR 173657 and LF16-0687.

d WAC, (647). The WAC, and VPAC, re- lumbosacral spinal cord (652). VIP receptors
ptors bind VIP and PACAP with similar af- have been localized within cerebral microves-
whereas the PAC, receptor binds sels (653). In the peripheral nervous system,
preferentially (646). VIP is present in pre- and postganglionic fi-
east three distinct receptors for PACAP bers and in nerve terminals of the autonomic
d VIP have been cloned and expressed nervous system in humans. Autonomic VIP
7). The WAC, receptor was first isolated nerve fibers tend to be widely but somewhat
m rat lung (648), the WAC, receptor ini- nonuniformly distributed among blood vessels
ly was cloned from the rat olfactory lobe (639,654,655). Whereas both VIP and PACAP
9), whereas the PAC, receptor was cloned can be found in the hypothalamus, only VIP is
nally from a rat carcinoma cell line (650). synthesized in the pituitary gland (637). The
intracellular events that occur subse- overall localization and distribution of PACAP
ent to ligand binding by these receptors pre- andVIP within the central nervous system are
minantly involve stimulation of CAMP quite different (645). In the periphery, VIP
stimulation of G, protein (646). Addi- and PACAP are often colocalized to the same
second-messenger systems such as pro- cells (645). VIP and PACAP preferentially are
ion of inositol triphosphate and calcium associated with cerebral blood vessels (656-
ilization by stimulation of phospholipase 658), vagal projections to the heart (659), and
are activated by PAC, receptors (651). with nerve fibers innervating the smaller di-
Among CNS regions involved in cardiovas- ameter blood vessels (660, 661).
function, VIP is present within the nu- VIP is a potent vasodilator (634, 662). VIP
of the tractus solitarius and in the inter- and PACAP elicit vasodilatation in cerebral
olateral spinal cord, especially within the blood vessels (663). Electrical stimulation of the
228 Endogenous Vasoactive Peptides

Table 4.3 VIP Receptors and Ligands


Receptor Agonista Antagonist
PACl Maxadilan (667) PACAP (6-27) (671)
WAC, [Lys15, Arg16, L ~ u ~ ~ ] VGRFs-27-
IP~-~ [Ac-His1, d h e 2 , Lys15, Arg16]VIP3-7 GRFGZ7-
NH2 (668) NH, (672)
WAC, Ro 25-1553 (669)
RO25-1392 (670)
"GW,growth hormone releasing factor,

cerebral cortex or mesencephalic reticular adi- (Table 4.4). It was identified in 1973 and
vating system, which innervates the cortex, originally characterized for its actions as a hy-
causes local release of VIP and vasodilatation of pothalamic inhibitor of pituitary growth hor-
arterioles and venules at the cortical surface mone release (673). Subsequently, somatosta-
(664). Importantly, VIP directly causes artery tin has been found to be a regulatory hormone
vasodilatation in the absence of endothelial that inhibits the release of a variety of peptide
cells, suggesting that VIP acts directly on the hormones, including glucagon, growth hor-
smooth muscle (640). Moreover, in the cat hind- mone, insulin, and gastrin (6741, and inhibits
limb, VIP- and PACAP-induced vasodilatation cell proliferation (675). Somatostatin is a po-
does not require nitric oxide, prostaglandins, or tent vasoconstrictor and has a negative inotro-
Kf , channels (665). Although VIP evokes a pic action on noradrenaline-mediated atrial
depressor response in the cat, the hemodynamic
muscle contractions in humans (676,677). Its
pattern of responses to PACAP administration
antiarrhythmic action is thought to be caused,
is more complex, initially manifesting as a de-
pressor response that is subsequently followed in part, by a reduction in calcium influx across
by a more prolonged rise in arterial pressure the sarcoplasmic reticulum of atrial myocytes
(665). VIP circulates in the plasma and VIP is (678). However, somatostatin's actions may
released into coronary vessels during vagal stim- also result from a reduction in calcium influx
ulation to cause coronary artery dilation (666). in atrioventricular node cells (679).Because of
VIP concentrations in the coronary sinus have its action as a potent vasoconstrictor, soma-
been shown to be elevated during coronary ar- tostatin may have a useful role in stopping
tery occlusion and reperfusion (666). uncontrolled bleeding with esophageal varices
Several peptides have been proposed as (680-682). Within the CNS, somatostatin-
VIP receptor agonists and antagonists (Table containing cell bodies and/or afferent fibers
4.3). However, the development of selective, are present in the rostral portion of the ven-
high affinity agonists and antagonists for VIP trolateral medulla (VLM) and nucleus of the
and PACAP receptors is still in an early stage tractus solitarius (NTS) and project to the in-
as none of the available peptidic fragments termediolateral column of the spinal cord
possesses sufficient absolute specificity and se- (683, 684). Direct stimulation of the VLM af-
lectivity. There are no currently available an- fects blood pressure. Peripherally, somatosta-
tagonists for the WAC, receptor. Further tin-containing nerve fibers are generally of
progress in our understanding of the VIP1 limited distribution. Somatostatin is present
PACAP receptor family awaits the develop- in subpopulations of pre- and postganglionic
ment of nonpeptidic antagonists with both autonomic fibers, and is evident in autonomic
high selectivity and specificity. ganglia including mesenteric and superior cer-
vical ganglia (685). Somatostatin immunore-
activity is localized within the fibers innervat-
12 OTHER PEPTIDES
ing the heart as well as the intrinsic
12.1 Somatostatin
parasympathetic neurons in the heart (677,
686). Five distinct seven transmembrane G-
Somatostatin is a 14 amino acid peptide that protein-coupled receptor subtypes for soma-
has two cysteines linked by a disulfide bridge tostatin have been cloned and characterized
12 Other Peptides

Table 4.4 Structures of Somatostatin, Gastrin-Releasing Peptide, Neurotensin, and


Somatostatin
Ala-Gly-Cys-Lys-Asn-Phe-Phe-Trp-Lys-Thr-Phe-Thr-Ser-Cys-OH

Gastrin-releasing peptide
Try-Pro-Arg-Gly-Asn-His-Trp-Ala-Val-Gly-His-Le~-Met-NH~
I

Neurotensin
pGlu-Leu-Tyr-Glu-Asn-Lys-Pro-Arg-Arg-Pro-TpIle-Leu-OH

Relaxin
Glu-Phe-Leu-Ala-Val-Tyr-Pro-Arg-Arg-Lys-Lys
I

s-s S S
I I
Cys-Gly-Arg-Glu-Leu-Val-Arg-Ala-Gln-Ile-Ala-Ile-Cys-Gly-Met-Ser
I
Leu-Lys-Ile-Val-Asp-Asp-Lys-Trp-Lys-Ala-Ala-Val-Ala 1
'hr

(687,688).Several potent peptidic somatosta- ally the role of gastrin-releasing peptide in


tin analogs have been identified, including oc- vascular function has been uncertain (6971,
treotide (689), used clinically to relieve symp- this peptide is related to bombesin, which is
toms associated with gastro-entero pancreatic a potent vasoactive substance in amphibians
endocrine tumors and to s t o ~A -
bleeding from (694,698). Bombesin, neuromedin B, or gas-
gastro-esophagealvarices in patients with cir- trin-releasing peptide causes increases in
rhosis (690, 691). In addition, selective non- phosphoinositol turnover and elevations in
peptidic agonists have been identified for the intracellular calcium in isolated rat endo-
hive receptor subtypes (692,693). thelial cells (700). These actions are medi-
ated through seven transmembrane G-pro-
12.2 Gastrin-Releasing Peptide
tein-coupled receptors, which are described
Human gastrin-releasing peptide contains as bombesin-like peptide receptor subtypes
27 amino acids (694) and belongs to a family 1, 2, and 3 (701-705). A variety of selective
of peptides, including bombesin and neuro- peptide ligands for these receptors have
medin-B, that share homology in their C- been developed (706). In addition, several
rminal sequences (Table 4.4). Bombesin, a selective nonpeptidic antagonists have been
tetradecapeptide, causes vascular relaxation identified (707, 708). Infusion of a peptidic
E in the gut (699). Gastrin-releasing peptide is bombesin antagonist into a man with pulmo-
' oresent in some nerve fibers innervating the - nary hypertension led to acute hemody-
:respiratory system (695) and in the gastro- namic effects, including a decrease in sys-
intestinal tract (696). Although tradition- tolic pressure (709).
Endogenous Vasoactive Peptides

12.3 Neurotensin termed A and B, and is linked through three


disulfide bonds (732, 733). Two molecular
Neurotensin (NT), a 13 amino acid peptide
forms of relaxin have been identified, H1 and
originally isolated from bovine hypothalamic
H2, and are encoded by two distinct genes
extracts in 1973 (710), is found in brain, gas-
(734,735). Relaxin is derived from a precursor
trointestinal, and cardiovascular tissues (Ta-
protein, preprorelaxin, after proteolytic diges-
ble 4.4) (711-718). NT acts as a neurotrans-
tion of a signal and connecting peptide (736).
mitter and neuromodulator through specific
The highest concentrations of relaxin are
receptors. Three NT receptor subtypes have
found in the female reproductive system (7371,
thus far been identified and cloned (7191, two
although relaxin is also produced in males, pri-
belonging to the family of seven transmem-
marily in the prostate gland (738). Interest-
brane G-protein-coupled receptors. A variety
ingly, relaxin also can be synthesized by atrial
of peptidic analogs of NT have been prepared,
cardiocytes (739). To date, specific receptors
and these indicate that only the last six amino
that bind relaxin have not been identified and
acids [NT,-,,I are needed for biological activ- characterized. Knockout mice have confirmed
ity (720). A nonpeptidic NT antagonist has
the well-known pregnancy-related effects of
been identified and used as a tool to study the
relaxin, such as preparation of the birth canal
central effects of NT receptor blockade (721).
for delivery and mammary gland development
In the CNS, NT acts as a neuromodulator as- (737-740). Relaxin also modulates collagen
sociated with dopamine (721) and can modu-
deposition (741, 742) and has antifibrotic ef-
late the activity of various cholinergic neurons fects in a number of different animal models
and corticotropin-releasing factor cells (722-
(743, 744). With regard to the cardiovascular
724). In the periphery, NT is involved in the
system, relaxin is a potent vasodilator that
control of gastrointestinal and cardiovascular acts through a nitric oxide-dependentlcGMP
systems (718). NT has been shown to cause
pathway (735, 745, 746). Chronic administra-
vasoconstriction in a number of vessels (725). tion of relaxin produces renal hypertiltration
Intravenous or intraperitoneal injections of
and vasodilation mediated, in part, by activa-
NT in guinea pigs elicit dose-dependent in-
tion of the endothelin ETBreceptor (747).Re-
creases in blood pressure and heart rate (726,
laxin also has been shown to inhibit platelet
727), resulting in part from activation of the aggregation (748). Thus, agents that modulate
sympathetic nervous system innervating re- or simulate the physiologic effects of relaxin
sistance blood vessels and the heart (728).
may prove useful as novel therapies for vascu-
However, administration of an NT analog to
lar (749) and renal diseases (744).
normal rats led to hypotension (729).

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Contents
1 Introduction, 252
2 Erythropoietin, 255
2.1 Physical Properties, 255
2.2 Bioactivity, 256
2.3 Therapeutic Indications, 256
2.4 Side Effects, 257
2.5 Pharmacokinetics, 257
2.6 Preparations, 258
3 Granulocyte-Macrophage Colony-Stimulating
Factor, 258
3.1 Physical Properties, 258
3.2 Bioactivity, 259
3.3 Therapeutic Indications, 259
3.4 Side Effects, 260
3.5 Pharmacokinetics, 260
3.6 Preparations, 261
4 Granulocyte-ColonyStimulating Factor, 261
4.1 Physical Properties, 261
4.2 Bioactivity, 262
4.3 Therapeutic Indications, 262
4.4 Side Effects, 263
4.5 Pharmacokinetics, 263
4.6 Preparations, 264
5 Interleukin-11,264
5.1 Physical Properties, 264
5.2 Bioactivity, 265
5.3 Therapeutic Indications, 265
5.4 Side Effects, 266
5.5 Pharmacokinetics, 266
5.6 Preparations, 266
6 Stem Cell Factor, 266
6.1 Physical Properties, 266
6.2 Bioactivity, 267
6.3 Therapeutic Indications, 268
6.4 Side Effects, 268
6.5 Pharmacokinetics, 268
6.6 Preparations, 268
7 Investigational Agents, 268
7.1 Interleukin-6,268
7.2 Thrombopoietin, 270
Hematopoietic Agents

7.3 Interleukin-3,271
8 Summary and Conclusion, 272

1 INTRODUCTION ingly restricted in their developmental path-


way. As the maturational state between the
Hematopoiesis is a life-long process that in- hematopoietic stem cell and the progenitor
volves the continuous formation and turnover of cell progresses, the capacity for self-renewal
blood cells. Maintaining adequate blood cell pro- declines, and subsequent divisions will even-
duction, as well as being able to meet increased tually yield an end-stage, fully differentiated
demand (sickle cell anemia, infection), is in part cell type that has lost the capacity to prolifer-
under the control of a group of hormone-like gly- ate. The balance between self-renewal and
coproteins referred to collectively as cytokines maturational divisions of the hematopoietic
-
that includes the hernatopoietic growth factors stem cells and progenitor cells allows one he-
and the interleukins. The hematopoietic growth matopoietic stem cell to yield approximately
factors are as follows: erythroprotein (EPO), 1000 mature cells (1).
thrombopoietin (TPO), stem cell factor (SCF; Key experiments by Jacobsen et al. (2,3)in
also known as steel factor, kit ligand, and mast the early 1950s, demonstrating restoration of
cell growth factor), and the colony-stimulating hematopoiesis with spleen and/or bone mar-
factors (CSF): grandocyte/macrophage-CSF, row-derived cells in irradiated animals, com-
granulocyte-CSF, and macrophage-CSF (also bined with Till and McCulloch's (4) work dem-
known as CSF-1). Originally the term interleu- onstrating that a single bone marrow-derived
kin (IL) was operationally defined and implied cell could form a macroscopic nodule in the
that production of and the response to the mol- spleen composed of rapidly proliferating he-
ecule was restricted to leukocytes. As the cell matopoietic cells, showed the in vivo existence
and molecular biology of the interleukins has of a hematopoietic stem cell. The pivotal devel-
expanded, it is clear the original definition is not opment of an in vitro assay system by Bradley
always applicable, e.g., many nonleukocytes pro- and Metcalf (5), Ichikawa et al. (6), and
duce andlor respond to the interleukins. How- Pluznik and Sachs (7) with refinements by
ever the term interleukin has been retained by " Dexter et al. (8) and Whitlock et al. (9) for
the International Congress of Immunology. culturing hematopoietic cells, allowed many of
Consequently as new hematopoietic growth fac- the developmental pathways and the regula-
tors are identified, an interleukin number is as- tory molecules involved in hematopoietic ho-
signed once the amino acid sequence has been meostasis to be identified. In these assay sys-
determined; currently there are 23 interleukins. tems, hematopoietic stem and progenitor cells
Under steady-state conditions, 2 x 1Ol1 are cultured in a semi-solid matrix, and in the
blood cells are produced and destroyed per presence of cytokines, colonies of cells initi-
day. Key to maintaining a high rate of blood ated from a single cell develop. The Dexter and
cell production is the hematopoietic stem cell, Whitlock-Witte modifications involve co-cul-
which gives rise to all mature circulating cells: turing hematopoietic stem and progenitor
erythrocytes, platelets, lymphocytes, mono- cells with bone marrow-derived stromal cell
cytes/macrophages, and neutrophilic, eosino- monolayers. Based on morphological andlor
philic, and basophilic granulocytes (Fig. 5.1), histochemical criteria, the composition of cells
Between the pluripotential hematopoietic within the colony is determined, and the phe-
stem cells that gives rise to either myeloid or notype of the stem/progenitor cell that gener-
lymphoid cells and the end-stage mature cir- ated the colony (the colony forming unit or
culating cells are a hierarchy of progenitor CFU) and therefore the target of cytokineb)
cells that differ in degree of lineage restriction, action can be identified. Colony assays have
Hematopoietic stem cells self-renew (give rise proven to be powerful screening tools for iden-
to identical daughter cells) and/or divide and tifying myeloid specific cytokines and some-
give rise to progenitor cells that are increas- what less fruitful in identifying cytokines re-
Pluripotential

I IL-6 Stem cells

'
I SCF
IL-1
I" IL-11

\
Lymphoid
progenitor progenitor
IL-1 @-
CFU-GEMM

BFU-E CFU-Meg CFU-GM


r . - I
EPO

v
1
B-lymphoblast T-lymphoblast
[Diffez~ng

0.. .
...

Reticulocyte
Megakaryocyte Monocyte precursor cell B-cell T-cell

I Mature
cells

Activated T-cell
Red blood cells Plasma cell
Eosinophils
' (Erythrocytes) Macrophages

: Figure 5.1. Schematic of blood cell development. Blood cell development is a hierarchical process with
self-renewal and maturational divisions marring as a continuum. A pluripotential stem cell will divide, and
, the daughter cells will either be identical in functional capacity (self-renewal) or the daughter cell will be
. more mature (maturational division). The number of cell divisions, hence, the number of different cell
slightly
-
typesbetween the hematopoieticstem cell and the genration of myeloid or lymphoidprogenitors,is not known,
nor isthe point at which phenotypically distinct lymphoid and myeloid progenitorsare generated. The dashed
lines in the figure are meant to refled this. As progenitors, cells can undergo self-renewal and maturation
divisions; however, as the cells progress toward the end-stage mature cell, the capacity for self-renewal is lost,
andprimarily,maturational divisions occur. The colony-formingunit ( 0
and burst-formingunit (BFU)are
morphological distinctions. GEMM, granulocyte, erythroid, monocyte, megakaryocyte; E, erythroid; Meg,
megakaryocyte; GM, grand-, monocyte. These refer to the cell types present in the colonies.
254 Hematopoietic Agents

GM-CSF + G-CSF b G-CSF + IL-6

GM-CSF + IL-3 I+ IL-3 + IL-1

G-CSF

I I I I I I I I I I I I I I I I I I
0 20 40 60 80 100 120 140 160 0 20 40 60 80 100 120 140 160
CFU-GM CFU-GM

Figure 5.2. Synergistic effects between human growth factors on CFU-GM formation of myeloid
progenitors. Bone marrow-derived myeloid progenitor cells were incubated with the indicated cyto-
kines under standard cell culture conditions. Seven days later, the number of CFU-GM present in the
cell cultures were quantitated. Ftesults are presented as the mean of duplicate determinations + SD
and are representative of four separate experiments. This figure was reproduced with permission
from Jacobsen et al. (11).

quired for the development and maturation of itors proliferate in response to GM-CSF. In
the earliest lymphoid-restricted progenitor contrast, GM-CSF treatment of neutrophils
cells and end stage B- and T-lymphocytes. In and macrophages, mature end-stage cells that
general, identification of lymphoid-specific cy- have lost the capacity to proliferate, enhances
tokines has relied more heavily on examining their functional activity. In both instances,
the ability of the cytokineb) to promote the GM-CSF treatment leads to target cell activa-
proliferation of developmentally restricted T- tion, and the difference in biological effects
and B-cell lines. The molecular mechanism(s) elicited by GM-CSF reflects the functional ca-
in which cytokines effect hematopoietic cell pacity of the target cell at that point in differ-
lineage restriction, if they do, is not clear and entiation pathway. A second key activity some
is an area of intense research. However, re- cytokines share is their ability to synergize.
sults obtained with in vitro assay systems have Synergistic responses observed between cyto-
elucidated several key features of cytokine ac- kines range from greater that additive re-
tion (see Ref. 10 for an in-depth discussion). sponses when used in combination versus in-
Several properties of cytokines, including dividually to a single cytokine with no
multiple cell targets, synergistic responses, apparent effect increasing the activity of a
and overlapping activities, have important bi- functional cytokine. Experimental data repre-
ological implications. First, as schematically sentative of these two types of synergistic in-
depicted in Fig. 5.1, many cytokines share the teractions is presented in Fig. 5.2. The target
ability to affect the activity of multiple cell cell population is highly purified mouse bone
types, and dependent on the maturational marrow-derived progenitor cells (Lin BM
state of the cell type, may elicit different re- cells) and the ability of several cytokines alone
sponses. Targets for GM-CSF include the mul- and in combination to promote granulocyte1
tipotential myeloid progenitor cell (CFU) that macrophage progenitor cell growth (CFU-
gives rise to the granulocyte, erythroid, mono- GM) is being quantitated. As presented in A,
cyte/macrophage, and megakaryocyte cell lin- IL-3, GM-CSF, or GCSF alone support CF'U-GM
eages (CFU-GEMM), and the more restricted growth; however, greater than additive effects
CFU-GM progenitor that generates the gran- are observed when the cytokines are added in
ulocyte and monocyte/macrophage cell lin- combination. Neither IL-1 or IL-6 alone sup-
eages. The CFU-GEMM and CFU-GM progen- port CFU-GM growth (Fig. 5.2); however if
2 Erythropoietin

GM-CSF or IL-3 plus IL-1 or IL-6 is assayed,


progenitor cell growth is greater compared
with growth with GM-CSF or IL-3 alone (com- EPO, a key regulator of erythropoiesis, was
pare A and B). In contrast, IL-1 or IL-6 has the first hematopoietic growth factor activity
little effect on G-GSF-stimulated progenitor identified. In 1906, Carnot and Deflandre (15)
cell growth. Molecular mechanisms that give reported that serum derived from an anemic
rise to the synergistic interactions detected animal, when introduced into a normal ani-
with the various cytokine responses are being mal, led to increased numbers of circulating
actively explored in numerous laboratories. red blood cells and proposed that an activity
Identified mechanisms include one cytokine present in the serum, hemopoietin, mediated
increasing the expression of another cytokine the effect. Subsequent studies demonstrated
receptor (11)and one cytokine inducing tran- that anemia itself lead to an increased blood
scription of genes whose transcripts are stabi- level of hemopoietin and that hemopoietin was
lized by another cytokine (12). The third key produced by the kidney (16, 17). Biologically
feature of cytokines, also depicted in Fig. 5.1, active EPO was first purified from urine (18),
is the apparent overlapping activities seen and oligonucleotide probes, based on the EPO
with cytokines. In some instances, there are amino acid sequence, were used to clone the
quantitative differences in the responses elic- human gene (19,20). In 1985, the first patient
ited. IL-3, GM-CSF, or SCF in the presence of received recombinant EPO (21).
EPO will support CFU-GEMM growth; how-
2.1 Physical Properties
ever, colonies grown in the presence of EPO
plus SCF contain significantly more cells. Al- The protein-coding region of the EPO gene is
ternatively, a common activity can be the re- composed of five exons and four introns, and
sult of one cytokine inducing the expression of the human gene is located at chromosome
the cytokine that mediates the response. IL-3 7pter-q22 (22). The 1.6-kilobase (kb) tran-
and M-CSF independently support CFU-GM script encodes a 193 amino acid protein of
growth; however, IL-3 can induce M-CSF gene which the first 27 amino acids (leader-se-
expression in CFU-GM (13). Consequently, in quence) and a carboxy terminal arginine are
both cases M-CSF may mediate the response. removed before secretion. The mature protein
Whether the overlapping activities detected in contains two internal disulfide bonds and has
in vitro colony assays implies functional re- a calculated molecular mass of 18.4 kilodal-
dundancy in vivo is less clear. Studies in ani- tons (kDa). However, EPO is also glycosylated
mals have revealed overlapping as well as dis- (adding three N-linked and 1 0-linked carbo-
tinct activities for the cytokines (see Ref. 14 hydrate chain) before secretion; thus, circulat-
for a comprehensive discussion). Physiological ing forms of EPO are larger (34-39 kDa) (23,
parameters such as access to the cytokine, lo- 24). During fetdneonatal growth, EPO is pro-
cal cytokine concentration, and presence of duced in the liver, but near birth, production
other cytokines will also influence effects seen shifts to the kidney. Peritubular cells (fibro-
in vivo. In the following sections, four cyto- blast-like type I interstitial cells) that are
kines approved by the Food and Drug Admin- present in the kidney cortex and outer me-
istration (FDA) for use in humans are de- dulla are the primary sites of EPO production;
scribed; this is followed by a limited discussion within the liver, a subset of hepatocytes, in
of one cytokine, stem cell factor, that cur- addition to the fibroblastoid fat storing Ito
rently has orphan drug status, and a brief cells, retain the capacity to produce EPO (25).
description of two cytokines currently under- Under normal physiological conditions, circu-
going clinical evaluation. The field of cytokine lating EPO levels are between 10-20 mU/mL
research is ever expanding as new interleu- in plasma (approximately 0.1 nM). In response
kins are identified each year. As our under- to tissue hypoxia or anemia, circulating EPO
standing of the cell and molecular biology of levels can increase 100- to 1000-fold. The cel-
cytokine action on hematopoiesis increases, lular and molecular mechanism(s) that senses
the clinical use of these agents will become and signals for increased EPO gene expression
more refined. is unclear. There are data that suggest that
Hematopoietic Agents

the putative oxygen sensor in EPO-producing carboxy-terminal Trp-Ser-x-Trp-Ser motif (x


cells is a ferroprotein (26). The ferroprotein = nonconserved amino acid). The EPO recep-
may use a hemoglobin-like mechanism in tor contains a CRH region, but no Ig or FNIII
which a heme containing iron would revers- domains. In response to EPO binding, EPO
ibly bind oxygen (27) or alternatively use a receptors homodimerize and may also het-
nonmitochondrial electron transport mecha- erodimerize with other cytokine receptors,
nism that involves an iron moiety (28,29). In- such as the receptor for stem cell factor (37).
dependent of the mechanism through which Receptor homo- or heterodimerization leads
the cell senses changes in oxygen status, to receptor activation and activation of intra-
production of hypoxia inducible factor-la cellular signaling pathways (38,39). EPO-me-
(HIFla) is increased. HIFla heterodimerizes diated activation of the EPO receptor leads to
with HIFlP (also known as ARNT), which is phosphorylation of tyrosine residues on the
constitutively expressed. HIFla/HIFlP het- EPO receptor (40) and activation of phospha-
erodimers bind to hypoxia inducible elements tidylinositol 3-kinase (41, 42) as well as pro-
(HREs) present in the promoter region of the tein kinase CE (43). EPO-mediated activation
EPO gene, leading to an increase in EPO gene of the tyrosine kinase Jak2, leading to activa-
transcription. Once there has been an appro- tion of the STAT5 transcription factor, has
priate increase in red blood cell mass, EPO also been observed (44, 45). EPO-mediated
production declines. changes in gene expression include increased
expression of the c-myc gene (46, 47) and in-
2.2 Bioactivity
creased expression of erythroid specific genes
EPO induces erythrocyte production by stim- (48).
ulating the proliferation and differentiation of
2.3 Therapeutic Indications
erythroid progenitors termed burst forming
units-erythroid (BFU-E) (24) and the prolifer- EPO is used in the treatment of disease-re-
ation and differentiated activity of more ma- lated anemias that are defined by a reduced
ture erythroid precursors (proerythroblast, red blood cell volume for which a blood trans-
erythroblast) (30). EPO can stimulate mega- -
fusion is antici~atedor needed. EPO would
karyocyte colony formation in vitro (31, 32) not be used for correctable anemias, e.g., iron,
and platelet production in mice (33). However, folate, or vitamin B,, deficiencies. Current
in humans, EPO administration has had in- FDA-approved uses for EPO are for anemia
consistent effects on platelet levels. Conse- associated with chronic renal failure. anemia
quently the physiological relevance of the in in acquired immune deficiency syndrome
vitro and animal studies indicating that EPO (AIDS) patients receiving azidothymidine
induced changes in platelet formation is un- (AZT), anemia in cancer patients receiving
clear. EPO effects are mediated through a cell chemotherapy (49), and for allogenic blood
surface receptor composed of a single mem- transfusions in patients undergoing elective,
brane-spanning domain (34,35). The EPO re- noncardiac, nonvascular surgery (50-52).
ceptor is a member of the cytokine receptor Anemia in patients with chronic renal failure
superfamily (CKR-SF) that includes receptors is chiefly the consequence of nonfunctioning
for interleukins 2-7, G-CSF, GM-CSF, TPO, kidneys failing to produce enough EPO (53,
and the receptors for two nonhematopoietic 54). Other contributing factors include the fol-
ligands, prolactin and growth hormone (36). lowing: hemolysis, blood loss, aluminum tox-
The CKR-SF is distinguished by common do- icity, hyperparathyroidism, and folate defi-
mains present in the extracellular portion of ciency. In poorly dialyzed patients with end-
the receptors: a cytokine receptor homologous stage renal disease, accumulation of uremic
(CRH) region, and in some but not all recep- toxins (polyamines)in the blood can produce a
tors, an immunoglobulin-like (Ig) domains bone marrow suppression (55). The etiology of
and/or a fibronectin type 111-like (FNIII) do- the AIDS-related anemia is unknown; contrib-
mains. The CRH region contains two con- uting factors include diminished production of
served sequence motifs: four conserved cys- EPO as well as an AZT mediated down-regu-
teine residues in the amino terminal half and a lation of EPO receptors on bone marrow pro-
tor cells (56). One of the dose-limiting tox- receiving EPO for the treatment of anemia as-
es associated with AZT therapy in AIDS sociated with chronic renal failure is the devel-
emia and nearly one-half of all opment or reoccurrence of hypertension. Hy-
treated patients require red blood cell pertensive episodes, usually in the first 3
sfusions. Anemia in patients receiving months of EPO therapy, may be related to an
is associated with increased cir- increase in total peripheral resistance that oc-
g levels of EPO, and the underlying curs in response to reversal of the anemia-in-
gy may include the following: impaired duced vasodilatation and the increase in vis-
to EPO (57) or elevated circu- cosity of whole blood (60). Other side effects
kines that negatively regu- observed include clotting at the sit of vascular
poiesis (e.g., tumor necrosis fat- access in renal dialysis patients, development
al evaluation is the use of iron deficiency that is related to increased
anemias associated with chronic dis- use of stores, and seizures possibly linked to
e (e.g., rheumatoid arthritis); anemia of increases in blood pressure (see Ref. 61 for an
anemia, myelodysplas- extensive list).
syndromes, and in response to surgical 2.5 Pharmacokinetics
EPO stirnulatory effects on erythroid pro- Native EPO is a mixture of a and P forms that
nitor proliferation and differentiation gen- have identical protein content and effects in
ates increased numbers of erythroblasts and vivo but differ in carbohydrate content; the a
ticulocytes, leading to increased numbers of form contains more N-acetylneuraminic acid.
s. In general, erythro- Glycosylationis required for biological activity
properties (size and volume) are unaf- in vitro and in vivo and prolongs the EPO half-
ed. The increased hematocrit is paralleled life in vivo. Increased sugar chain-branching
an increased hemoglobin level that can lead (tetra- versus bi-antennary) reduces the rate
a decline in plasma iron and ferritin concen- of clearance; a decrease in half-life is detected
arget of EPO action is following removal of sialic acid residues be-
erythroid cell lineage, and meaningful cause of rapid clearance by the liver (62, 63).
Commercially available EPO (recombinant
able effects on plate- human; rHuEPO) preparations are obtained
noted. The changes from Chinese hamster ovary cells engineered
be a consequence of to express the human gene. Because a mam-
sult of a reactive malian cell line is used for production,
ombocytosis elicited in response to the iron rHuEPO is glycosylated; commercial rHuEPO
ncreased hemoglo- preparations may differ in the degree of glyco-
sylation and sugar chain-branching. Glycosyl-
ation may explain the nonimmunogenicity of
rHuEPO preparation; local skin irritations
t blood loss, and infection or inflammatory that are occasionally seen may relate to the
use of human albumin in the preparations.
c benefit of EPO therapy is the reduced rHuEPO is administered parenterally (e.g.,
for, and in some cases elimination of, intravenous infusion, subcutaneous injection,
transfusions. The reversal of the anemia or intraperitoneal in patients undergoing peri-
toneal dialysis). There is no clinical advantage
ty of life parameters (e.g., senses of well to the intravenous versus subcutaneous route
of administration unless a venous access de-
vice is in place. RHuEPO distributes into a
single compartment, and the volume of distri-
e effectsassociated with EPO therapy seem bution approximates or slightly exceeds
be a consequence of reversing the anemia plasma volume. There is limited information
opposed to a response to EPO itself. The on elimination kinetics, but rHuEPO seems to
ost common adverse effect seen in patients decline in a first-order fashion. The mean
Hematopoietic Agents

plasma elimination half-life ranges from 4 to approximately 2.5 kb in length, is composed of


16 h in healthy individuals and individuals four exons and three introns, and is expressed
with chronic renal failure (64). Desialylated as a 1-kb transcript in T-lymphocytes, macro-
EPO is generated and cleared by the liver; ap- phages, mast cells, endothelial cells, osteo-
proximately 10% of the administered dose ap- blasts, and fibroblasts (71, 76, 77). Under
pears unchanged in the urine. RHuEPO (in- basal conditions, there is little, if any, GM-
travenous or subcutaneous) is normally CSF gene expression. In response to immune
administered two to three times weekly. The or inflammatory stimulation/mediators such
onset of rHuEPO action is within 1-2 weeks, as tumor necrosis factor or IL-1, steady-state
with the desired effects seen in 8-12 weeks. levels of GM-CSF transcripts increase dramat-
ically. Changes in the steady-state level of GM-
2.6 Preparations CSF are mediated at the transcriptional as
well as the post-transcriptional level. Tran-
There are two commercially available prepa-
scriptional changes in GM-CSF gene expres-
rations of rHuEPO currently available: epo-
sion are mediated through a combination of
etin alfa (EPOGEN, Amgen) and PROCRIT
trans acting factors including NF-KB,Elk-1,
(Ortho Biotech). Both preparations are de-
and AP-1 binding to cognate cis acting ele-
rived from the Chinese hamster ovary cells en-
ments located in the region of the GM-CSF
gineered to express the human EPO cDNA, gene that flanks the 5' end of the coding region
are nearly identical preparations, and are pre-
(78-81). Post-transcriptional changes in GM-
pared for parenteral administration for IV or CSF in mRNA levels are mediated through
subcutaneous use in sterile preservative-free
AU-rich elements located in the 3' untrans-
solutions.
lated region of the GM-CSF mRNA. The AU-
rich sequences serve as binding sites for an
3 GRANULOCYTE-MACROPHAGE RNA activity that may be inactivated in re-
COLONY-STIMULATING FACTOR sponse to IL-1 or tumor necrosis factor-a! (for
reviews see refs. 77,82). The human GM-CSF
A biological activity that supported the in vitro protein is a monomer that contains two inter-
growth of neutrophil and mononuclear cells nal disulfide bonds and is composed of 144
was initially detected in culture media condi- amino acids; the first 17 amino acids compose
tioned by phytohemagglutin-P stimulated pe- the leader sequence. The crystal structure
ripheral blood lymphocytes (65). Based on this for GM-CSF solved to 2.4 A revealed a two
study and others (66-69), human granulo- stranded-antiparallel P sheet with an open
cyte-macrophage colony-stimulating factor bundle of four a helices (83). The predicted
(GM-CSF) was purified from culture super- molecular mass for the GM-CSF is also a gly-
nates obtained from a T-lymphoblast cell line coprotein; consequently, the secreted forms of
infected with human T-cell leukemia virus-I1 the GM-CSF protein range in size from 18 to
(70). At that time, the novel approach of ex- 22 kDa. In contrast to EPO, where complete
pression cloning was used to isolate the hu- removal of the carbohydrate portion elimi-
man GM-CSF cDNA from a cDNA library con- nates biological activity in vitro because of loss
structed with mRNA isolated from lectin of structure, removal of the carbohydrate on
stimulated T-lymphoblasts and expressed in GM-CSF is associated with an increase in spe-
COS-1 cells (71). The first phase I and I1 clin- cific activity (84). The carbohydrate portion of
ical trials with GM-CSF were conducted in GM-CSF has been speculated to interfere with
1987 (72, 73). GM-CSF receptor binding and/or receptor ac-
tivation. In normal healthy adults, circulating
levels of GM-CSF are near or below the limits
3.1 Physical Properties
of detection (0.1 ng/mL by enzyme-linked im-
The human GM-CSF gene has been mapped to munosorbent assay). Increased circulating
chromosome 5q21-q32 within 500 kb of sev- levels of GM-CSF have been seen in some, but
eral other cytokine genes including IL-3, IL-4, not all burn patients (851, in transplant recip-
and IL-5 (74,75). The human GM-CSF gene is ients preceding an infection (861, and in can-
3 Granulocyte-Macrophage Colony-Stimulating Factor

cer patients with malignant disease and high GM-CSF binding to its a-chain can signal for
an increase in glucose uptake (93). The high
affinity signaling receptors are composed of a
is consistent with the concept that GM-CSF ligand specific a-chain plus a transmembrane
most likely works at the local level in a para- PC-chain,which are shared by 11-3, IL-5, and
wine or autocrine fashion. Consistent with GM-CSF. The &-chain contains a longer cyto-
this model, elevated levels of GM-CSF have plasmic tail compared with the a-chain. Cur-
rent models predict that IL-3, IL-5, or GM-
with chronic inflammatory disease (88). Inter- CSF binds to high affinity aPCheterodimers;
estingly, in eosinophils, a GM-CSF target, ob- this results in a ligand-dependent interaction
tained from asthmatics, the GM-CSF protein between the a- and &-chain cytoplasmic re-
is found in granules (89). In these patients, the gions that initiate the signaling process. Puta-
local release of GM-CSF could enhance in the tive post-receptor signaling pathways acti-
inflammatory response during an asthmatic vated in response to GM-CSF binding include
changes in ion fluxes, inositol phosphate
mobilization, activation of protein kinase C,
and the mitogen-activated protein kinases
inant GM-CSF supports the (MAPK; for review see Ref. 94). GM-CSF-in-
differentiation of granulo- duced changes in gene expression have been
macrophage progenitors (CFU-GM) and linked to activation of the tyrosine kinases
tivity of mature macro- J a k l and Jak2, leading to activation of the
s, neutrophils, and eosinophils (90). En- STAT5 transcription factor (95, 96), and acti-
d functional activities of mature cells in- vation of the MAPK cascade, leading to activa-
ng: tumoricidal activity, tion of the Elk and Egr-1 transcription factors
tibody-dependent cell-mediated cytoxicity, (97). Consistent with a role in promoting cel-
ion, phagocytic activity, se- lular proliferation, one response to GM-CSF is
kines (e.g., GM-CSF stim- increased expression of growth-related genes
CSF-1 production by macrophages). In such as c-fos, c-jun, and c-myc.
-3, IL-6, and SCF, GM-
3.3 Therapeutic Indications
liferation and differenti-
e myeloid progenitors FDA-approved uses for recombinant GM-CSF
ination with EPO, are to accelerate myeloid recovery in patients
-CSF will support the proliferation and with non-Hodgkin's lymphoma, acute lym-
rentiation of erythroid progenitors and phoblastic leukemia, or Hodgkin's disease un-
rentiation of cells in dergoing autologous bone marrow transplan-
(see Fig. 5.1). GM- tation; to prolong the survival of adults who
F effects are initiated at the plasma mem- have undergone allogenic or autologous bone
me in response to GM-CSF-mediated re- marrow transplantation and in whom engraft-
ptor activation. The GM-CSF receptor is a ment is delayed or has failed; to accelerate
he cytokine recep- neutrophil recovery in patients with acute
myeloid leukemia that have received chemo-
therapy; and to mobilize hematopoietic pro-
heterodimers, and genitor cells into circulation for collection by
3, IL-5, or GM-CSF binds to an IL-3-, leukapheresis. In general, patients who have
5-, or GM-CSF-specific ligand-binding received high dose chemotherapy with or
hain. Like other members of the CKR-SF, without the subsequent replacement of hema-
topoietic stem and progenitor cells (bone mar-
row, peripheral blood, or umbilical cord blood
ion of the defin- transplant) have an initial period of neutrope-
atures of the CKR-SF family members). nia that is associated with an increased risk of
general, the low affinity trans-membrane developing a life-threatening infections. In
hain does not transduce a signal; however, clinical trials, patients receiving GM-CSF
Hematopoietic Agents

therapy had a more rapid neutrophil recovery, 3.4 Side Effects


fewer days of antibiotic treatment, fewer in-
Sargramostim (Leukine, Immunex), recombi-
fectious episodes, and spent fewer days in the
nant human GM-CSF (rHuGM-CSF), is pro-
hospital (98-101). Under investigation is the duced in yeast and is usually well tolerated at
use of GM-CSF to increase neutrophil counts clinically useful doses. rHuGM-CSF differs
in patients with AIDS; congenital, cyclic, or from native GM-CSF by the substitution of a
acquired neutropenias; myelodysplastic syn- leucine for a proline at position 23, and poten-
drome; or severe aplastic anemia. Also under tially, a difference in the nature and amount
-
evaluation is the use of GM-CSF to recruit uui- of carbohydrate present. The most common
escent malignant hematopoietic stedprogen- side effects observed with sargramostim in
itor cells into cycle, thus rendering them re- placebo-controlled studies were diarrhea,
sponsive to subsequent chemotherapy. GM- asthenia, rash, and malaise. With intrave-
CSF mobilizes hematopoietic stedprogenitor nous opposed to subcutaneous administra-
cells from the bone marrow into circulation, tion, edema, capillary leak syndrome, pleural,
and this effect of GM-CSF has been used when andlor pericardial effusion have been seen
peripheral blood is harvested for use in autol- (109). The fluid retention and capillary leak
ogous peripheral blood stem cell transplants syndrome may be the result of GM-CSF-in-
(102-104). duced production of tumor necrosis factor by
The initial response to GM-CSF adminis- neutrophils. A first dose effect has been seen
tration is a transient drop in circulating neu- with patients receiving sargramostim; more
severe first dose effects are seen with mol- 4
trophils and monocytes caused by margin-
ation and sequestration of neutrophil and gramostim (rHuGM-CSF expressed in bacte-
ria) and regramostim (rHuGM-CSFexpressed
monocytes in the lung (105); within 2-6 h,
in Chinese hamster ovary cells). First dose ef-
neutrophil and monocyte counts return. GM-
fects can include transient flushing, tachycar-
CSF responses are dose-dependent and bipha-
dia, hypotension, musculoskeletal pain, nau-
sic. Increased neutrophil counts are seen with
sea, vomiting, dyspnea, and a fall in arterial
low GM-CSF doses; at higher doses, increased
oxygen saturation. The latter two responses
numbers of monocyte/macrophages and eosin-
are thought to be caused by sequestration of
ophils are seen. After initial transient changes,
neutrophils in the pulmonary circulation. The
there is a steady increase in numbers of circu-
potential for drug-drug interactions may ex-
lating neutrophils, followed by a plateau 3-7
days after initiation of GM-CSF therapy. A
ist if rHuGM-CSF is given simultaneously
with drugs known to have myeloproliferative
second increase in circulating neutrophils is
effects, e.g., lithium and corticosteroids.
seen 4-5 days after the initial plateau. Redis-
tribution of mature cells from the marrow. a
3.5 Pharmacokinetics
shortening maturation time, and retention of
neutrophils in the vasculature (decreased ex- Sargramostim and regramostim are produced
tra vascular migration and increased demar- as glycoproteins. Both GM-CSF preparations
gination) accounts for the first phase of neu- differ from each other and from endogenous
trophil production. The second phase is GM-CSF in the degree of glycosylation. Anal-
caused by GM-CSF recruiting into cycle and ysis of regramostim and molgramostim (non-
decreasing the cell cycle time of the stedpro- glycosylated rHuGM-CSF) in humans after
genitor cell compartment (106); this also leads subcutaneous administration revealed that
to increased number of circulating stedpro- nonglycosylated GM-CSF was absorbed more
genitor cells (107,108). GM-CSF has a primar- rapidly and to a greater extent, but was also
ily myeloid effect; in general, lympho&te, re- cleared more rapidly than glycosylated
ticulocyte, and erythrocyte counts remain rHuGM-CSF. Whether the carbohydrate moi-
unchanged with variable effects on platelet ety accounts for the differences observed or
counts. Neutrophil counts return to pretreat- whether there are additional differences be-
ment levels 2-10 days after GM-CSF therapy
is discontinued.
4 Granulocyte-Colony Stimulating Factor

ing Sargramostim (rHuGM-CSF produced in degenerate oligonucleotides probes were used


yeast) developed antibodies that recognize the to identify and clone the human G-CSF cDNA
rHuGM-CSF. Mapping studies revealed that (115-117). The first clinical trails with recom-
the antibodies were recognizing epitopes that binant human G-CSF were performed in can-
are glycosylated in native GM-CSF (110). cer patients who had received chemotherapy
rHuGM-CSF is administered parenterally (in- (118-120).
travenous infusion, subcutaneously). When
4.1 Physical Properties
administered subcutaneously, rHuGM-CSF is
absorbed rapidly with peak serum concentra- The human G-CSF gene locus spans 2.3 kb,
tions occurring within 2-4 h. The information contains five exons and four introns, and has
available on sargramostim suggests it distrib- been mapped to chromosome 17q21-22 (117,
es into two compartments with first-order 121, 122). G-CSF is expressed as a 2-kb tran-
etics. It is unclear into which tissues Sar- script (122) in monocytes, macrophages, neu-
gramostim distributes or how it is metabolized trophils, fibroblasts, and endothelial cells. In
andlor eliminated. Serum concentrations de- general, basal G-CSF gene expression is low;
cline in a biphasic manner (109). Sargramos- in response to inflammatory/immune media-
tim is most commonly administered as an tors (e.g., tumor necrosis factor, IL-l, endo-
can also be adminis- toxin, M-CSF, and GM-CSF), steady-state lev-
and myelopoietic ef- els of G-CSF mRNA increase because of
1
obtained following either route of admin- enhanced G-CSF gene transcription and in-
! ration are comparable. creased stability of the G-CSF mRNA (123). In
parallel to GM-CSF, changes in G-CSF gene
transcription are mediated by trans acting fac-
1 tors, including NF-ILGICEBP and NF-KB,
- amostim is the only FDA-approved form
binding to their cognate cis acting elements,
- uGM-CSF available in the United States.
located 5' to the G-CSF protein coding region
- a powder, which also con-
(124, 125). The G-CSF mRNA also contains
I ns mannitol, sucrose, and tromethamine, in
AU-rich elements that contribute to the regu-
3 water for p a r e n t e d lation of G-CSF mRNA stability. Structurally,
f lgramostim (Gramal, Pro- G-CSF, like GM-CSF, has a four a-helical bun-
e -Gm, Gautier; Leucomax, dle topology (126, 127). The human G-CSF
- vartis, Sandoz, Schering-Plough, UpJohn,
protein contains two internal disulfide bonds
Y one-Poulenc Rorer) is available outside the
and is composed of 204 (or 207, see below)
e amino acids; before secretion, a 30-amino
acid leader sequence is removed. Native
4 GRANULOCME-COLONY STIMULATING G-CSF is a glycoprotein with a molecular mass
of approximately 20 kDa; without the carbo-
d hydrate, the predicted molecular mass is
LS A biological activity that promoted neutro- closer to 18.6 kDa. There are two alternatively
LS philic differentiation of a mouse myelomono- spliced variants of G-CSF; one encodes a 177
1- cytic cell line was first identified in the serum amino acid mature peptide and the other en-
I- of endotoxin-treated mice (111). Subse- codes a 174 amino acid mature peptide. Two
31 quently, a similar activity present in media splice donor sites are present at the 5' end of
%t conditioned by lungs obtained from endo- intron 2 with one acceptor site at the 3' end of
re toxin-treated mice formation was identified intron 2 (117). The splice donor sites are
30 ulocyte-colony stimulat- present in tandem, nine base pairs apart. Con-
?d activity (112). Initially human granulo- sequently, the larger protein contains three
)i- -colony stimulating factor (G-CSF) activ- additional amino acids (val-ser-glu) between
Dr was detected and purified from media Leu35 and Val36. The 174 amino acid form of
e- dder carcinoma and a G-CSF predominates in vivo and is approxi-
is ous carcinoma cell line (113, 114). mately 20-fold more active than the 177 amino
v- -CSF protein sequence, acid form. In normal healthy adults with a
Hematopoietic Agents

normal neutrophil count, circulating levels of plasma membrane spanning domain orienta-
G-CSF are <78 pg/mL as measured in en- tion, a carboxy terminal Ig followed by a CRH
zyme-linked immunoassays (128). Increased region and three FNIII domains. Mutagenesis
circulating levels of G-CSF have been detected studies have revealed that the carboxy termi-
in some patients with leukemia (129), in pa- nal Ig domain and the CRH region are re-
tients with cytoxic chemotherapy-induced quired for G-CSF binding and subsequent re-
neutropenia (129),and during the acute phase ceptor oligomerization (137, 138). Immediate
of an infection (130). down-stream effectors of ligand-activated G-
CSF receptor include the tyrosine kinases,
4.2 Bioactivity
J a k l and Jak2 (139, 140), that in turn phos-
Insight into the role of endogenous G-CSF can phorylate and activate the transcription factor
be gained from the phenotype seen in mice in STAT3, thus leading to changes in gene ex-
which the endogenous G-CSF gene has been pression ((141); for in depth review see ref.
inactivated ("knock-outs") (131). G-CSF (95)). Increased CAMP levels and release of
knock-out mice have chronic neutropenia that membrane-associated arachidonic acid are
is reversed after administration of G-CSF, also detected after G-CSF receptor activation
which suggests one function of endogenous G- (142, 143).
CSF is to maintain normal neutrophil levels.
4.3 Therapeutic Indications
When challenged with infectious agents, e.g.,
Listeria monocytogenes, the G-CSF knock-out Current FDA-approved indications for G-CSF
mice developed more severe infections, had a theraw- " are to decrease the incidence of infec-
higher mortality rate, and did not respond tion in patients with nonmyeloid malignancies
with neutrophilia. The latter observation indi- receiving myelosuppressive chemotherapy
cates endogenous G-CSF may mediate the regiments that are associated with a signifi-
characteristic neutrophilia seen in response to cant incidence of severe neutro~eniawith fe-
infections. In vitro, recombinant G-CSF is a ver and to reduce the duration df neutropenia
lineage restricted growth factor that affects and neutropenia related clinical sequelae, e.g.,
the proliferation, differentiation, and activa- febrile neutropenia in patients with nonmy-
tion of progenitors cells committed to the eloid malignancies undergoing myeloablative
granulocytic/monocytic lineage (CFU-GM; see chemotherapy followed by bone-marrow
Fig. 5.1). In combination with other cytokines, transplantation. The myelosuppression asso-
e.g., IL-3 or GM-CSF, G-CSF can support the ciated with certain chemotherapy regimens
proliferation of more primitive progenitors and the myeloablative chemotherapy before
(132-135). G-CSF stimulates the functional bone marrow transplant places patients at
activities of developing and mature neutro- risk for developing life-threatening infections
phils including chemotaxis, phagocytosis, an- and can limit the length, frequency, andlor
tibody-dependent cellular cytoxity, and en- dose of chemotherapy. Under these condi-
hanced production of superoxide anion and tions, G-CSF therapy can, in general, decrease
hydrogen peroxide (see Refs. 123, 136 for re- the duration of severe neutropenia, infectious
views). G-CSF-mediated effects are initiated episodes, and infective therapy and hospital-
at the plasma membrane in response to G-CSF ization. Ongoing studies are aimed at deter-
mediated receptor activation. The G-CSF re- mining whether prophylactic G-CSF therapy
ceptor is a member of the cytokine receptor is more effective than waiting for the neutro-
superfamily (CKR-SF) that includes receptors penia andlor neutropenic sequelae to occur.
for interleukins 2-7, GM-CSF, TPO, EPO, and G-CSF therapy is approved for patients with
the receptors for two nonhematopoietic li- severe chronic neutropenias (congenital neu-
gands, prolactin and growth hormone (36). As tropenia, cyclic neutropenia, or idiopathic
discussed previously (see Section 2.2), the neutropenia). These patients have decreased
CKR-SF is distinguished by common domains numbers of circulating neutrophils and are
present in extracellular portion of the recep- more susceptible to bacterial infections.
tor. The G-CSF receptor contains in its extra- G-CSF therapy reduces the incidence and du.
cellular domain, in a carboxy terminal to ration of neutropenia related sequelae, e.g.,
4 Granulocyte-Colony Stimulating Factor

fever, infection, and oropharyngeal ulcers, 4.4 Side Effects


d also leads to improved quality of life.
In general, recombinant human G-CSF is well
ether long-term G-CSF therapy should be tolerated and is not associated with the capil-
ed in these patients is under evaluation. In lary leak syndrome seen after administration
parison with GM-CSF therapy, G-CSF of other cytokines, e.g., rHuGM-CSF. Mild-to-
erapy may be more clinically effective, be- moderate bone pain, specifically in bones rich
se G-CSF does not also cause an increase in in marrow, e.g., sternum, spine, pelvis, and
e number of circulating eosinophils. G-CSF long bones, is a common side effect (520% pa-
also used to mobilize hematopoietic stem tients) and is most likely related to increased
progenitor cells into the peripheral blood blood cell production. Reversible elevations in
r collection by leukapheresis. The use of G- serum lactate dehydrogenase, alkaline phos-
F therapy to increase neutrophil counts in phatase, uric acid, and leukocyte alkaline
tients with myelodysplastic syndrome and phosphatase are seen and have been related to
treat the neutropenia in adults with acute increased neutrophil turnover (148). Some pa-
emia undergoing myelosuppressive che- tients with myelodysplastic syndrome have
otherapy is also under clinical evaluation. developed leukemia while receiving G-CSF
ese particular uses of G-CSF therapy are (149, 150). Whether the leukemia was the re-
ntroversial because in both instances there sult of G-CSF-mediated acceleration of the
he potential for G-CSF to serve as a mito- disease process or expansion of leukemic cells
and thus stimulate expansion of leukemia already present is not known. Exacerbation of
t cells. G-CSF is under evaluation in pa- pre-existing inflammatory conditions (e.g., ec-
ents with AIDS or AIDS-related complex to zema, cutaneous vasculitis) has been seen in
some patients (150). More notable in patients
et the granulocytopenia associated with
treated for extended periods of time with G-
ne therapy. CSF is splenic enlargement, which may be
Within the first hour after intravenous or caused by extramedullary hematopoiesis
cutaneous administration of G-CSF, there (151).
drop in the number of circulating neutro-
s and monocytes because of margination 4.5 Pharrnacokinetics
neutrophils/monocytes to blood vessel
.Within 24 h, the numbers of circulating The currently available commercial prepara-
rophil begin to increase because of demar- tion of recombinant human G-CSF (Filgras-
ation of cells and release of maturing neu- tim, Neupogen; Amgen Inc.) is obtained from
hils from the bone marrow (144). The re- Escherichia coli engineered to express the hu-
man G-CSF cDNA. For expression in E. coli, a
nse to G-CSF is dose-dependent, and
methionine group was added to the amino ter-
utrophil numbers generally plateau within minus of the human G-CSF protein conse-
week. G-CSF treatment leads to a significant quently the recombinant G-CSF is referred to
ase in the rate of neutrophil production as recombinant methionyl human GCSF (r-
consequence of recruiting more myeloid metHuGCSF). Baderially expressed r-metHuG-
genitors into cycle, stimulating progenitor CSF is not glycosylated, whereas endogenous
proliferation, accelerating the maturation G-CSF has an 0-linked carbohydrate moiety
e, and enhancing bone marrow release of at threonine 133. r-metHuG-CSF is functional
g neutrophils (145). Transient changes in vitro and in vivo, which suggests that glyco-
he circulating levels of other hematopoietic sylation per se is not required for biological
8 have been seen in during G-CSF therapy activity. In vitro and in vivo studies with re-
ing increased numbers of circulating combinant human G-CSF expressed in a
old progenitors, monocytes, and lympho- mammalian cell line (Chinese hamster ovary
s, and decreased numbers of platelets cells; Lenograstim, Granocyte; Chugai Phar-
8, 146, 147). After discontinuation of G- maceutical Company), suggest that glycosyla-
herapy, circulating neutrophils numbers tion may increase the stability and potency of
n to pretreatment levels within 4-7 days. G-CSF. R-metHuG-CSF can be administered
Hematopoietic Agents

subcutaneously or intravenously; subcutane- characterization of IL-11 uncovered its ability


ously administered r-metHuG-CSF is rapidly to synergize with other cytokines to promote
absorbed with peak serum concentrations oc- hematopoietic stem and progenitor cell
curring within 4-5 h. Absorption and clear- growth. IL-11 is a member of the gp130 family
ance rates are not dose-dependent and follow of cytokines that includes IL-6, leukemia in-
first-order pharmacokinetic modeling (152). r- hibitory factor, oncostatin M, ciliary neurotro-
metHuG-CSF is rapidly distributed; the high- phic factor, and cardiotropin-1 (for in depth
est concentrations appear in the bone marrow, review ofthe gp130 family see Ref. 156).IL-11,
adrenal glands, kidney, and liver. The volume like other gp130 family members, seems to be
of distribution after subcutaneous or intrave- a mediator of the acute phase response (157).
nous administration averages 150 mL/kg. It is
not known how r-metHuG-CSFis metabolized
or eliminated from the body. The serum half- 5.1 Physical Properties
life of r-metHuG-CSF is approximately 3-4 h The human IL-11 gene is located at chromo-
and varies between patients; contributing fac-
some 19q13.3-13.4 and is composed of five ex-
tors include differences in disease states and
ons and four introns (158). The IL-11 gene is
actual numbers of neutrophils that may con-
tribute to r-metHu-G-CSF metabolism. expressed by a wide variety of cell types in-
cluding fibroblasts, trophoblasts, chondro-
4.6 Preparations cytes, and endothelial cells. IL-11 transcripts
are found in a wide variety of organs in the
Filgrastim (Neupogen; Amgen Inc.) is cur- adult including thymus, spleen, bone marrow,
rently the only FDA-approved form of lung, large and small intestine, kidney, brain,
r-metHuG-CSF available in the United States. testis, and ovary. IL-11 gene transcripts are
r-MetHuG-CSF is packaged in liquid form in either 1.5 or 2.5 kb in size, and the differences
ready-to use vials for either subcutaneous or
in transcript sizes are the result of difference
intravenous administration. Granocyte (Rhone-
in polyadenylation site selection. However,
Poulenc), Granulokine (Roche), Gran (Kirin),
and Grimatin (Sankyo) are available outside both IL-11 transcripts encode the same sized
the United States. Currently SDlOl, a sus- proteins. IL-11 gene expression is under tran-
tained duration Filgrastim, is undergoing clin- scriptional control with cis acting elements for
ical evaluation (153). To generate SD/Ol, a the transcription factors AP1 and CIEBP lo-
polyethylene glycol (PEG) molecule was added cated in the IL-11 gene promoter. Cytokines
to the amino terminus of filgrastim. In com- like tumor necrosis factor-a and IL-1 that ini-
parison with Filgrastim, the plasma clearance tiate the acute phase response enhance IL-11
of SD/O1 was decreased, and the plasma half- gene transcription (159, 1601, as well as mito-
life of SD/Ol was increased. Early clinical eval- gens such as epidermal growth factor. The
uations did not uncover serious toxicities. A presence of AU rich regions in the 3' flanking
significant advantage of SDIO1over Filgrastim region of the IL-11 mRNA suggest that
therapy is that the daily Filgrastim injections changes in IL-11 expression may also be medi-
could be replaced with weekly or biweekly ated at the post-transcriptional level (158).
SD/O1 injections. Unlike most cytokines, IL-11 does not contain
any cysteine residues or potential 0-or
N-linked glycosylation sites; consequently, af-
ter removal of a 21-amino acid signal se-
Interleukin-11 (IL-11) was originally discov- quence, mature IL-ll, with an approximate
ered in an in vitro screen for activities pro- molecular mass of 23 kDa, is secreted from
duced by bone marrow stromal cells that cells. Whereas IL-11 is widely expressed, the
would support the proliferation of an 1L-6-de- IL-11 protein is rarely detected in blood, which
pendent mouse plasmacytoma cell line (154). is consistent with the concept that IL-11, like
Kawashima et al. (155) cloned IL-11 based on GM-CSF, most likely works at the local level in
its ability to inhibit adipogenesis. Further a paracrine or autocrine fashion.
5 Interleukin-1 1

5.2 Bioactivity tential N-linked glycosylation sites in its ex-


tracellular domain. Two isoforms of the
Analysis of mice in which the IL-11 receptor human IL-11 receptor a-subunit have been
a-subunit has been genetically deleted re- identified that differ with one lacking a cyto-
vealed a requirement for IL-11 in embryonic plasmic domain. Transcripts that would en-
implantation
. in which the blastocysts im- code a soluble form of the IL-11 receptor
plant, but the decidualization response neces- a-subunit have also been described. Post-re-
sary for development of the chorioallantoic ceptor signaling pathways activated in re-
placentas fails; thus, the embryos fail to de- sponse to IL-11 include the Jak-STAT path-
velop (161-163). Hematopoietic indices, in- way, MAP kinases, src family of tyrosine
cluding hematocrit, platelet count, and circu- kinases, and phospholipase D (177).
lating white blood cell numbers, appear
5.3 Therapeutic Indications
normal in these animals. Thus, IL-11 does not
seem to be essential for blood cell develop- The only currently FDA-approved use for
ment, proliferation, or maturation. Like IL-6, rhIL-11 is for the prevention of chemother-
IL-11 alone has little if any effect on hemato- apy-induced thrombocytopenia in patients
poietic cell growth in in vitro assays. However, with nonmyeloid malignancies (solid tumors,
IL-11 will synergize with other cytokines, like lymphomas). In general, the greatest benefit
SCF, to support hematopoietic stem and pro- from rhIL-11 therapy is the acceleration of
genitor cell growth. In combination with IL-3 platelet recovery, thus reducing the require-
and IL-6, IL-11 will also support BFU-E, CFU- ment for platelet transfusions. Thus, in
Meg, and megakaryocyte maturation (164- adults, administration of rhIL-11 is initiated
170). IL-11 has effects outside the hematopoi- within 24 h of the last cycle of chemotherapy
etic system, including induction of acute phase with daily administration usually spanning
protein expression in hepatocytes (1711, inhi- 10-21 days until platelet counts of >50,00O/pL
bition of adipogenesis in cultured cell lines are reached. rhIL-11 is not as effective once
(172), and stimulation of embryonic hip- patients become severely thrombocytopenic.
pocampal-derived neuronal differentiation In animal models, IL-11 exerts a protective
(173). Of clinical significance are the effects of effect on intestinal and oral mucosal integrity
IL-11 on intestinal epithelium where the spe- that is normallv" lost after intense irradiation

cific effect of IL-11 depends on the growth and chemotherapy or is compromised in


state of the epithelium. In animal models in gastrointestinal-inflammatory disease states
which the epithelium is damaged as a conse- (178-180).In animal models, IL-11 treatment
quence of the chemotherapy andlor radiation, is associated with increased crypt cell (181)
IL-11 seems to promote the proliferation of and enterocyte proliferation (182, 183). Thus,
the damaged epithelium and simultaneously rHuIL-11 is currently in clinical trials for the
preventing the proliferation of non-damaged treatment of cytotoxic-associated mucositis
epithelium (174). (184) and Crohn's disease (185).
IL-11 effects are initiated at the plasma In preclinical studies in rodents, larger an-
membrane in response to IL-11-mediated re- imals, and nonhuman primates, subcutaneous
ceptor activation. In parallel to other mem- or intravenous rHuIL-11 administration led
bers of the gp130 family of cytokines, IL-11 to dose-dependent increases in megakaryocyte
binds to a ligand-specific a-subunit, and the size and maturation and increased platelet
IL-11 ligand-IL-11 receptor a-subunit com- production. IL-11 had little, if any, effect on
plex interacts with gp130, the signaling sub- the circulating levels of mature red or white
unit shared in common by the different gp130 blood cells; however, increased numbers of cir-
family members (92,175). IL-11 has three dis- culating myeloid progenitors were detected
tinct receptor interacting sites, one site recog- (186). In phase I clinical trials, rHu-IL-11 ad-
nizes specifically the IL-11 receptor a-subunit ministration before chemotherapy led to a
and the other two sites recognize gp130 (176). dose-dependent increase in the number of
The IL-11 receptor a-subunit is a single mem- circulating platelets with no changes detected
brane spanning protein, containing two po- in the numbers of circulating mature white
Hematopoietic Agents

blood cells or myeloid progenitors. In mice, 5.6 Preparations


rHuIL-11 has been shown to inhibit the
Neumegais packaged in 5-mg amounts in a
release in an NF-KB-dependent manner of single-use vial as a sterile, lyophilized powder
pro-inflammatory cytokines (tumor necrosis for subcutaneous administration.
factor-a, IL-1, IL-12) from activated macro-
phages. A reduction in the circulating levels of
pro-inflammatory cytokines combined with the 6 STEM CELL FACTOR
ability of IL-11 to promote the growth of certain
intestinal epithelial cell types may facilitate the Stem cell factor (SCF) is also known as c-kit
repair of damaged mucosal epithelium (180). ligand, mast cell growth factor, or steel factor.
Instrumental in the identification of SCF were
mice with mutations at the steel locus. The
5.4 Side Effects
steel locus in mice was known to encode a gene
In general, rHuIL-11 is well tolerated, and the essential for development of neural crest-de-
majority of side effects detected (extremity rived melanocytes, primordial germ cells, and
edema, dyspnea, and pleural effusions) are hematopoietic stem cells (190); mutations in
thought to be the result of fluid retention and the human locus have not been identified. In
increased plasma volume caused by sodium re- 1990, several groups reported, using a wide
tention (187). Other side effects include in- variety of experimental approaches, the iden-
creased serum levels of acute phase proteins, tification and cloning of the product of the
fatigue, myalgias, and arthralgias (188, 189). steel locus, which encoded a cytokine eventu-
ally termed SCF (191-198).

5.5 Pharmacokinetics 6.1 Physical Properties


Oprelvekin is the active ingredient in the cur- The human SCF gene is located at chromo-
rently available commercial preparation of some 12q22-q24,spans 50 kb, and is composed
recombinant human IL-11 (Neumega; Genet- of eight exons and seven introns (195). The
ics Institute). Recombinant human IL-11 SCF gene is expressed in a variety of cells in-
(rHuIL-11)is obtained from E. coli engineered cluding fibroblasts, bone marrow stromal
to express the human IL-11 cDNA and is non- cells, vascular endothelial cells, keratinocytes,
glycosylated. rHuIL-11 differs from the natu- intestinal epithelial cells, Sertoli's cells, gram
rally occurring molecule in that it lacks an ulosa cells, and various embryonic tissues
amino terminal proline residue, which to date (199-201). Differential splicing generates two
has had no effect on bioactivity either in vitro different SCF mRNAs, one with and one with-
or in uiuo. rHuIL-11 is administered parenter- out exon 6, which encodes a proteolytic cleav-
ally (intravenously, subcutaneously). The se- age site. In the absence of the proteolytic
rum half-life of rHuIL-ll is approximately 7 h. cleavage site, SCF is retained as a transmem-
In comparing intravenous versus subcutane- brane protein at the cell surface that consists
ous administration, subcutaneous adminis- of an extracellular domain (157 amino acids),a
tration limits bioavailability to approximately transmembrane domain (27 amino acids), and
65%. The reason for the decrease in bioavail- a cytoplasmic domain (36 amino acids). SCF
ability is unknown and could be the result of containing the proteolytic cleavage site is syn-
degradation at the site of injection andlor as a thesized as a 248 amino acid protein that is
consequence of entry into the lymphatics, cleaved to generate a 164 amino acid secreted
which bind to target cells in lymph nodes protein (202). Both transcripts are present in
(188). Interestingly, rHuIL-11 seems to be cells that express SCF and both forms, se-
eliminated by metabolism. With daily dosing, creted and membrane-bound, are biologically
an increase in platelet counts can be detected active. Membrane-bound SCF seems to main-
5-9 days after initiation of treatment and will tain its receptor c-kit in a prolonged activation
continue to increase for an additional week state, presumably because of less rapid down-
after withdrawal of treatment. regulation of c-kit protein (203,204). The reg-
6 Stem Cell Factor

ulation of SCF mRNA splicing and SCF pro- cludes release of mast cell mediators such as
tein proteolysis is not well understood. Mature histamine that impact the clinical use of SCF.
soluble SCF contains two internal disulphide SCF effects are initiated at the plasma
bonds, five N-linked sites of glycosylation, and membrane in response to SCF-mediated re-
a molecular mass of 36 kDa. Under normal ceptor activation. The SCF receptor is a mem-
physiological conditions, circulating SCF lev- ber of the split tyrosine kinase family of
els range between 2 and 5 ng/mL (205). The growth factor receptors and is composed of
cellular and molecular mechanisms that medi- five immunoglobulin-like repeats in its extra-
ate changes in SCF gene expression are not cellular domain, a hydrophobic membrane
well understood and may vary by cell type (see spanning domain, and an intracellular ty-
ref. 206 for an in-depth discussion). For exam- rosine kinase domain. The first three immu-
ple, treatment of bone marrow-derived stro- noglobulin repeats constitute the SCF binding
mal-cell pro-inflammatory cytokines, such as domain and the fourth immunoglobulin re-
tumor necrosis factor-1 and IL-1, leads to a peat is required for receptor homodimeriza-
decrease in the steady-state level of SCF tion. The role of the fifth immunoglobulin re-
mRNA (207), whereas IL-1 treatment of hu- peat is not clear. Point mutations in the SCF
man umbilical cord endothelial cells results in receptor kinase domain, ligand-binding do-
an increase SCF mRNA levels (208). main, and the domain immediately proximal
to the membrane spanning domain have been
identified in hematopoietic cells from patients
Analysis of mice containing mutations in ei- with mastocytosis, idiopathic myelofibrosis,
ther the steel locus or the dominant white acute leukemia, and gastrointestinal stromal
spotting (W) locus, which encodes the SCF re- tumors (213).
ceptor, revealed that SCF is important for he- The SCF receptor, like the receptors for
matopoietic cell development and survival, platelet-derived growth factor and colony
l- melanocyte survival, and proliferation and the stimulating factor-1, is a member of the split
d liferation of primordial germ tyrosine kinase family (also known as the type
e ing feature of SCF I11 receptor tyrosine kinases; for an in-depth
1- roposed role for transmem- review see ref. 214). Characteristic of the
d g development where it is growth factor receptor family is an insert re-
8, stulated to be involved in guiding primor- gion within the cytoplasmic tyrosine kinase.
1- and melanocytes In response to SCF binding, c-kit receptors ho-
$8 While addition of modimerize and the tyrosine kinase intrinsic
I0 lanocytes will induce a pro- to the receptor is activated, leading to auto- or
h- rative response (209) and is capable of cross-phosphorylation of the receptor itself,
v- owth of cells within the with several of the phosphorylated tyrosine
;ic (210),to date the majority of studies on residues clustered in the insert region. Re-
n- e focused on the role of ceptor auto/transphosphorylation generates
its s. In vitro, SCF supports binding sites for proteins containing Src ho-
,a proliferation and differentiation of the mology 2 (Sh2) domains. Among the Sh2 bind-
nd nitors that give rise to CFU-GEMM (see ing proteins known to bind c-kit are phospho-
2F .I).Whether the actual target cell is the lipase Cy-1 and a variety of adaptor proteins
'n- atopoietic stem cell is not clear. In the ab- including growth factor receptor-bound 2 and
is ce of other cytokines, SCF will not support SOS, upstream regulators of ras, which in
;ed h in vitro. However SCF turn regulate raf-1-MAPK cascade activity.
in -CSF, and EPO to Additionally several kinases, including Src ki-
se- f CFU-GEMM, BFU-E, nases, the p83a subunit of phosphatidylinosi-
J~Y (211). A significant to1 3'-kinase, and the tyrosine kinase Jak.2 are
in- is stimulation of the found in association with activated c-kit recep-
ion tiation of mast cell tors. The precise role that the individual sig-
m- recursors and functional activity of mature naling pathways play in directing cellular re-
eg- mast cells (212). The latter effect of SCF in- sponses to SCF is under investigation in many
Hematopoietic Agents

laboratories (for an in-depth review see ref. pigmentation, and uricaria. The responses oc-
206). SCF-dependent Jak activation has been cur within 90-120 min after injection, can last
linked to STATla- and STATBdependent up to 48 h, and are reversible. Multisystem
changes in gene expression (215, 216). PI-3 systemic anaphylactoid-type reactions includ-
kinase has been shown to be important for ing skin reactions and respiratory distress
mast cell adhesion to fibronectin (217), have also been observed (221). To counter
whereas PLCy-1-dependent changes have these side effects, antihistamines, an H,-re-
been associated with changes in cell adhesion ceptor antagonist, and p-adrenoreceptor stim-
and chemotaxis. ulants are administered prophylactically
(213).
6.3 Therapeutic Indications
6.5 Pharmacokinetics
Based on its ability in vitro to support the pro-
liferation and survival of hematopoietic cells, SCF is administered parenterally by subcuta-
SCF has been evaluated clinically for in vivo neous injection. Limited information is avail-
and ex vivo expansion of hematopoietic stem able regarding the pharmacokinetics of SCF.
cells, to mobilize hematopoieticstem cells, and After daily subcutaneous administration, in-
in the survival of hematopoietic stedprogen- creased white cell counts were detected within
itors in aplastic anemia. r-metHuSCF cur- 7-15 days (221). In breast cancer patients, a
rently has orphan drug status for use in com- 2-week course of daily SCF injections resulted
bination with G-CSF in patients who have in increased numbers of neutrophils, mono-
undergone myelosuppressiveor myeloablative cytes, lymphocytes, and reticulocytes (222).
therapy. Under these conditions, the combina-
tion of G-CSF plus SCF seems to enhance the 6.6 Preparations
number of hematopoietic stendprogenitor Recombinant human SCF is obtained from E.
cells recovered in peripheral blood and to ac- coli engineered to express the human SCF
celerate engraftment times for neutrophils gene. For expression in E. coli, a methionine
and platelets (213). group was added to the amino terminus of the
SCF alone exhibits little if any effect on the human SCF protein; consequently, the recom-
growth of hematopoietic cells ex vivo. However binant SCF is referred to as recombinant me-
the combination of SCF and other cytokines thionyl human SCF (r-metHuSCF) (223). Re-
such as IL-3, IL-6, and EPO significantly en- combinant human SCF is manufactured by
hances ex vivo expansion of hematopoietic Amgen and is distributed under the name An-
stem/progenitor cells. Administration of SCF cestim.
will lead to a significant increase in the num-
bers of circulating hematopoietic stemlpro-
genitor cells in vivo (218, 2191, an effect that 7 INVESTIGATIONAL AGENTS
presumably reflects the presence of circulat-
ing cytokines. In patients who received The clinical experience gained and successes
r-metHuSCF before chemotherapy, dose-re- achieved with EPO, GM-CSF, G-CSF, IL-11,
lated increases in neutrophils, and at higher and SCF have enhanced the pursuit for clini-
r-metHuSCF doses, increases in platelets, re- cally effective cytokines. As new cytokines
ticulocytes, and circulating progenitors are identified and their associated biological
(BFU-E, CFU-GM) were detected (220). activities are characterized, many will enter
clinical trials. In the following section, the bi-
6.4 Side Effects ological properties and potential clinical appli-
cations of cytokines currently undergoing
The most frequently observed side effects as- clinical evaluation are described.
sociated with SCF therapy are consistent with
the knowledge that mast cells have receptors
for SCF. Adverse responses occur locally at the
site-of-injection and include wheal formation Interleukin-6 (IL-6) is a member of a larger
with edema, erythema, pruritus, skin hyper- cytokine family that includes leukemia inhib-
7 Investigational Agents

itory factor, IL-11, oncostatin M, ciliary neu- clude the tyrosine kinases Jakl, Jak2, and
hic factor, and cardiotropin-1. This cyto- Tyk2 that in turn lead to activate the tran-
e family, collectively termed the gp130 scription factors STAT1 and STAT3 (92, 95,
ly of cytokines, is a key mediator of the 156). Coordinate with the IL-6-dependent ac-
e phase response, which is a systemic re- tivation of the Jaks, activation of the MAPK
ion to inflammation and tissue damage cascade occurs, resulting in activation of the
(157). The human IL-6 gene has been mapped NF-IL6 transcription factor. Together NF-IL6
to chromosome 6p21-p14 and is composed of and STAT3 enhance the expression of genes
hve exons and four introns (224, 225). The whose products mediate the acute phase re-
I t 6 gene is expressed by a wide variety of sponse.
k u e s including fibroblasts, keratinocytes,
Recombinant human IL-6 (rHuIL-6)is pro-
smooth muscle cells, astrocytes, endothelial
duced in bacteria; in contrast to r-metHuG-
cells, T- and B-lymphocytes, pancreatic islet
cells, and nearly all tissue-associated macro- CSF and r-metSCF, the bacterial expression
phage. IL-6 gene expression is primarily under system employed does not result in the addi-
transcriptional control and can be activated by tion of an amino terminal methionine (231).In
n wide variety of pro-inflammatory mediators animal models, administration of rHuIL-6
including 11-1, tumor necrosis factor-a, inter- leads to increased numbers of circulating
ferons, viruses, and bacterial products (226). platelets. In individuals with normal marrow,
After removal of a signal sequence, mature rHuIL-6 administration leads to a dose-depen-
fL-6 is composed of 183 amino acids, has two dent increase in numbers of circulating plate-
sites of N-linked glycosylation, and has a mo- lets that peak 10-14 days after initiation of
h l a r mass of 20-26 kDa. IL-6 alone has no therapy with no changes in circulating white
effect on hematopoietic progenitor cell growth cell numbers (232, 233). Dose-independent
in in vitro assays. IL-6 synergizes with other side effects detected in phase 1 trials included
kines to promote hematopoietic stem and fever, chills, and mild fatigue; at higher doses,
nitor cell growth, T-cell proliferation acute phase proteins were detected in the sera
11 activity, and of significant clinical and dose-limiting hepatic, neurologic, and car-
rest, CFU-Meg proliferation and mega- diac effects (232,234). At the onset of rHuIL-6
ocytic maturation. In addition to affect- therapy, an anemia can develop that resolves
ematopoiesis, IL-6 will inhibit the growth after discontinuation of therapy. The etiology
ous human tumor cell lines grown in of the anemia is unknown, but it may be the
d in animal model systems (227). IL-6 result of red blood cell sequestration in the
s are initiated at the plasma membrane
spleen or changes in plasma volume. rHuIL-6
sponse to IL-6-mediated receptor activa-
alone and in combination with other cytokines
. As described previously for IL-11, an- is being evaluated as a thrombopoietic agent
er gp130 family member (Section 5.21, re-
tors for the gp130 family of cytokines are in patients. The ability of IL-6 to inhibit the
posed of two subunits: a ligand-specific growth of certain human tumor cell lines in
nit and a common signaling subunit, addition to its ability to synergize with IL-2 to
ed gp130 (91,951. The extracellular por- activate cytotoxic T-lymphocytes lead to the
of the IL-6 ligand-specific subunit con- evaluation of rHuIL-6 as an antitumor agent
an Ig-like domain and two FNIII do- (232). However, preliminary results were not
. In response to IL-6 binding to its promising (233). rHuIL-6 is currently manu-
-specific subunit, the complex interacts factured for distribution in the United States
either a gp130 monomer that dimerizes by Novartis. rHuIL-6 is administered paren-
th another gp130 monomer or with gp130 terally (subcutaneously, intravenously). After
mers directly. The end result is a cova- subcutaneous administration, serum levels of
t linkage between two gp130 molecules, rHuIL-6 peak between 2 and 7 h, with an elim-
in turn initiates the cellular signaling ination half-life of 4 h after subcutaneous ad-
S(S) (228-230). Post-receptor signal- ministration and 20 min after intravenous ad-
ays activated in response to IL-6 in- ministration.
Hematopoietic Agents

7.2 Thrombopoietin (TPO-R) is similar to the receptors for EPO


and G-CSF; the extracellular domain contains
Thrombopoietin (TPO) is also referred to as
four conserved cysteine residues in the amino-
megakaryocyte growth and development fac-
terminal half and carboxy terminal Trp-Ser-x-
tor (MGDF). The human TPO gene spans 6 Trp-Ser motif (x = nonconserved amino acid).
kb, is located at chromosome 3q26, and con- Two different c-mpl transcripts that encode
tains five exons and four introns (235, 236). proteins with similar extracellular but differ-
The TPO coding region is interrupted by in- ent intracellular sequences have been identi-
trons present in the same location as those fied (245). The functional significance of the
present in the EPO coding region; the amino two TPO receptor isoforms is not clear. Puta-
terminal half of TPO shares 23% identity and tive downstream effectors of ligand-activated
50% homology with the EPO protein. How- TPO receptors are Jak2 tyrosine kinase, ras-
ever, the TPO protein contains an additional raf-1-MAPK cascade, and phosphoinositol
181 amino acids, enriched for sequences that 3-kinase (246, 247).
direct N- and 0-linked glycosylation, which is Preclinical evaluation of recombinant
not present in the EPO protein. The similari- mouse TPO revealed a dose-dependent in-
ties in gene structure and sequence homology crease in the numbers of splenic and marrow
suggest that the TPO and EPO genes may be CFU-Meg, increased megakaryocyte matura-
derived from a common ancestral gene (237). tion, and increased numbers of circulating
The TPO gene is expressed as a 1.8-kb tran- platelets (240, 246), with no change in the
script in a variety of tissues, with highest lev- numbers of circulating red and white blood
els present in the liver and smaller amounts in cells. Currently recombinant human throm-
kidney, smooth muscle, and spleen (238-240). - bopoietin (rHuTPO) has orphan drug status
The precise cell within these tissues that ex- for use in accelerating platelet recovery in pa-
presses TPO is not known, but TPO tran- tients undergoing hematopoietic stem cell
scripts can be detected in fibroblasts and en- transplantation. Potential applications for
dothelial cells, cells commonly found in many TPO include HIV-related thrombocytopenia,
tissues (237). After removal of a 21 amino acid thrombocytolytic thrombocytopenias, and im-
signal sequence, mature TPO, composed of provement of ability of peripheral blood pro-
332 amino acids, is glycosylated before secre- genitors to reestablish bone marrow function
tion. TPO may undergo a proteolytic cleavage (248-250). Evaluation of rHuTPO in clinical
that would lead to removal of the carbohy- trials after parenteral administration (sub-
drate-rich carboxy terminus before secretion cutaneous or intravenous) revealed a stimu-
(241). In vitro, TPO alone stimulates CFU- lation of thrombopoiesis within 4 days (peak
Meg proliferation and maturation and has no response at 12 days) of intravenous admin-
effect on the growth of other myeloid progen- istration and within 6 days of subcutaneous
itors. TPO will synergize with cytokines that administration (peak response at 12-18
normally have no effect on megakaryopoieses days) (251,252). Adverse reactions includea
(e.g., IL-11, SCF, or EPO) to promote CFU- slight 3-5% increase in the incidence of
Meg growth (242). TPO effects are initiated at asymptomatic thrombocytosis, deep-vein
the plasma membrane in response to TPO-me- thrombosis, pulmonary embolism, and throm-
diated receptor activation. The TPO receptor bophlebitis. Two forms of thrombopoietin are
is the product of the c-mpl cellular proto-onco- currently under investigation for clinical use:
gene, which is expressed in early hematopoi- recombinant human TPO (Genetech), which
etic progenitors, megakaryocytes, platelets, is full-length TPO and a truncated form of hu-
and endothelial cells (243). Consistent with man megakaryocyte growth and development
the biological activity of TPO, mice genetically factor (lacks the carboxyl terminal 169 amino
engineered to not express the c-mpl gene (c- acids residues) that is conjugated with poly-
mpl knockouts) have a dramatic decrease in ethylene glycol (PEG-rHuMDGF; Amgen).
the numbers of circulating platelets with no The clinical use of the PEG-rHuMGDFis cur-
changes in the circulating levels of red or rently unclear; it has been shown to induce the
white blood cells (244). Structurally, c-mpl formation of antibodies that neutralize native
7 Investigational Agents

thrombopoietin, resulting in the development administration leads to a dose-dependent in-


of thrombocytopenia. Antibodies to rHuTPO crease in neutrophils, basophils, eosinophils,
have not been detected. and platelet numbers (261-263); during the
onset of rHuIL-3 therapy, increased numbers
of early myeloid progenitors (CFU-GEMM,
The human interleukin-3 (IL-3) gene is lo- CFU-GM) appear in the peripheral blood
cated at chromosome 5q23-31,9 kb upstream (264). rHuIL-3 has been evaluated in patients
from the GM-CSF gene, and is composed of that have undergone cancer chemotherapy,
five exons and four introns (251,253). Expres- after bone marrow transplantation, in bone
sion of the IL-3 gene is under transcriptional marrow failure and for hematopoietic progen-
and post-transcriptional control and is ex- itor cell mobilization from the bone marrow
pressed as a 1-kb transcript in activated T- (265). The clinical use of rHuIL-3 as a single
lymphocytes, mast cells, and eosinophils agent is limited, because dose-limiting side ef-
(253). After removal of a 19 amino acid signal fects (severe headache, fever, malaise, fatigue,
sequence, mature IL-3, composed of 133 arthralgias) appear at doses lower than those
amino acids, is glycosylated before secretion. necessary to achieve a clinical advantage.
In normal healthy adults, circulating levels of With the goal of minimizing the side effects
IL-3 are near or below the limits of detection associated with IL-3 therapy, a synthetic cyto-
(<25 pg/mL serum) (254). The estimated mo- kine, Synthokine, was developed. Synthokine
lecular mass of IL-3 is 14-30 kDa. In vitro IL-3 is in essence a greatly modified version of na-
synergizes with other cytokines (e.g., GM- tive IL-3 with N- and C-terminal deletions and
CSF, EPO) to support the proliferation and numerous amino acid substitutions in the re-
differentiation of multilineage progenitors maining portion of the protein (266). Syntho-
(CFU-GEMM) and committed progenitors kine binds with high affinity to the IL-3 recep-
(CFU-GM, CFU-Meg, BFU-E) (255,256). IL-3 tor and is more potent than IL-3 in promoting
alone will stimulate the functional activity of the hematopoietic cell proliferation and in col-
more mature monocytes (including produc- ony formation assays (267). Phase 1/11studies
tion of other cytokines) and eosinophils (157- are underway, and preliminary results indi-
260). IL-3 effects are initiated at the plasma cate that Synthokine may be useful for en-
membrane in response to IL-3-mediated re- hancing platelet and neutrophils recovery in
ceptor activation. The IL-3 receptor is a mem- myeloablated patients (268).
ber of a cytokine receptor subfamily that in- The potent synergistic effect of IL-3 and
cludes the receptors for GM-CSF and IL-5. GM-CSF on hematopoietic progenitor cell
Like GM-CSF, IL-3 binds to an IL-3-specific growth in a variety of in vitro and in vivo as-
a-chain that does not transduce a signal. The says led to the development of a genetically
high affinity-signaling receptors are composed engineered GM-CSF-IL-3 fusion protein
of a ligand-specific a-chain plus a transmem- (PIXY321).PIXY321 contains full-length GM-
brane &-chain, shared in common by IL-3, CSF and IL-3 proteins separated from each
IL-5, and GM-CSF. Current models predict other by an amino acid linker (GGGGS), that
that IL-3, IL-5, or GM-CSF binds to a high was included to allow sufficient space for pro-
affinity a& heterodimer that results in a li- tein folding. PIXY321 binds to either the GM-
gand-dependent interaction between the cyto- CSF or the IL-3 receptor with equal or greater
plasmic regions that initiates the signaling affinity as the native cytokine and will support
process (91, 156). Post-receptor signaling BFU-E, CFU-GM, CFU-GEMM, and CFU-
pathways activated in response to IL-3 include Meg growth (269, 270). PIXY321 is more po-
the Jak tyrosine kinases, which in turn, lead to tent that equimolar mixture of IL-3 and GM-
activation of the transcription factor STAT5 CSF in in vitro colony assays. In contrast to
(95,961. IL-3 therapy, where side effects limit adminis-
Recombinant human IL-3 (rHuIL-3)is pro- tration of sufficient rHuIL-3, fewer side effects
duced in bacteria (nonglycosylated) and in were observed for PIXY321. However, results
mammalian cells (glycosylated).In individuals of phase I11 clinical trials revealed that
with normal bone marrow function, rHuIL-3 PIXY321 is no more effective than rHuGM-
Hematopoietic Agents

CSF; consequently, development of PIXY321 therapy has allowed higher doses of chemo-
as a therapeutic was halted. However, build- therapeutic agents (approaching and includ-
ing on the possibility of generating biologically ing myeloablative amounts) to be used and/or
active chimeric proteins, a family of chimeric more cycles of standard chemotherapy, thus
IL-3-G-CSF proteins, collectively termed my- increasing the overall effectiveness of chemo-
elopoietins, have been generated. Based on therapeutic regimens (274). Mobilization of
promising in vivo studies in animals (2711, stem/progenitor cells from the bone marrow in
myelopoietins are being evaluated for their response to cytokine therapy (e.g., GM-CSF,
ability to stimulate neutrophils and platelet IL-3, SCF, IL-11) has several therapeutic im-
recovery, as well as to mobilize hematopoietic plications. First, cytokine therapy followed by
cell progenitors (272). harvesting of peripheral blood progenitors be-
fore administration of myeloablative doses of
chemotherapy allows for the use of autologous
8 SUMMARY AND CONCLUSION versus allogenic stedprogenitor cell trans-
plants, thus diminishing the potential compli-
The development of cytokines as therapeutic cations of allogenic transplants (e.g., identify-
agents has had a major impact on the treat- ing HLA matched donor, graft-versus-host
ment of renal disease and certain cancers. disease). Recent evidence suggests a period of
EPO therapy has led to a significant decline in myelosuppression follows exogenous cytokine
the number of transfusions that patients with therapy (275, 276). Therapeutically, the my-
end-stage renal disease need to receive. Con- elosuppression results in a myeloprotective ef-
sequently, the number of transfusion-related fect during the ensuing period of chemother-
complications, e.g., iron overload, transfer of apy. The next phase of cytokine therapies are
infectious agents like hepatitis B and C, and likely to involve mobilization of stedprogeni-
human immunodeficiency virus has declined. tors followed by ex vivo expansion stemlpro-
Additionally, assessment of "quality of life" genitors before readministration. A clear long-
measures revealed significantly less fatigue term goal will be to use the expanded stem/
and severity of physical symptoms in patients progenitor cells as vehicles for gene therapy.
receiving EPO therapy (273). The develop- An unforeseen benefit of clinical trails with
ment of cytokines as therapeutic agents has the cytokines has been the effects observed on
had a major impact on the management of pa- other organ systems. Clearly the protective ef-
tients receiving chemotherapy. In the past, fect on intestinal and oral mucosal integrity
amount and/or duration of certain chemother- seen with IL-11 is of significant clinical inter-
apeutic regimens was limited by severe myelo- est. In addition to the myelosuppressive/my-
suppression. Life-threatening infections and/or eloablative effects of irradiation and chemo-
bleeding episodes were treated with antibiot- therapy, the intestinal and oral mucosa are
ics and transfusions, placing patients at risk of often severely damaged. Colony stimulating
developing antibiotic resistance/sensitization factor-1 (CSF-1) is a cytokine that promotes
and/or acquiring blood-born viral-related in- the proliferation and differentiation of mono-
fections. Despite these ameliorative measures, cytelmacrophage progenitors and the activity
the severity of the myelosuppression often of mature macrophages, and can synergize
limited the dose and/or the number of cycles of with other cytokines to support the prolifera-
chemotherapy patients received, thus dimin- tion of more primitive progenitors. CSF-1 has
ishing its overall effectiveness. rHuG-CSF been evaluated in several clinical settings, e.g.,
therapy, as well as rHuGM-CSF therapy, min- treatment of fungal infections, antitumor ac-
imizes the neutropenic periods and has led to tivity, and osteopetrosis, with mixed results
decreased antibiotic usage and shorter periods (277). However, CSF-1 has also been evalu-
of hospitalization. RHuEPO has diminished ated in animal models of arteriosclerosis. In
the requirement for blood cell transfusions, response to rHuCSF-1, cholesterol levels in
and current indications are that TPO will de- normal and hypercholesterolimic animals de-
crease the requirement for platelet transfu- clined, and atherosclerotic lesion progression
sion. Moreover, the development of cytokine was slowed and the regression enhanced
I

1 References

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nticoagulants, Antithrombotics,
nd Hemostatics

Contents
1 Introduction, 284
2 Clinical Use of Agents, 284
2.1 Current Drugs on the Market, 284
2.2 Adverse Effects, 291
2.2.1 Bleeding Risks for Antithrombotic and
Fibrinolytic Agents, 291
2.2.2 Other Adverse Effects for
Antithrombotic Drugs, 294
2.2.3 Adverse Effects for Hemostatic Prepa-
rations, 294
2.3 Absorption, Distribution, Metabolism,
Elimination, 295
2.4 Typical Treatment Regimens for Common
Thrombotic Conditions, 298
2.4.1Venous Thromboses, 298
2.4.2Arterial Thromboses, 298
3 Physiology, Biochemistry, and Pharmacology, 299
3.1 Molecular Mechanisms of Thrombosis and
Fibrinolysis, 299
3.2 Mechanisms and Sites of Action of the
Classes of Marketed Antithrombotic and
Antiplatelet Agents, 308
3.2.1 Heparin and Other Anionic Poly-
saccharides, 308
3.2.2 Warfarin and Other Vitamin K-
dependent Inhibitors, 310
3.2.3 Direct Thrombin Inhibitors: A
Historical Perspective, From Concept
to Drug, 310
3.2.3.1Small-Molecule Direct
Thrombin Inhibitors, 311
3.2.3.2Hirudin and Hirudin-Like
Thrombin Inhibitors, 316
3.2.4 Platelet GPIIbDIIa Antagonists, 318
3.2.5 Platelet ADP Receptor Antagonists, 320
3.2.6 Aspirin and Dipyridamole, 321
y Donald J. Abraham 3.3 Thrombolytic Agents: Mechanisms
N 0-471-37029-0 0 2003 John Wiley & Sons, Inc. and Improvements, 322
283
Anticoagulants, Antithrombotics, and Hemostatics

4 Antithrombotic Agents Having Alternative 4.2.2 Platelet PAR-1 and -4 Receptor


Mechanisms of Action, 323 Antagonists, 326
4.1 Inhibitors of Coagulation Factors, 323 4.2.3 Other Platetlet Targets: Serotonin,
4.1.1Direct Active Site Inhibitors of FXa,324 PGI,, and PAF Receptors, 327
4.1.2Inhibitors of FIXa, 325 4.3 Potential Profibrinolytics: Inhibition
4.1.3 Inhibitors of FVIIa and TFIFVIIa, 325 of Factor XIIIa, TAFIa, PAI-1,
4.2 Antiplatelet Agents with Alternative Modes and a2-Antiplasmin, 329
of Action, 326 5 The Future of Antithrombotic Therapy, 331
4.2.1 Agents That Interfere with the
6 Abbreviations, 332
Thromboxane Receptor and
Thromboxane Synthase, 326

1 INTRODUCTION most prevalent side effects of several currently


used antithrombotic drugs.
Maintenance of proper blood flow is a complex Both thrombi and hemostatic plugs are
and highly regulated physiological process, composed primarily of two structural ele-
with multiple complementary and opposing ments: activated platelets that aggregate
mechanisms of control. Normally in the vascu- within the blood vessel, and a protein fibrin
lature, a precise balance is achieved, permit- network that stabilizes the platelet mass (Fig.
ting free flow while also allowing the trigger of 6.1). Thrombin, a serine protease, is one of the
nearly instantaneous clot formation at sites of central modulators of clot formation. It is the
vascular injury to prevent hemorrhage. In- most potent activator of platelets and acts as
deed, blood coagulation evolved in humans the processing enzyme for the production of
and animals as a protective mechanism monomeric protein fibrin units from the pre-
against bleeding. As protective blood clots (he- cursor protein fibrinogen. Once formed, fibrin
mostatic plugs) form and grow after injury, monomers self-assemble into long fibrin
mechanisms exist to maintain or dissolve strands that can be crosslinked by the trans-
them as needed while allowing normal flow in glutaminase Factor XIIIa (FXIIIa), to further
the remainder of the vasculature. Imbalances stabilize the platelet-fibrin mass. FXIIIa is
in the complex regulatory network of coagula- generated by thrombin through activation of
tion and anticoagulation, however, can lead to the zymogen form of the enzyme FXIII.
a variety of pathological consequences, such as Thrombin is itself produced by way of a cas-
hemorrhage or obstructive clot (thrombus) cade involving many other enzymes and cofac-
formation in veins or arteries, leading to tors, and platelets can be activated by many
stroke, pulmonary embolism, heart attack, agents other than thrombin. In addition, there
and other serious conditions. These imbal- are multiple feedback loops and endogenous
ances may result from genetic or acquired con- inhibitors that can either accelerate or
ditions (1). dampen these processes. Plasmin, another
This multifaceted regulatory framework of serine protease, acts to proteolytically degrade
coagulation and anticoagulation inherently fibrin, thus promoting dissolution of the clot.
presents numerous opportunities for inter- This abbreviated description of coagulation
vention with drugs to modulate one or more and thrombolysis is expanded on later in this
pathways, to achieve a therapeutic effect chapter.
when needed. However, one of the overriding
challenges with current or future drug strate- 2 CLINICAL USE OF AGENTS
gies for antithrombotic, thrombolytic (clot-
dissolving), and hemostatic therapy is the pre-
2.1 Current Drugs on the Market
cise correction of the pathologic imbalance
without overcompensating and thus creating Drugs currently marketed as antithrombotics
safety issues. For example, excessive bleeding, include anticoagulant agents that directly or
occasionally life-threatening, can be one of the indirectly interfere with the activity of throm-
2 Clinical Use of Agents

Other coagulation factors

Thrombin

FXlll - FXllla

Stabilized clot

Figure 6.1. Simplified scheme


Clot lysis of clot formation and lysis.

andlor its precursors, and also include an- tide anticoagulant hirudin, originally isolated
atelet agents that interfere with platelet from the salivary glands of a species of leech.
egation (Table 6.1). Hepa- Bivalirudin (41, a 20 amino acid peptide, is a
the low molecular weight heparins highly truncated analog of lepirudinldesirudin
are heterogeneous mixtures of poly- which has certain other engineered modifica-
olysaccharides. A certain fraction of tions in the peptide sequence. Like lepirudin
orate a unique pentasac- and desirudin, bivalirudin binds simulta-
de sequence (la,Fig. 6.2), which binds to neously to the active and fibrinogen binding
endogenous protein antithrombin. This sites of thrombin, thus inactivating it. Ar-
tivate thrombin, factor gatroban (5) reversibly inactivates thrombin
(FXa), the serine protease that activates by binding to just the active site region. The
other coagulation en- platelet antagonists, abciximab (a monoclonal
lb) consists of just this antibody), eptifibatide, and tirofiban disrupt
ccharide sequence. Warfarin (2) indi- aggregation by binding to the platelet glyco-
imits the activity of thrombin and sev- protein (GP) IIIbIIIa receptor in place of the
her coagulation proteins by inhibiting a receptor's endogenous ligand, fibrinogen. Ti-
osttransla- rofiban (6)and the cyclic heptapeptide eptifi-
.Warfarin batide (7) both contain a similar pharmaco-
lant currently mar- phore: an optimally spaced basic nitrogen and
Lepirudin (3a;Fig. 6.3) is a recombi- carboxylate group (Fig. 6.4), mimicking the
5 amino acid polypeptide that can inac- Arg-Gly-Asp (RGD) binding motif of fibrino-
thrombin directly by binding gen. The structures of the other marketed
taneously to its active site and fibrinogen antiplatelet agents (ticlopidine, clopidogrel,
, which is aspirin, and dipyridamole) shown in Fig. 6.4
entical to lepirudin operate through other mechanisms, discussed
the exception of two amino acids at the later.
inus. Structurally, lepirudin and de- Marketed thrombolytic drugs are enzymes
of the natural pep- or nonenzyme proteins that generate plasmin
Table 6.1 Marketed Antithrombotic Drugs
Generic Name Trade Mechanism of Route of
(structure) Name Marketed by Chemical Class Form. Wt." Action Administration
Anticoagulants
Heparin (la) Heparin Eli Lilly; Wyeth- Anionic 15,000 (ave.) Antithrombin IV, SC
Ayerst polysaccharide cofactor
Danaparoid Orgaran Organon Anionic 5500 (ave.) Antithrombin SC
polysaccharide cofactor
(heparhoid)
Dalteparin Fragmin Pharmacia & Upjohn Anionic 5000 (ave.) Antithrombin SC
polysaccharide cofactor
N
w (LMwHb)
Tinzaparin Innohep Bristol-Myers Squibb Anionic 5500-7500 (ave.) Antithrombin SC
polysaccharide cofactor
1-(
Enoxaparin Lovenox Aventis Anionic 4500 (ave.) Antithrombin SC
polysaccharide cofactor
=
()
Fondaparinux (lb) Arixtra Sanofi-Synthelabol Anionic 1728.1 Antithrombin SC
Organon pentasaccharide cofactor
Warfarin (2) Coumadin Bristol-Myers Squibb Coumarin 330.3 Vitamin K Oral, IV
antagonist
Lepirudin (3a) Refludan Aventis Polypeptide 6979.5 Direct "bivalent" IV
(recombinant thrombin
hirudin analog) inhibitor
Bivalirudin (4) Angiomax The Medicines Polypeptide 2180.3 Direct "bivalent" IV
Company (recombinant thrombin
hirudin analog) inhibitor
Argatroban (5) Novastan GlaxoSmith-Kline; Small-molecule Arg- 526.7 Direct active-site IV
Texas based substrate thrombin
Biotechnology analog inhibitor
Abciximab ReoPro Eli Lilly Monoclonal antibody 47,615 GP IIbKIIa IV
(recombinant Fab) receptor antag.
Eptifibatide (6) Integrilin Millennium Cyclic heptapeptide 832.0 GP IIb/IIIa IV
RGD mimetic receptor antag.
Tirofiban (7) Aggrastat Merck Nonpeptide RGD 495.1 GP IIb/IIIa IV
mimetic receptor antag.
Ticlopidine (8) Ticlid Roche Thienopyridine 300.3 P2Y,, receptor Oral
antagonist
Clopidogrel(9) Plavix Bristol-Myers Thienopyridine 419.9 P2Y,, receptor Oral
Squibb; Sanofi- antagonist
Synthelabo
Aspirin (10) Ecotrin; GlaxoSmith-Kline; Acetyl salicylic acid 180.2 COX inhibitor Oral
Regimen Bayer (AM)
Bayer
Dipyridamole (11) Persantine Boehringer Pyrimidinopyrimidine 504.6 PDE inhibitor Oral
Ingelheim
Aspirin + Aggrenox Boehringer - - Combination Oral
dipyridamole Ingelheim
V
"Formula weight, includes salt form and hydrates, where appropriate.
bLMWH, low molecular weight heparin.
Anticoagulants, Antithrombotics, and Hemostatics

(la) Heparin and low molecular weight heparin (LMWH)


(partial structure showing the pentasaccharide motif which
binds to antithrombin)

(1b) Fondaparinux

(2) Warfarin
(racemate)

Figure 6.2. Heparin and LhWH (partial structures), fondaparinux, and warfarin.

+H3N-X1X2MDCTESGQNLCLCEGSNVCGQGNKClLGSDGEKNQCVTGEGTPKPQSHNDGDFEEIPEEYLQ-CO2-
(321) Lepirudin

I X1X2 = L-T

(3b) Desirudin
XI x2= v-v
(Europe)

(4) Bivalirudin

(5) Argatroban

Figure 6.3. Structures of lepirudin, desirudin, and bivalirudin (single-letter amino acid nomencla-
ture; DF, D-Phe), and argatroban.
(6) Eptifibatide

(7)Tirofiban
\
CHa

(8)~iclopidine (R = H)
(9) Clopidogrel (R = C02CH3)

(10) Aspirin

(11) Dipyridamole

Figure 6.4. Structures of marketed antiplatelet agents (abciximab not shown).

n plasminogen, its zymogen precursor (Ta- restore flow. Fibrinolytic therapy is also occa-
6.2). Because all of these agents promote sionally used to treat dangerous thrombi that
llissolution of fibrin, they are also referred to form in the venous system, especially the legs.
fibrinolytics. This type of venous thrombosis [deep vein
A thrombotic occlusion of a coronarv or ce- thrombosis (DVT)] often carries the risk that
bra1 artery results in potentially life-threat- the clot, or pieces thereof, travel to the lung,
hg acute myocardial infarction or ischemic resulting in life-threatening pulmonary embo-
lke, respectively, and fibrinolytic therapy lism (PE). Fibrinolytic therapy is usually ac-
be used to rapidly dissolve the clot and companied by and followed up with adminis-
2 Clinical Use of Agents

(6) Eptifibatide

-
CONH~

(7)Tirofiban

(8)Ticlopidine(R = H)
(9) Clopidogrel (R = C02CH3)

(10) Aspirin

(11) Dipyridamole

Figure 6.4. Structures of marketed antiplatelet agents (abcixiimab not shown).

plasminogen, its zymogen precursor (Ta- restore flow. Fibrinolytic therapy is also occa-
2). Because all of these agents promote sionally used to treat dangerous thrombi that
ution of fibrin, they are also referred to form in the venous system, especially the legs.
This type of venous thrombosis [deep vein
A thrombotic occlusion of a coronary or ce- thrombosis (DVTII often carries the risk that
-

the clot, or pieces thereof, travel to the lung,


resulting in life-threatening pulmonary embo-
lism (PE). Fibrinolytic therapy is usually ac-
e used to rapidly dissolve the clot and companied by and followed up with adminis-
290 Anticoagulants, Antithrombotics, and Hemostatics

Table 6.2 Marketed Thrombolytic (Fibrinolytic) Agentsa


Generic
Name Trade Name Marketed by Description Clot Selectivity
Alteplase Activase Genentech Recombinant natural tPAb Selective
Tenecteplase TNKase Genentech Recombinant modified tPA Selective
Reteplase Retavase Centocor Recombinant modified tPA Nonselective
Streptokinase Streptase AstraZeneca Purified bacterial protein Nonselective
"All are administered IV.
btPA,tissue plasminogen activator.

tration of anticoagulant or antiplatelet agents, clot-triggering protein, and other thrombo-


depending on the pathology. genic material such as collagen and von Wille-
Anticoagulant and antiplatelet agents, brand factor (see Section 3.1). The condition
sometimes in combination, are also used in "unstable angina" is associated with the pa-
patient populations with existing thrombi or thology of rupture-prone atherosclerotic
who are predisposed to thrombus formation, plaques, whereas stable angina is associated
for example, individuals with atrial fibrillation with the presence of plaques, which are not in
or stable or unstable angina, and in patients immediate danger of rupturing.
with artificial heart valves. Also, anticoagu- The currently marketed anticoagulant, an-
lant and antiplatelet agents are used as pro- tiplatelet, and fibrinolytic agents are, in gen-
phylaxis in many surgeries, especially hip and eral, efficacious for the indications in which
knee replacement surgery and coronary artery they are used. On the other hand, many suffer
bypass grafts, and as prophylaxis in other pro- from limitations. As mentioned, unwanted
cedures such as coronary angioplasty. Antico- bleeding can be an issue for many of these
agulant therapy is also used for treatment of agents and therefore intensive patient moni-
DVT and PE. toring may be necessary. Several of the cur-
Although thrombi can form in either veins rent anticoagulant and antiplatelet agents are
or arteries, the structural nature of the clots in limited by the requirement for intravenous
each of these settings differs and therefore the (IV) administration, which necessitates a hos-
optimal agents for drug treatment differ as pital stay. All of the fibrinolytics are adminis-
well. Flow conditions largely dictate the ratio tered by IV. Many of these agents are not
of fibrin to platelets in the growing thrombus. broad-use drugs suitable for a variety of
Arterial thrombi, formed under high flow con- thrombotic indications. The limitations of
ditions, are platelet-rich aggregates, whereas some of these agents might eventually be over-
venous thrombi, formed under more static
conditions, are composed largely of fibrin with
trapped red blood cells and relatively few
platelets. However, as an arterial thrombus
occludes the vessel, a condition of stasis re-
sults, promoting the original platelet-rich clot
to grow a more fibrin-rich component. In this
regard, clinical studies have shown that both
anticoagulant and antiplatelet agents are wall
efficacious for arterial thrombosis, whereas
anticoagulant drugs are superior to antiplate-
let agents for venous thrombosis. Arterial
thrombi are typically the result of the rupture
of atherosclerotic plaques, pathological pock-
ets of lipid and macrophage foam cells that fibrous cap
form beneath the vessel endothelium surface
(Fig. 6.5). Rupture exposes tissue factor, a Figure 6.5. Atherosclerotic plaque rupture.
2 Clinical Use of Agents

come as ongoing clinical study continues to 2.2 Adverse Effects


explore treatment variables (e.g., use of alter-
native dosing regimens, use of different com- 2.2.1 Bleeding Risks for Antithrombotic and
binations of existing agents, or examination of Fibrinolytic Agents. Bleeding is the principal
their use in different thrombotic indications). risk associated with antithrombotic and fi-
At the same time, a great deal of recent re- brinolytic therapy (2). Bleeding episodes can
search effort is directed toward the discovery be major and life-threatening as in cases of
and development of next-generation antico- intracraneal bleeding, or less severe (minor or
agulants and antiplatelet agents. This re- nuisance bleeding) as in bleeding gum or nose
search seeks new agents that operate by either
bleeds. Because thrombotic conditions being -
treated can range from acute and life-threat-
established mechanisms of action or by novel
ening to relatively stable situations, wherein
mechanisms that current drugs do not target. only low level preventive antithrombotic ther-
Whether any of these potential new agents ul- apy is recommended, the bleeding risks need
timately displaces the drugs in Tables 6.1 and to be weighed against the benefit of therapy.
6.2, only time and clinical experience will tell. The bleeding risk is highly dependent on a
In any case, the Holy Grail of antithrombotic number of variables, including the drug being
drug research is the discovery of safe agents used; the thrombotic condition being treated
requiring no patient monitoring and having (e.g., stroke vs. myocardial infarction); level
single-agent efficacy over a broad range of and duration of therapy, whether other anti-
thrombotic indications, and, especially for thrombotic agents are used in combination;
chronic treatment, having good oral bioavail- age of patient; or underlying predispositions
ability. As of 2002, we do not yet have such a to bleeding (e.g., past episodes of gastrointes-
drug on the market. tinal bleeding or recent surgery or trauma)
Many human bleeding disorders result (2). Typically the dose of therapeutic agent is
from inherited defects of certain protein fac- adjusted to the level necessary to achieve the
tors involved in coagulation or platelet func- desired therapeutic effect without causing un-
tion and individuals with these diseases may acceptable bleeding. In the case of anticoagu-
not be able to achieve adequate hemostasis lant therapy, the level or "intensity" is often
without therapy. Hemophilia A and B are carefully monitored using various in vitro
characterized by genetic defects in coagula- tests, which measure the length of time for
tion factors VIII and M,respectively, whereas blood clotting. For example, common clotting
tests measure prothrombin time (PT, often
von Willebrand disease is characterized by
used during warfarin therapy) and activated
functionally defective von Willebrand factor, a
partial thromboplastin time (aPTT, often
glycoprotein required for platelet adhesion to used during heparin therapy). The interna-
the endothelium. Effective therapy for correc- tional normalized ratio (INR) is calculated
tion of hemostasis in at-risk individuals dur- from the PT and is meant to be a better com-
ing acute bleeding episodes or in anticipation parator among individuals/sites when slightly
of surgery can be achieved by replacement or different in vitro clotting reagents are used.
augmentation of one or more of the natural The following describes a few examples of
factors. For this purpose, marketed hemostat- treatments and settings where major bleeding
ics include plasma fractions that are concen- was quantified during anticoagulant therapy.
trated in multiple factors, as well as natural or As part of the International Stroke Trial, pa-
recombinant forms of individual factors (Ta- tients with acute ischemic stroke associated
ble 6.3). with atrial fibrillation were treated with sub-
The remainder of this chapter examines in cutaneously (SC) administered heparin (two
t more detail the current agents
- on the market different doses), aspirin, heparin + aspirin, or
that affect thrombosis and hemostasis, and neither. Heparin at the higher dose caused
then describes newer antithrombotic agents bleeding complications that offset the anti-
under development that operate by either thrombotic benefit. The overall frequency of
known or novel mechanisms. hemorrhagic stroke in the heparin-treated
292 Anticoagulants, Antithrombotics, and Hemostatics

Table 6.3 Marketed Hemostatic Preparationsa


Trade Name Marketed by Description
Factor WII Preparations
Monoclate Aventis Behring FVIII, purified from human plasma
Hemofil Baxter Healthcare FVIII, purified from human plasma
Koate Bayer Biological FVIII, purified from human plasma
Kogenate/Helixate Bayer Biological; FVIII, recombinant
Aventis Behring
Recombinate Baxter Healthcare FVIII, recombinant
ReFacto Genetics Institute FVIII, recombinant

Factor ZX Preparations
Mononine Aventis Behring FIX, purified from human plasma
Beneh Genetics Institute FIX, recombinant

Other
NovoNordisk FVIIa, recombinant

Mixtures of Coagulation Factors


Humate Aventis Behring FVIII + von Willibrand Factor; purified from human
plasma
Bebulin Baxter Healthcare FIX + FIIc + FX, purified from human plasma
Konyne Bayer Biological FIX + FII + FX, purified from human plasma
Proplex Baxter Healthcare FIX + FII + FX + FVII; purified from human plasma
Feibab Baxter Healthcare FIX + FII + FX + FVIIa; purified from human plasma
Autoplexb Nabi FXa + FVII + FVIIa + other factors; purified from human
plasma
"All are administered IV.
bFor patients with inhibitors (antibodies) to FVIII.
%I1 (Factor 11) is prothrombin, the zymogenic form of thrombin (FIIa).

groups after 14 days was 2.1% compared to the LMWH enoxaparin in these surgeries led
0.4% for the patients not treated with heparin to corresponding bleeding rates of 2.3 and
(3). Use of a 7-day IV course of danaparoid for 0.2%, respectively. Although enoxaparin
acute ischemic stroke increased the incidence showed a lower bleeding rate in the knee sur-
of major bleeding (5.2% vs. 1.8% for placebo) gery setting, fondaparinux was more effica-
(4). On the other hand, in several trials involv- cious for both types of surgeries (5a,b).
ing patients with ischemic coronoary artery In a stroke prevention trial in patients who
disease, treatment with heparin or LMWHs had experienced a transient ischemic attack
did not seem to increase the risk of major (TIA) or a minor ischemic stroke, warfarin
bleeding (2). In a combined analysis of a num- therapy resulted in an 8.1% rate of maijor
ber of studies of patients treated for venous bleeding complications (including fatal bleed-
thromboembolism (VTE),use of IV heparin ing) compared to 0.9% for aspirin (6).On the
was associated with major bleeding rates of other hand, major bleeding rates associated
0-7% (fatal bleeding 0-2%) and use of SC with warfarin therapy in patients with atrial
LMWHs was associated with major bleeding fibrillation or in patients after a myocardial
rates of 0 3 % (fatal bleeding 0- 0.8%) (2). Use infarction were relatively lower (7). In a num-
of the pentasaccharide fondaparinux for pro- ber of studies comparing oral warfarin with
phylaxis of DVT in patients undergoing hip or SC heparin or SC LMWHs for the treatment of
knee surgery resulted in major bleeding rates venous thromboembolism, there was a ten-
of 2.2 and 2.1%, respectively, whereas use of dency toward higher rates of bleeding with
Clinical Use of Agents

combination resulted in increased bleeding,


agulation with warfarin was compared with whereas dipyridamole alone was indistin-
guishable from placebo (12,14).
In one study with the thienopyridine anti-
sults in higher bleeding rates than with platelet agent clopidogrel, clinical bleeding
event rates were slightly lower compared to
ompared to heparin, lepirudin increased those of medium dose aspirin (15), whereas in
-threatening major bleed- two other studies that compared aspirin alone
in patients being treated for unstable an- to clopidogrel plus aspirin, the clopidogrel-
a (UA) or myocardial infarction (MI), but it treated patients experienced either the same
or slightly higher major bleeding rates com-
ding risk in the setting of
pared to thsoe of aspirin (16). The related
omboprophylaxis for elective hip replace-
thienopyridine antiplatelet drug ticlopidine
ent. In both of these settings, however, lepi-
has been associated with increased bleeding,
more efficacious than ei- including spontaneous posttraumatic and
heparin or LMWH (9). Use of bivalirudin perioperative bleeding (17).
patients with coronary angioplasty resulted Abciximab, a monoclonal antibody to the
hemorrhagic complications, but at a re- platelet GPIIbiIIIa receptor, was studied in
d rate compared to that of heparin (10). A coronary angioplasty patients in combination
th the small-molecule direct thombin with standard doses of heparin. Significant in-
r argatroban in patients undergoing creases in major bleeding were attributed to
ary intervention (PCI; abciximab, though a later study using reduced
onary angioplasty) showed a slightly doses of heparin showed a reduction of the
er rate of major bleeds compared to histor- bleeding risk (18).A trial of the heptapeptide
heparin rates (11). GPIIbiIIIa antagonist eptifibatide in patients
Bleeding complications are also associated undergoing PC1 showed a modest increase in
latelet agents. Although hemorrhagic complications (19). Moreover, in
relatively safe for most the setting of PCI, the nonpeptide small mol-
of major bleeding risk ecule GPIIbiIIIa antagonist tirofiban, in com-
), it can cause gastro- bination with heparin or aspirin, was associ-
stinal bleeding even at low doses. This ef- ated with an increase in bleeding compared to
is believed to be attributed to combined that of heparin or aspirin alone (20).
The use of thrombolytic (fibrinolytic)
agents in the settings of acute myocardial in-
) effect, and inhibi- farction, ischemic stroke, and VTE carries the
. Functional cellular risk of major bleeding complications, with the
1 in the gastric mucosa is important for most severe being intracraneal hemorrhage
taining mucosal integrity (13). Low dose (hemorrhagic stroke). Three trials employing
is generally better streptokinase for acute ischemic stroke were
own to be as effica- halted prematurely by safety committees be-
s as higher doses for antiplatelet therapy. cause of unfavorable rates of early mortality
contrast, the anti-inflammatory activity of and intracranial bleeding (21). Trials using
hibition of cellular tissue plasminogen activator (tPA) for acute
-2 requires larger doses of aspirin. In gen- stroke reported intracranial bleeding rates of
the risk associated with aspirin therapy is 3-7% (21). Thrombolytic therapy for MI re-
ptable in individuals who are at greater sulted in noncranial major bleeding rates of
of major thrombotic events (12). 1.1%compared to 0.4% for placebo, and re-
sulted in an excess of 3.7 hemorrhagic strokes
alone, appears to have minimal bleeding per 1000 patients compared to that of placebo
(22). Patients who receive thrombolytic ther-
apy for VTE have a 1-2% risk of intracranial
bleeding (23).
Anticoagulants, Antithrombotics, and Hemostatics

Combining fibrinolytic therapy with anti- with this condition. Additionally, studies in
coagulation carries significant risk for bleed- patients with confirmed HIT demonstrated
ing (21). For example, recruitment of patients that the pentasaccharide fondaparinux is not
with acute coronary syndromes (unstable an- associated with in vitro cross-reactivity to hep-
gina or MI) in a trial combining streptokinase arin antibodies, and therefore this agent may
or tPA with heparin or hirudin (desirudin) present low risk for HIT (5c).
was stopped early because of hemorrhagic For all three marketed platelet GPIIb/IIIa
stroke, with rates varying from 0.9% (tPA + antagonists, there have been reports of throm-
heparin) to 3.2% (streptokinase + hirudin). It bocytopenia. In a trial with abciximab, ap-
was found that aPTT values were predictive of proximately 6%of patients developed antibod-
risk of hemorrhagic stroke in patients receiv- ies to the variable regions of abciximab (28),a
ing these combination therapies (24). human chimeric antibody, and about 1-2% of
It is hoped that some of the newer agents patients developed low platelet counts as a re-
(see below) will show improvements in thera- sult (12). Withdrawal of the drug is usually
peutic window and monitoring burden. sufficient for recovery (12). Eptifibatide treat-
ment, although not associated with an in-
2.2.2 Other Adverse Effects for Antithrom- creased frequency of thrombocytopenia over-
botic Drugs. Thrombocytopenia (low platelet all, might be associated with a small increase
count) c& occur during treatment with sev- in cases of profound thrombocytopenia (12,
eral anticoagulant and antiplatelet drugs. He- 19). Thrombocytopenia arising from treat-
parin-induced thrombocytopenia (HIT) is a ment with tirofiban has been reported in a
consequence of an antibody response to a com- small percentage of patients and has been pos-
plex between heparin and endogenous platelet tulated to be the result of an immunologic re-
activating factor 4 (PF4). The resulting anti- sponse to the tirofiban-GPIIbDIIa complex
body-heparin-PF4 complex is thought to in- (29,301. It has been recommended that all pa-
teract with Fc receptors on platelets, leading tients receiving parented GPIIb/IIIa antago-
to platelet activation and thrombin genera- nists be monitored for development of throm-
tion (25, 26). As a result, a general platelet bocytopenia within 24 h of the initiation of
aggregation response ensues with platelet therapy (30).
depletion, and an independent thrombotic Thrombotic thrombocytopenic purpura
pathology, called HITTS (heparin-induced (TTP) is a relatively rare condition that has
thrombocytopenia with thrombosis), often de- been reported in patients treated with ticlopi-
velops. HIT usually develops between day 5 dine and, to a much lesser extent, with clopi-
and day 15 of heparin therapy and affects up to dogrel (31, 32). TTP is characterized by
3% of patients (27). In the absence of overt thrombocytopenia, microangiopathic hemo-
thrombosis, cessation of heparin therapy is of- lytic anemia (fragmented red blood cells), and
ten sufficient to relieve HIT. However, to other symptoms. For ticlopicine, the esti-
manage the thrombotic condition for which mated incidence may be as high as one case in
the heparin was originally indicated as well as every 2000-4000 patients exposed (17). For
any thrombotic complications resulting from clopidogrel, TPP has been reported at a rate of
HIT itself, a switch to a different anticoagu- about 1 case per 250,000 patients exposed
lant (e.g., lepirudin) is often necessary (25). (33).
Given that a minimum of 12-14 saccharide
units is required to form the antibody complex 2.2.3 Adverse Effects for Hemostatic P r e p
with PF4, LMWHs tend to cause HIT less fre- rations. Although the infusion of mixtures of
quently than does heparin (27). However, as a endogenous coagulation factors, especially
result of the observation of in vitro cross-reac- mixtures containing coagulation factor M
tivity to heparin antibodies, LMWHs are con- (FIX), into patients with bleeding disorders
traindicated in patients with known heparin- has historically carried the risk of thrombotic
associated antibodies. On the other hand, the complications (34), such complications are
"heparinoid" danaparoid seems not to induce currently not regarded as clinically important.
HIT and therefore is used to treat individuals Additionally, the historical risk of infection re-
2 Clinical Use of Agents

sulting from viral (e.g., HIV or hepatitis) con- heparin. Heparin is cleared by two distinct
tamination of plasma products or purified co- mechanisms (25, 38). In the first mechanism,
agulation factors is now reduced as a result of which is saturable, heparin binds to macro-
rigorous screening and heat-treating of puri- phages and endothelial cells where it is de-
fied plasma products as well as to the use of graded. The second, slower, and nonsaturable
recombinant coagulation factors. Neverthe- mechanism is renal clearance. Heparin phar-
less, it is generally recommended that individ- macokinetics is therefore not linear, another
uals with hemophilia be vaccinated for hepati- reason for patient monitoring. In the case of
tis A and B. Additionally, parvovirus B19, a the LMWHs, however, protein and cell bind-
non-lipid-enveloped DNA virus, cannot cur- ing for these shorter polysaccharides is sub-
rently be removed from plasma products and, stantially less compared to that of heparin,
although infected adults are usually asyrnp- and pharmacokinetics are therefore less vari-
tomatic, this virus can affect pregnant women able and more predictable. Elimination is al-
or immune-compromised individuals (35). most exclusively by the renal route, with elim-
The presence of low levels of potentially im- ination half-lives of about 3-5 h, except for
munogenic blood group isoagglutins in the danaparoid, which has a half-life of about 24 h.
case of plasma-purified products, and murine LMWHs are given by the SC route usually
or other nonhuman proteins in the case of re- once per day, and patient monitoring, with the
combinant products, is generally regarded as exception of individuals with renal impair-
not a clinically significant risk. However, a ment, is not routine. This superior pharmaco-
well-recognized complication of treatment of kinetic profile for LMWHs is a well-recognized
hemophilia A using replacement FVIII ther- advantage of LMWHs over heparin and one of
apy is the development by the patient of neu- the principal reasons for the increasing use of
tralizing antibodies to the infused FVIII pro- LMWHs instead of heparin (25). Like the
tein (36). Strategies for overcoming this LMWHs, the pentasaccharide fondaparinux
complication include treatment with higher displays a linear, predictable dose-dependent
levels of FVIII when the antibody response is pharmacokinetic profile. It is 100% bioavail-
not overwhelming, or augmentation with able after SC injection, with a half-life of
other coagulation factors, such as FVIIa or 14-16 h, and is eliminated primarily by the
products that contain mixtures of coagulation kidneys (39).
factors (see Table 6.3). The term "oral anticoagulation" currently
refers to warfarin therapy. Warfarin is a race-
2.3 Absorption, Distribution, Metabolism,
mic mixture and no advantage has been estab-
lished in administering a single enantiomer.
Not surprisingly, because of its size and Although R-warfarin is several times more ac-
charge, heparin is not effective by oral admin- tive as an anticoagulant than the S-form, the
is given by intermittent IV in- R-enantiomer has about double the clearance
sion, or SC injection. When rate compared to that of the S-form. Warfarin
e IV route, the full pharmacological is essentially completely absorbed after oral
ost immediately. Given SC, administration, reaching peak levels within
heparin peak plasma levels are achieved only 4 h, and has a mean half-life of about 40 h. It is
er 2-4 h, and the bioavailability is lower almost entirely protein bound (99%).Because
that of the IV route. Subcutaneous ad- of its mechanism of action. the full.~harmaco-
n is either twice or three times dynamic response to a single dose of warfarin
. Recently, however, heparin formula- takes approximately 2-5 days (40). Warfarin
intended to facilitate gastrointestinal ab- interferes with the critical vitamin K-medi-
tion of heparin ("oral" heparin) are start- ated y-carboxylation of glutamic acid residues
estigated in humans (37). Heparin near the N-terminus of several procoagulant
ds plasma proteins and this ac- enzymes (prothrombin, FX, FIX,and FVII) as
s for some of the variability of anticoag- well as two anticoagulant proteins (protein S
response among patients. Patient mon- and protein C). Lacking this critical modifica-
itoring, usually using aPTT, is standard with tion, these enzymes and proteins are essen-
296 Anticoagulants, Antithrombotics, and Hemostatics

Table 6.4 Plasma Concentrations and Half-Lives of Coagulation Factors


Coagulation Factor Plasma Concentration ( p M l Plasma Half-Life (h)
Serine Proteases
FII (prothrombin)
Fx
FIX
FXI
FVII
Protein C

FV
FVIII
Protein S

tially functionally inactive. Because each of When dosed as an IV bolus, the pharmaco-
these proteins has a different half-life in kinetics of the polypeptide thrombin inhibitor
plasma (Table 6.4), the simultaneous inhibi- lepirudin is described by a two-compartment
tion of their combined biosynthesis leads to a model with an initial half-life of 10 min fol-
gradual increase in the anticoagulant effect lowed by a terminal elimination half-life of
with time. As an example, prothrombin has a about 1.3 h (43). By SC administration, how-
half-life of 50 h, and the anticoagulant effect ever, the PK follows a one-compartment
stemming from the loss of this clotting en- model, with a maximum plasma concentration
zyme after initiation of warfarin therapy is not achieved after about 2 h. It is eliminated pri-
fully realized until the new lower steady state marily by the kidneys as a mixture of intact
for this protein is achieved. drug and metabolites. Renal impairment can
The cytochrome P450 CYP2C9 is the major lengthen the elimination half-life up to 150 h
enzyme responsible for converting S-warfarin (44). Because lepirudin binds nearly irrevers-
to its inactive oxidative metabolites. Warfarin ibly to thrombin, and there is no antidote, pa-
is also metabolized by reductases to products tients are typically monitored (aPTT).
with minimal anticoagulant activity. Almost The related peptide bivalirudin has a half-
the entire dose of warfarin is excreted as me- life of 36 min after IV infusion and, unlike
tabolites in the urine. One source of patient lepirudin, is proteolytically cleaved by throm-
variability to warfarin is the presence of two bin (between Arg-Pro) (45) and possibly by the
variant CYP2C9 allelles in 10-20% of the Cau- liver, which accounts for a significant fraction
casian population, but present in less than 5% of its clearance (46).SC administration results
of African-Americans or Asians (41). Further- in a more sustained pharmacokinetic profile.
more, there is a long and expanding list of Argatroban has an elimination half-life of
drugs that interfere with warfarin activity (40, 39-51 min after an IV bolus (47) and is exten-
42), either by competing for its major meta- sively metabolized in the liver to four metab-
bolic pathways (e.g., CYP2C9) or displacing it olites by hydroxylation and aromatization of
from protein binding sites. Either of these in- the tetrahydroquinoline ring. The major me-
terfering mechanisms can potentially lead to tabolite has been shown to be weaker than
hemorrhagic complications. Other factors, argatroban as an anticoagulant. Even though
such as hepatic dysfunction or relative abun- it was demonstrated in vitro that CYP3A4/5
dance or deficiency of vitamin K in the diet, converted argatroban to these four metabo-
can also influence the effect of warfarin. Reg- lites, the lack of an in vivo effect by erythro-
ular patient monitoring through measure- mycin (a potent CYP3A415 inhibitor) on
ment of PT (commonly expressed as the INR argatroban pharmacokinetics suggests metab-
value) is required during warfarin therapy. olism by additional routes (48).
2 Clinical Use of Agents

C02CH3

0 (yf-p
C02CH3

-
clopidogrel
i
@ - 6'-r
k C02CH3

EN C02H c02H

I clopidogrel active metabolite

DS ADP (P2Yj2)
platelet receptor

Figure 6.6. Metabolism and propose ~echanismof action of clopidogrel.

After an IV bolus of abciximab (the mono- likely converted in vivo to an analogous active
clonal antibody to the platelet GPIIb/IIIa re- metabolite. After oral dosing of 400 mg of clo-
ceptor) the free plasma concentrations de- pidogrel, the inhibition of platelet aggregation
crease rapidly over 30 min, probably reflecting was detectable after 2 h and remained rela-
its distribution to the platelet receptors. Peak tively stable up to 48 h. On repeated daily dos-
effects on receptor blockade were observed af- ing of 50-100 mg clopidogrel, platelet aggre-
2 h (first sampling) with platelet function gation was inhibited from the second day of
turning to >50% of baseline after about treatment and reached steady state after 4-7
(49). The pharmacokinetic profile of days. After an oral dose of 14C-labeledclopi-
c heptapeptide GPIIb/IIIa blocker ep- dogrel, in humans, approximately 50% of the
after IV bolus dosing is linear and radioactivity was excreted in the urine and ap-
ose proportional. Plasma protein binding is
proximately 46% in the feces in the 5 days af-
ow (25%).Renal clearance predominates and
ter dosing (33,54).
he terminal half-life is about 2.5 h (50). Ef-
cts on platelet function return to normal Aspirin is rapidly absorbed in the stomach
thin 6-12 h. The nonpeptide GPIIbIIIIa and upper intestine (40-50% oral bioavailabil-
ocker tirofiban has a half-life of 2 h and is ity) and peak plasma levels occur 30-40 min
leared mainly by the kidneys, and metabo- after ingestion. Inhibition of platelet function
m appears to be minimal (51). After cessa- is evident by 1 h. Because aspirin irreversibly
on of tirofiban therapy, platelet function re- inactivates platelet COX-1 by acylation of an
rned to normal within about 4 h (52). active site serine, and because platelets do not
The two ADP receptor antagonists clopi- synthesize new proteins, this effect lasts the
el and ticlopidine present an interesting lifetime of the platelet (7-10 days). Because
, in that the parent compounds are inac- platelet COX-1 is acetylated in the presys-
ve as platelet aggregation inhibitors and temic circulation, the antiplatelet effect of as-
st undergo hepatic conversion to active me- pirin is largely independent of systemic bio-
lites. In the case of clopidogrel, the struc- availability, and the rapid plasma clearance of
e of the active metabolite has been deter- aspirin (15-20 min) does not impact its thera-
ned to be a sulfhydryl compound, which is peutic duration (12). Complete inactivation of
tulated to react by disulfide bond forma- platelet COX-1 is achieved when 160 mg of
to a cystein residue within the platelet aspirin is taken daily. This dose is lower than
P receptor (Fig. 6.6) (53). Ticlopidine is that required for the anti-inflammatory ef-
Anticoagulants, Antithrombotics, and Hemostatics

fects of aspirin (i.e., levels required to thera- ized because of UA, heparin or LMWH treat-
peutically affect COX-2). ment (at least 48 h) is added. Lepirudin can be
2.4 Typical Treatment Regimens for substituted for heparin and is indicated in-
Common Thrombotic Conditions stead of heparin for patients with a history of
HIT. Patients experiencing acute myocardial
Employing the currently marketed anticoagu- infarction (AMI)are typically immediately put
lant, antiplatelet, and fibrinolytic agents, the on aspirin and treated IV with a fibrinolytic.
following are representative treatment regi- Fibrinolytics for MI can be contraindicated in
mens for some of the major classes of throm- certain cases, however, as in patients with a
botic conditions (55). Of course, treatment history of intracranial hemorrhage or current
modalities will vary based on the individual
active bleeding. Depending on individual cir-
circumstances of the patient and the biases of
cumstances, either streptokinase, alteplase, or
the treating physician.
TNK-ase can typically be administered. Ad-
2.4.1 Venous Thromboses. For the preven- junctive therapy with IV heparin (or IV lepi-
tion of VTE in the setting of major orthopedic rudin in individuals with a history of HIT) is
surgeries such as hip and knee replacement, recommended. For acute MI patients not re-
either LMWH or warfarin is often employed ceiving fibrinolytic therapy, aspirin plus IV
(heparin being an alternative) with dosing ini- heparin followed by a course of warfarin is typ-
tiated before the surgery. Existing venous ically recommended.
thromboembolic disease (e.g., DVT or PE) can Fibrinolytic therapy with tPA is recom-
result from stasis of blood or abnormalties of mended in eligible patients suffering from
the vessel wall. Treatment with LMWH is rec- acute ischemic stroke. Treatment with tPA
ommended (unfractionated heparin being an must be initiated within 3 h of clearly defined
alternative) for the first 5 days, with overlap- symptom onset. Patients with a history of in-
ping oral anticoagulation (warfarin), which is tracranial bleeding, recent surgery, or several
then continued for at least 3 months. Patients other conditions are not eligible for thrombo-
with very serious embolism or thromboses and lytic therapy because of the high risk of fatal
who are at low risk for bleeding may undergo hemorrhage. For these excluded patients, lV
treatment with thrombolytic agents. or SC heparin or SC LMWH or danaparoid can
Atrial fibrillation is the most common sus- be administered, although clinical trials have
tained cardiac arrhythmia and can lead to the been inconclusive regarding the benefit of
generation of clots resulting from atrial blood these agents in this setting. For stroke preve
stasis. These clots can be carried to the brain
tion in individuals who have experienced
and precipitate ischemic stroke. For long-term
previous stroke or TIA, aspirin, aspirin pl
prevention of this thrombotic condition in low
risk patients, aspirin (325 mglday) is recom- dipyridamole (which is more effective than
mended, whereas in moderate risk groups, ei- pirin alone), or clopidogrel are typic
ther aspirin or warfarin is recommended, and recommended.
in high risk groups, warfarin is recommended. The term PC1 refers to various revascular
ization procedures, such as balloon
2.4.2 Arterial Thromboses. Coronary ar- plasty or stent placement, which are usu
tery disease (CAD) encompasses stable and performed as part of the treatment for
unstable angina and acute myocardial infarc- Antithrombotic therapy is administered d
tion. For primary prevention of CAD, the ing these procedures to prevent thrombo
recommended treatments for low, medium, complications (e.g., abrupt closure or late
and high risk patients are aspirin, low dose stenosis). Pretreatment is with aspirin, clo
warfarin, and aspirin plus low dose warfarin, dogrel, or aspirin plus clopidogrel (which
respectively. Aspirin is recommended for the more effective than either agent separ
management of stable angina. For unstable An IV GPIIbAIIa agent (abciximab, ep
angina, long-term aspirin therapy (75-162.5 batide, or tirofiban) along with IV heparin
mg) is recommended and for patients hospital- often used in patients undergoing PCIs, es
3 Physiology, Biochemistry, and Pharmacology

vation are simultaneously triggered to pre-


vent excessive clot growth, and the processes
involved in clot dissolution are also activated.
Prothrombin+ FVa + C + PLS Thus clot growth and dissolution is a highly
regulated and delicately balanced set of pro-
cesses that under normal conditions quickly
FX + FVllla + C + PLS generates a clot of proper size and maintains it
for only the time it is needed.
The coagulation cascade (Fig. 6.7) involves
two converging sequences of enzymatic steps
involving successive activations of serine pro-
teases in an amplifying manner, ultimately
Tissue factor (TF) + PLS FXla generating a burst of active thrombin. Thus,
after vascular injury or atherosclerotic plaque
rupture, membrane-bound tissue factor (TF)
is exposed to the vascular lumin and nonco-
valently associates with the circulating serine
protease Factor VIIa (FVIIa), which in the
Vessel endothelial cell surface presence of calcium on the phospholipid sur-
faces of subendothelial cells forms the "extrin-
e 6.7. Coagulation cascade. C, CaC2; PLS,
sic tenase complex" (Fig. 6.8a). The proteo-
lytic activity of FVIIa in this complex with TF
is significantly enhanced compared to the ac-
cially those at high risk. Bivalirudin is indi- tivity of free FVIIa. In this complex, FVIIa
cated in place of heparin for patients with a cleaves a peptide sequence on the inactive zy-
mogen FX,forming the proteolytically active
enzyme FXa, which in turn cleaves a peptide
sequence on the inactive precursor prothrom-
PHYSIOLOGY, BIOCHEMISTRY,
bin to generate active thrombin. This throm-
ND PHARMACOLOGY bin-activating step occurs most efficiently
when FXa combines with the protein cofactor
.I Molecular Mechanisms of Thrombosis
FVa on platelet phospholipid surfaces in the
presence of calcium, forming the "prothrom-
binase complex" (Fig. 6.8b). An alternative
pathway leading to generation of FXa and
olytic agents, a more detailed picture of thrombin involves the proteolytic activation of
ot formation and lysis is now presented. FIX to FIXa by the serine protease FXIa (Fig.
lots consist of both fibrin strands and plate- 6.7). The resultant FIXa associates with the
s in a ratio dependent on the local vascular protein cofactor FVIIIa in the presence of cal-
ronment (e.g., blood flow), as previously cium on platelet phospholipid surfaces, form-
sed. At the site of vascular injury plate- ing the "intrinsic tenase complex" (Fig. 6.8~).
ind to the injured surface, become acti- As part of this complex, FIXa efficiently acti-
, and aggregate. In parallel, the coagula- vates FX to FXa by the same proteolytic cleav-
cascade (Fig. 6.7) is triggered, leading to age as was effected by FVIIa. Finally, in a
crossover mechanism linking the two path-
es the formation of insoluble fibrin strands ways, the FVIIa-dependent extrinsic tenase
r activates platelets. In turn, acti- -
comdex can also activate FIX to FMa. The
lets create local environments re- historical terms "intrinsic," referring to the
FXI-mediated pathway, and "extrinsic," refer-
ring to the TF-mediated pathway, are still
st thrombin generation and platelet acti- used, although mainly for convenience.
Anticoagulants, Antithrombotics, and Hemostatics

(b)
Prothrombin

FXa
or FlXa n '1 \ 1 Thrombin

Exposed subendothelial Activated platelet


phospholipid surface phospholipid surface
Extrinsic tenase complex Prothrombinase complex

Activated platelet Exposed subendothelial


phospholipid surface phospholipid surface
Intrinsic tenase complex Tissue factor pathway inhibitor

Figure 6.8. (a) Extrinsic tenase complex; (b)prothrombinase complex; (c) intrinsic tenase complex;
(d) tissue factor pathway inhibitor complex.

The successive enzymatic catalytic steps in FXa, and prothrombin. For these calcium-de-
the coagulation cascade result in a great am- pendent complexes, binding of the proteins to
plification of the overall thrombin "signal." the anionic phospholipid surfaces is mediated
Consequently, only small amounts of up- by a cluster of 10-12 y-carboxyglutamic acid
stream zymogens are required compared to (Gla) residues near the N-terminus of each
those further downstream. For example, the protein. These Gla domains anchor these pro-
concentration of FVII in plasma is only 0.01 teins to the phospholipid surface, although the
a, whereas FX is 0.14 $4 and prothrombin precise mechanism of binding is still being in-
is 1.4 (Table 6.4). FVIIa, FXIa, FKa, FXa, vestigated. It is believed, however, that cal-
and thrombin are all trypsinlike serine pro- cium ions form salt bridges within the Gla do-
teases, and the cofactors TF, F'VIIIa, and FVa main, thereby inducing an ordered structure
are nonenzymatic proteins, which serve to al- that results in an outward display of certain
losterically enhance the activities of their lipophilic residues. According to this model,
serine protease partners (F'VIIa, FMa, and binding of the Gla domain with the phospho-
FXa, respectively). The phospholipid surfaces lipid surface results from a combination of li-
for assembly of the intrinsic tenase and pro- pophilic and Gla-calcium-mediated electro-
thrombinase complexes are expressed on acti- static interactions (57). The Gla residues on
vated platelets and serve to "concentrate" and these proteins are biosynthesized posttransla-
preorganize the factors and cofactors for effi- tionally in a vitamin K-dependent manner by
cient proteolytic processing. Calcium is re- y-carboxylation of glutamic acid residues and
quired in these complexes for proper binding therefore these enzymes and their zymogens
and orientation of FVII/FVIIa, FWFMa, FX/ are collectively referred to as the vitamin K-
_.
3 Physiology, Biochemistry, and Pharmacology 301

7 Protein S
Thrombin (via APC) inactivates
@ FVllla and FVa:

1
Antithrombotic
negative feedbackloop
FVlll

E
Thrombomodulin FVllla

Thrombin
FXla

0
Thrombin leads to formation of
FVllla, FVa, FXla, and activated platelets: Figure 6.9. Thrombin
Prothrombotic positive and negative
positive feedback loop feedback loops.

dependent factors. Proteins C and S are also protein C, affording activated protein C (APC)
Gla domain-containing proteins. (59). APC in combination with its nonenzyrne
The coagulation cascade is regulated, both cofactor, protein S, proteolytically inactivates
positively and negatively, at a number of the essential procoagulant factors FVa and
points in the two pathways. Tissue factor FVIIIa. By so doing, APC downregulatesthrom-
pathway inhibitor (TFPI) is an endogeneous bin production, effectively acting as an anti-
protein that is a reversible inhibitor of the ex- coagulant. Thrombomodulin-bound thrombin
trinsic tenase complex and uses the active site also activates a carboxypeptidase, TAFI
of FXa to bridge to the active site of FVIIa, (thrombin-activatable fibrinolysis inhibitor,
rendering both proteases inactive (Fig. 6.8d). also called plasma carboxypeptidase B or U),
In a positive feedback loop, DZa can cleave which serves an opposing, antifibrinolytic
zymogen FVII, thus providing additional function by slowing the rate of plasmin-medi-
FVIIa. Most important, thrombin itself is in- ated clot lysis (see below) (60).
volved in a number of positive and negative
feedback loops, thus regulating its own pro-
duction (Fig. 6.9). In a positive feedback loop,
aves DU.to m a , thus activating Thrombin g"e
the intrinsic coagulation pathway toward
more thrombin generation. Thrombin also
cleaves the proteins FV and FVIII to afford the
active procoagulant cofactors FVa and FVIIIa, ]Pi3
q
respectively. Additionally, through cleavage of Non-thrombotic:
apeptide fragment on the platelet PARS (pro- Thrombin altered active site region
tease-activated receptors), thrombin activates does not process
platelets that then express prothrombogenic
phospholipid surfaces used to assemble the in-
trinsic tenase and prothrombinase complexes.
In a negative feedback loop, thrombin first
h the protein cofactor thrombo-
/yCtw&
Activate
protein C
fibrinogen, FV, FVIII, PAR

) in a manner that effectively al- Antithrombotic: Prothrombotic:


Decreases Decreases
thrombin's substrate specificity (Fig. 6.10) coagulation fibrinolysis
. In particular, TM-complexed thrombin
ger efficiently activates FV or FVIII or Figure 6.10. Thrombomodulin (TM) alters the ac-
on platelets, but does efficiently cleave tivities of thrombin.
Anticoagulants, Antithrombotics, and Hemostatics

,- - - - - - -

\
8 Exosite 2
;' +
++ I (binds heparin and
- - - - - - : dermatan sulfate)
1

-------------------
j S4,S3,S2
SER I
HIS j
ASP
S1 Exosite 1
,- - - - - - -
I Asp I (binds fibrinogen,
.------------------
A i
- - - - - -,
I
PAR-1, hirudin,
Active site
L ' thrombomodulin,and
heparin cofactor II)

Figure 6.11. Thrombin active site and exosites.

A schematic picture of thrombin is shown duces a conformational change within anti-


in Fig. 6.11. The active site contains the typi- thrombin, priming it for efficient protease in-
cal trypsinlike serine protease catalytic triade activation (25). Through its exosite 2 domain,
Ser-His-Asp with an adjacent S1 pocket con- thrombin then binds to part of a 13-saccharide
taining an Asp at the bottom, having a speci- stretch of the anionic heparin molecule adja-
ficity for binding Arg and Lys residues by salt cent to the pentasaccharide. In this manner,
bridges. The other substrate-specific binding thrombin's active site domain is brought into
sites (S2, S3, S4) typically bind hydrophobic contact with the inactivating domain of anti-
residues. Apart from the active site region, thrombin, forming an inactive covalent com-
thrombin has two distinct anion-binding re- plex. Heparin then releases the complex and
gions or "exosites" defined by patches of posi- can repeat the cycle of thrombin inactivation.
tive charge resulting from grouped LysIArg The antithrombin-heparin complex also effi-
residues. Exosite 1 interacts with negatively ciently inactivates FXa, leading to downregu-
charged domains on fibrinogen and platelet lation of thrombin generation. Unlike the
PAR-1, serving to orient these substrates for thrombin-antithrombin-heparin complex, a
processing at the thrombin active site. Exosite separate heparin-FXa interaction is not re-
1 also binds thrombomodulin and heparin co- quired for inactivation of DZa by antithrom-
factor 11, and additionally binds the anionic bin-heparin and thus the pentasaccharide in-
C-terminus of the peptide thrombin inhibitor teraction with antithrombin is all that is
hirudin as well as other hirudin-like peptides. needed for efficient inactivation of FXa.By
Exosite 2 typically binds polyanionic poly- a similar heparin-dependent mechanism,
saccharides such as heparin and dermatan thrombin is also inactivated by heparin cofac-
sulfate. This thrombin-heparin interaction tor I1 (HCII), another serpin. Unlike anti-
serves as the basis for another mechanism by thrombin, which can inactivate both thrombin
which thrombin activity can be regulated en- and FXa, HCII inactivates thrombin only and
dogenously, namely by its inhibition with an- binds to thrombin by a heparin bridge to ex-
tithrombin. osite 2 and by its N-terminus to exosite1 (62).
The protein antithrombin belongs to a Dermatin sulfate, another endogenous acidic
large class of endogenous enzyme inhibitors polysaccharide with anticoagulant activity,
called serpins (serine protease inhibitors), catalyzes only HCII, not antithrombin. Phys-
which includes al-antitrypsin, aBantiplas- iologically, antithrombin is the more relevant
min, and plasminogen activator inhibitor 1 serpin for regulation of intravascular throm-
(PAI-1) among others (61). The mechanism bin, whereas HCII is thought to be more irn-
for thrombin inhibition involves initial bind- portant in the extravascular inhibiton of
ing of a specific pentasaccharide sequence of thrombin and in the regulation of thrombin
heparin (Fig. 6.12) to antithrombin, which in- activity during pregnancy (63).
3 Physiology, Biochemistry, and Pharmacology

Antithrombin

+
Antithrombin Thrombin
Heparin
5 13

Antithrombin /
Antithrombin

Heparin
m
Figure 6.12. The interaction of thrombin and FXa with antithrombin and heparin. "5" represents
the heparin pentasaccharide sequence that binds to antithrombin.

Thrombin initiates synthesis


- of the fibrin crosslinked fibrin network meshes with the
network in clots, as shown in Fig. 6.13. Fibrin- aggregated platelet mass to form a highly sta-
ogen monomer, the precursor to fibrin, can be bilized clot.
characterized structually by two D-domains The role of platelets and their activators in
connected to a central E-domain, which con- clot formation is summarized in Fig. 6.14. Af-
tains a set of activation peptides, fibrinopep- ter vascular injury, including plaque rupture,
tides A and B (FPA and FPB). Thrombin binds proteins such as collagen and von Willebrand
to FPA and FPB through its exosite 1 and factor (VWF) are exposed from the vascular
cleaves off these peptides, thus exposing new subendothelium. The platelet collagen recep-
terminal sequences that promote self-aggre- tor (Ia) and VWF receptor (Ib) can anchor
gation of the resultant fibrin monomers, lead- platelets to these proteins and hence to the
ing to a hydrogen-bonded polymeric soluble vascular surface. Collagen can also serve to
fibrin strand. Thrombin also generates the ac- activate platelets. Platelet activation triggers
tive transglutaminase FXIIIa from the inac- conformational changes in the heterodimeric
ve zyrnogen FXIII. FXIIIa stabilizes the fi- GPIIb/IIIa receptor on the platelet surface,
n strand by crosslinking glutamine and which allows it to bind fibrinogen and VWF.
sine side-chains on adjacent D domains, Fibrinogen is a bidentate ligand with the ca-
rming insoluble fibrin polymer. Addition- pacity to bridge GPIIbIIIIa receptors across
ally, FXIIIa covalently links the serpin a2-an- two platelets, thus promoting general platelet
tiplasmin to the fibrin stands, thus providing aggregation above the endothelium surface.
fibrin strand some protection from the As previously mentioned, thrombin is a pow-
tolytic action of plasmin, the principal fi- erful activator of platelets. Adenosine diphos-
n-degrading enzyme. Furthermore, throm- phate (ADP),and thromboxane A, (TxA,) also
binds to fibrin within the clot and contin- activate platelets and, along with thrombin,
s to activate factors V, VIII, XI, and XI11 as mediate their activation by stimulation of spe-
ell as platelets, leading to localized propaga- cific G-protein-coupled receptors, which span
tion of the thrombus at the site of injury. The the platelet membrane (GPC receptors de-
Anticoagulants, Antithrombotics, and Hemostatics

1. thrombin cleavage of fibrinopeptides


A and B to form fibrin monomer
2. self-aggregation of fibrin monomers
v

Fibrin polymer (segment)


hydrogen bond
FXllla - FXlll
thrombin

- Covalent bond
plasmin

Fibrin degradation products

Figure 6.13. Assembly o f crosslinked fibrin, mediated by t h r o m b i n a n d FXIIIa, a n d i t s degradation,


mediated by plasmin.

noted by black squares in Fig. 6.14). Several by way of exosite 1 (Fig. 6.15). The thrombin
other, albeit weaker, agonists of platelet acti- active site then cleaves the sequence at Arg-
vation are recognized [e.g., epinephrine, sero- Ser, exposing a new N-terminal domain se-
tonin, and platelet-activating factor (PAF)] quence (SFLLRN) that folds back on the
that also employ GPC receptors. The two GPCR extracellular loop 2, acting as a teth-
GPCRs that thrombin employs for platelet ac- ered agonist, thereby activating its receptor.
tivation, platelet-activating receptors 1 and 4 PAR-4 functions in a similar manner using a
(PAR-1 and -4), are noteworthy because of different unmasked N-terminal sequence
their unusual mechanism for signal transduc- (GYPGQV) and appears to be less sensitive to
tion (64). In PAR-1, for example, the N-termi- thrombin, possibly because of the absence of a
nal extracellular domain incorporates an thrombin exosite-1 binding sequence on
acidic peptide sequence that binds thrombin PAR-4. Recent studies are consistent with the
3 Physiology, Biochemistry, and Pharmacology

Platelet

Phospho-
lipase C \
\ Fibrinogen

I Phospho-
lipase A,

Arachadonic acid
p2y12
llla

I
Thromboxane A,
thrornbox:
synthase

II
Anionic
ohosoholioid
,- - ,
surfaces '

E
-Injury

Vessel endothelial cell surface


Figure 6.14. Platelets and their activators and receptors.
factor

interpretation that low levels of thrombin ac- which then leads to an increase in intracellu-
"vate PAR-1 to provide an immediate state of lar calcium concentrations. Increased intra-
platelet activation, whereas higher levels of cellular calcium then stimulates phospho-
thombin, typically generated during the later lipase 4, liberating arachadonic acid from
stages of clot formation, activate PAR-4 and membrane phospholipids. Cyclooxygenase-1
act to sustain the late phase of the platelet- (COX-1) converts arachadonic acid to PGH,,
aggregation process (65). which is converted by thromboxane synthase
Dependingon the mode of agonism, various to thromboxane &, a potent platelet agonist,
~emicalpathways leading to platelet activa- which is secreted and leads to further activa-
In can be triggered. In a major pathway, tion through its own receptor. Two ADP re-
~ospholipaseC within platelets is activated, ceptors on the platelet surface modulate
Anticoagulants, Antithrombotics, and Hemostatics

1-
,
ESKATNATLDPR PNDKYEPFWEDEEKNES-

Thrombin cleavage site


t Thrombin exosite 1
binding domain
I

Figure 6.15. The activation of PAR-1 by thrombin.

let activation: P2Y, and P2Y1, (P2Y1, was platelets accelerates thrombin generation by
formerly called P2YAc, P2TA,, or P2,). Stim- 5-6 orders of magnitude, greatly facilitating
ulation of both is required for ADP-dependent fibrin formation. The significant role of plate-
platelet activation (66). Stimulation of P2Y1 lets in thrombin generation is suggested by
activates phospholipase C, whereas stimula- the observations that fibrin deposition at sites
tion of P2Y1, downregulates adenylate cyclase of vascular injury occurs after platelet adhe-
activity, leading to lower levels of CAMP, an sion and aggregation, and that fibrin forms in
inhibitor of platelet activation (see below). At close proximity to the deposited platelets (68).
the cellular level, stimulation of P2Yl gener- Platelet activation is inhibited by intracel-
ates an initial transient aggregation response, lular CAMP, which initiates a sequence of en-
whereas agonism of P2Y1, generates sus- zymatic steps leading to a reduction in platelet
tained aggregation (67). calcium concentration. Adenylate cyclase,
Activation of platelets typically leads to a which converts ATP to CAMP, is stimulated by
shape change wherein the platelets lose their prostaglandin D,, another product enzymati-
usual discoid shape and take on an irregular cally generated from arachadonic acid within
appearance. Activated platelets express an- the platelet.
ionic phospholipid surfaces and also receptors cGMP inhibits a major CAMP phosphodies-
for FVa on their outer membrane, which ac- terase that hydrolyzes CAMP, and thus ads
commodate formation of the intrinsic tenase indirectly as an inhibitor of platelet activation.
and prothrombinase complexes, leading to ad- Endothelial cells can participate in deactivat-
ditional thrombin generation. Granules ing platelets by releasing PGI, (prostaglandin
within activated platelets release their con- I,; prostacyclin), which stimulates platelet ad-
tents, among which are serotonin, ADP, fi- enylate cyclase to generate CAMP, and nitrous
brinogen, FV, and VWF, which serve to am- oxide (NO), which stimulates platelet guany-
plify platelet stimulation and clot formation. late cyclase to generate antiaggregatory
It has been estimated that the activation of cGMP.
3 Physiology, Biochemistry, and Pharmacology

Active complex
---------------------.
: t-PA + plasrninogen \
Fibrin -K
I

1
L--------------------d

Plasrnin + tPA
I Fibrin 1-K

Fibrin -K + K1-

Fibrin +K Figure 6.16. TAFI attenuates


the acceleration of fibrinolysis
Further fibrinolysis mediated by C-terminal lysine
is attenuated residues.

The process of clot dissolution involves lysine residues, which may mediate its binding
plasmin, a trypsinlike serine protease, which to fibrin, and also has a domain that has affin-
proteolytically degrades fibrin, generating ity for plasminogen. Details regarding the ex-
smaller fragments (FDPs, fibrin degradation act nature of the binding and interaction of
products) such as D-dimer (Fig. 6.13). Plasmin tPA and plasminogen on the fibrin surface are
is protolytically activated by tPA, another still being studied.
serine protease, from its inactive zymogen, During the proteolytic degradation of fibrin
plasminogen. This activation step can occur in by plasmin, C-terminal lysine residues are ex-
solution but is accelerated more than 100-fold posed on fibrin that induce an additional 2.5-
when both plasminogen and tPA bind in a co- fold rate acceleration of tPA-mediated activa-
ordinated manner on the surface of fibrin (Fig. tion of plasminogen compared to that of
6.16). Thus, fibrin can be viewed as a cofactor uncleaved fibrin (Fig. 6.16). TAFIa (the active
to plasminogen activation (60). Whereas form of TAFI) in a negative regulatory man-
solution-phase plasmin is inactivated almost ner attenuates this rate acceleration by cleav-
instantaneously by a,-antiplasmin, fibrin- ing off those C-terminal lysine residues. Con-
bound plasmin is partially protected from in- sequently, TAFIa effectively functions as an
activation. Plasminogen and plasmin contain antifibrinolytic agent by this unique mecha-
sites within their structures that have affinity nism and promotes persistence of formed fi-
for lysine residues, and it is these lysine bind- brin clots (60, 70). Another negative regula-
ing sites (LBS) that facilitate their binding to tory mechanism controlling fibrinolysis is the
the surface of fibrin. Also, the LBS mediates inactivation of tPA by the serpin PAI-1 (plas-
initial a2-antiplasmin binding to plasmin. minogen activator inhibitor 1).Recent studies
Thus both fibrin (substrate) and a2-antiplas- with PAI-1 and al-antitrypsin have revealed
min (inactivator) compete for the same LBS in the detailed mechanism by which these, and
plasmin. Simple lysine analogs, such as 6-ami- presumably all, serpins inactivate their target
nocaproic acid are known to inhibit plasmino- proteases (Fig. 6.17). In this mechanistic de-
gentplasmin binding to fibrin, and even have scription, the tPA active site serine cleaves the
been used therapeutically to treat individuals PAT-1 reactive center loop between the P1 Arg
with hemostatic disorders, such as hemophili- and P I ' methionine, forming a covalent ester
acs, although with variable outcomes (69). bond with the PAI-1 Arg carbonyl. Normally,
tPA contains a domain that has affinity for within the context of standard substrate turn-
Anticoagulants, Antithrombotics, and Hemostatics

center
loop Met

Insertion site

Figure 6.17. Mechanism of inactivation of


tPA by the serpin PAI-1.

over by serine proteases, such acylated serine lesser extent, heparin can also facilitate anti-
hydroxy groups are readily hydrolyzed in a thrombin-mediated inhibition of FIXa and
process mediated by the adjacent His-Asp res- FXIa. Pharmaceutical heparin [also called un-
idues of the catalytic triad. However, acylation fractionated heparin (UFH)] is isolated from
by the serpin triggers a dramatic translocation porcine intestinal mucosa. In addition to its
of the protease to the opposite side of the ser- limitations arising from pharmacokinetic
pin, with the N-terminal segment of the reac- variability, potential complications from
tive center loop fitting as a strand into an in- thrombocytopenia, and the requirement that
sertion site on the serpin. As a consequence of it be administered IV, heparin is not effective
this serpin-protease alignment, the protease
at inhibiting fibrin-bound thrombin, and re-
active site is severely deformed, resulting in
cent investigation~have provided a likely ex-
complete disconnection of the His-Asp away
planation (73). Thrombin binds to fibrin at its
from from the active site (acylated) serine.
Thus, normal turnover cannot take place be- exosite 1, and heparin can form a tight bridge
cause the catalytic triad is disrupted and the from the fibrin surface to exosite 2 of thrombin
serpin-protease complex is therefore stabi- (Fig. 6.18). This tight ternary complex does
lized (71). not allow the AT-heparin complex access to
Urokinase (uPA) like tPa has the ability to exosite 2 of thrombin, and therefore inhibition
convert plasminogen to plasmin, although un- of thrombin cannot proceed. UFH also cannot
like tPA, uPA has no affinity for fibrin. Phys- inactivate FXa in the prothrombinase com-
iologically, uPA mainly plays a role in extra- plex, and for these reasons UHF is not viewed '
cellular matrix degradation (72). as the ideal agent to prevent clot expansion
and propagation, which may explain why hiru-
3.2 Mechanisms and Sites of Action of the din (lepirudin) shows superior efficacy to that
Classes of Marketed Antithrombotic and of heparin in patients with unstable angina or
Antiplatelet Agents non-Q wave myocardial infarction (9b). Addi-
tionally, it has been noted that upon cessation
3.2.1 Heparin and Other Anionic Poly- of heparin treatment, a clustering of throm-
saccharides. As described in Section 3.1, en- botic events occurs in patients (9b, 25). This
dogenous heparin acts as an antithrombotic "heparin rebound" may be partially explained
by activating the serpin antithrombin (AT), by the ability of heparin to displace and mobi-
which covalently inactivates both thrombin lize endogenous TFPI from the vessel wall. It
and FXa by binding to their active sites. To a is believed that about one-third of the anti-
3 Physiology, Biochemistry, and Pharmacology 309

Antithrombin

Figure 6.18. Heparin-antithrom-


bin does not inhibit fibrinbound
thrombin.

hrombotic effect of heparin may be attributed LMWHs have been shown to be less efficient
o mobilized TFPI. At therapeutic doses, UFH than heparin at mobilizing endothelial TFPI
regressively depletes TFPI and when hepa- from the endothelium (77). As a consequence
orter average length, LMWHs ap-
r advantages over heparin in re-
rothrombotic state (74). duced incidences of heparin-induced thrombo-
The low molecular weight heparins (LMWH) cytopenia (78).
are prepared by chemical or enzymatic modi- Despite all of these differences, the princi-
fication of natural heparin and are about pal clinical differentiation between UFH and
one-third the length of heparin. Each of the the LMWHs remains the improved pharmaco-
marketed LMWHs is slightly different struc- kinetic profile displayed by LMWHs (25). Dan-
turally, depending on the manner in which it aparoid is a mixture mainly of the anionic
sulfate ( 4 4 % ) and
. Danaparoid has a
ir shorter average length, the LMWHs con- higher ratio of anti-FXa to antithrombin activ-
), likely attributable to the selec-
bit thrombin (>I7 saccharides, see Fig. tivity of heparan sulfate toward AT-mediated
ion (79). The dermatin sulfate
inactivate thrombin through
. Research is continuing in the
t mechanism, the LMWHs inhibit FXa in field of polysulfated polysaccharide anti-
10sof about 2:l to 4:l compared to that of thrombotics to discover new agents with ben-
ombin. Additionally, it has been reported efits in safety (less bleeding and thrombocyto-
okinetics, and utility (80).
arch is directed toward fur-
modifications of LMWHs.
wever, others report that LMWHs and even However, in a somewhat different approach, it
ndaparinux, the FXa-specific pentasaccha- was recognized that for selective inhibition of
-binding pentasaccharide
the prothrombinase complex (76). Also, (Fig. 6.la) is the minimally required struc-

0~0~-

Figure 6.19. Idraparinux.


Anticoagulants, Antithrombotics, and Hemostatics

t u r d subunit. Such FXa-selective pentasac- phenprocoumon, and acenocoumarol have


charides were synthesized and a few are under also been investigated as anticoagulants in hu-
clinical investigation. For example, fondapa- mans, although no significant advantages
rinux (Arixtra, Fig. 6.lb) and idraparinux were found. Some recent attempts have been
(SanOrg 34006, Fig. 6.19), both based on the made to prepare warfarin analogs with im-
specific heparin pentasaccharide, are indeed proved physical properties (e.g., reduced pro-
highly selective FXa inhibitors and have tein binding), which may have a safety benefit
shown antithrombotic efficacy in clinical stud- (83). However, little current research is di-
ies (81a). Arixtra was launched in the United rected toward the discovery of new analogs of
States in 2002 for the prophylaxis of DVT in warfarin or other indirect or direct inhibitors
patients undergoing hip and knee surgery. of Gla biosynthesis.
Whereas the pharmacokinetics of fondapa-
rinux allows once-a-day dosing, idraparinux 3.2.3 Direct Thrombin Inhibitors: A Histor-
can be administered once a week. Idraparinux ical Perspective, From Concept to Drug. The
binds to AT with a 10-fold greater affinity direct and selective active-site inhibition of
compared to that of fondaparinux, and it has thrombin, especially by small molecules that
been suggested that this greater affinity ac- have potential for oral bioavailablity, has long
counts for its longer plasma elimination half- been seen as an attractive opportunity for the
life and resultant longer pharmacodynamic ef- development of therapeutically useful antico-
fect (81b,c). agulant agents. Theoretically, many of the dis-
advantages of heparin, the LMWHs, and war-
3.2.2 Warfarin and Other Vitamin K-depen- farin would be avoided. For example, direct
dent Inhibitors. Warfarin's antithrombotic ef- thrombin inhibitors could inactivate fibrin-
fect is a consequence of its inhibition of the bound thrombin and could also avoid HIT,
vitamin K-dependent posttranslational y-car- both disadvantages of heparin, and also could
boxylation of glutamic acid residues in the avoid the unfavorable mechanism-based phar-
procoagulant zymogens FVII, FIX, FX, and macodynamics of warfarin. On the other
prothrombin (82).As discussed previously, for hand, there has been some recent concern that
these proteins a specific domain containing selective, reversible inhibitors of thrombin
10-13 y-carboxyglutamic acid residues is an could have the potential for a so-called throm-
essential requirement for permitting their bin rebound effect after cessation of dosing,
binding to phospholipid surfaces and hence especially if the inhibitors are cleared quickly
proper assembly into the active tenase and from the plasma. According to this view, after
prothrombinase complexes. Lacking Gla do- cessation of treatment with a reversible
mains, FVIIa, FMa, FXa,and thrombin are thrombin inhibitor, the inhibitor-thrombin
physiologically extremely poor procoagulants. complex dissociates, the drug is eliminated
Warfarin and its analogs (e.g., phenprocou- from the vasculature, and the resultant poolof
mon, acenocoumarol, and dicumarol) inhibit reactivated thrombin could, in theory, trigger
vitamin K epoxide reductase, an enzyme es- clinical thrombotic events. Moreover, revers-
sential for the reductive recyclingof vitamin K ible inhibition of thrombin during treatment
epoxide to vitamin K hydroquinone (Fig. would not be expected to impede the residual
6.20). Vitamin K hydroquinone is the cofactor generation of active thrombin resulting from
to y-glutamylcarboxylase, which affects the the ongoing activation of the coagulation
actual carboxylation of Glu residues. The ac- pathway. This newly generated thrombin
tivity of the anticoagulant Gla-domain-con- could add to the pool of reactivated thrombin
taining proteins, protein C and S, is also inhib- once the inhibitor is cleared from the blood.
ited by warfarin, affording a procoagulant Clinically, there has in fact been documenta-
activity. Still, the major effect achieved is an- tion of increases in thrombotic event rates af-
ticoagulation, primarily because of inhibition ter cessation of treatment with argatroban
of thrombin. The pharmacokinetic and safety and inogatran, although these events could
liabilities of warfarin have already been dis- not be decisively correlated to a thrombin re-
cussed (Sections 2.2.1 and 2.3). Dicumerol, bound phenomenon (85). Also, a trial that re-
3 Physiology, Biochemistry, and Pharmacology

C02H rglutamyl- C02H


glutamic acid carboxylase rcarboxy-glutamicacid
(Glu) (GW

OH 0

0
vitamin K
hydroquinone epoxide

vitamin K
epoxide reductase Vitamin K
I
(warfarin inhibition) KI R = C20 P ~ Y M
K2 R = polyprenyl

0 0

OH A' OH OH
R R' Dicumarol
Warfarin H CH2COCH3
Phenprocoumon H CH2CH3
Acenocoumarol NO2 CH2COCH3

. Vitamin K-mediated y-carboxylation of Glu residues in coagulation serine proteases,


hibitors of this process.

vel of thrombin genera- ray crystal structures of thrombin complexed


ion of napsagatran compared with active-site inhibitors have had a major im-
sion of heparin was not followed by any pact in the design of a diversity of highly potent
nd in thrombin activity after cessation of and selective active site-directedagents.
ion (86).At present, there does not seem 3.2.3.1 Small-Molecule Direct Thrombin
a general problem with a rebound phe- Inhibitors. Early insights leading to the dis-
enon for selective reversible thrombin in- covery of the first synthetic thrombin active-
rs, although the clinical use of such site inhibitors resulted from consideration of
the structures of the endogenous substrates of
the last 20 years, a vast amount of thrombin. Nearly all of the protein substrates
been directed toward the discovery for thrombin contain an Arg as the P1 residue,
ent of direct thrombin inhibi- with the P2 residue often being a Pro (Fig.
both small-molecule and hirudin-like. X- 6.21a). In 1978 Bajusz reported a series of tri-
312 Anticoagulants, Antithrombotics, and Hemostatin

-Gly-Pro-Arg-Val-

I
Cleavage site

Bajusz inhibitor H -CHO


PPACK H -COCH2CI
Efegatran -CH3 -CHO
DuP-714 -COCH3 -B(OH)2

Figure 6.21. (a) A thrombin cleavage site (fibrinogen A); (b) several covalent thrombin inhibitors.

peptide aldehydes modeled after the fibrino- beta-sheet interactions variously with Ser-214,
gen A peptide cleavage site and one of the Trp 215, and Gly 216; and stabilization of the
more potent compounds, based on clotting anionic charge (former Arg carbonyl) with H-
times, was the tripeptide aldehyde D-Phe-Pro- bond interactions within the so-called oxyan-
Arg-CHO (Fig. 6.21b) (87). At that time, and ion hole, defined by the Gly-193 and Ser-195
for many years thereafter, the paradigm of NHs (Fig. 6.22a). The processed protein is
joining a tripeptide-like structure to an elec- then released after normal catalytic deacyla-
trophilic moiety (an aldehyde, in Bajusz's pro- tion of the arginine-serine ester bond. In
totype) led to the preparation of a great diver- 1989 the published X-ray crystal structure of
sity of extremely potent thrombin active-site thrombin inhibited by PPACK revealed sev-
inhibitors, such as PPACK (D-PheProArg- eral critical molecular interactions (Fig.
chlommethylketone), efegatran, and DuP-714, 6.22b) (89). Both Ser-195 and His-57 were co-
to name just a few (Fig. 6.21b). Ki values for valently attached to the former chloroketone
many of the inhibitors in this class are in the trap, the Pro and D-Pheresidues occupied the
picomolar range (a). The electrophilic moiety es- S2 and S4 pockets, respectively, and several
sentially a d s as a "serine trap" and accounts for antiparallel beta-sheet hydrogen bond inter-
much of the potency of these inhibitors. Other actions could be observed. In the case of elec-
examples of such traps include trifluoromethyl- trophilic carbonyls such as aldehydes, and ac-
ketones, a-keto-esters, a-keto-amides, a-keto- tivated ketones, the analogous hemiacetal or
heterocycles, and boronic acids, among others. hemiketal bond is formed with Ser 189,
For endogenous substrates, the catalytic whereas in the case of boronic acid-based in-
triad serine of thrombin cleaves the arginine hibitors, a boronate ester is formed. As men-
amide bond in a manner typical of other serine tioned, many inhibitors in this class display
proteases (88). During this cleavage, an an- high potency, largely attributable to the effect
ionic tetrahedral transition state is formed by of forming a covalent bond at the active site of
attack of Ser-195 at the Arg carbonyl, forming thombin. It came to be appreciated, however,
a transient covalent bond. The remainder of that the serine trap concept had a number of
the substrate is stabilized by a number of potential drawbacks. For example, these in-
other interactions: hydrophobic contacts with hibitors typically exhibit slow binding kinetics
the enzyme S2, 53, and S4 pockets; a salt- that may not be sufficiently rapid to achieve
bridge interaction between the substrate argi- desired efficacy. After activation of the coagu-
nine and Arg 189 in the S1 pocket; antiparallel lation pathway, thrombin is generated in a
3 Physiology, Biochemistry, and Pharmacology 313

Asa 189

I
NH S1
"oxyanion hole"

Figure 6.22. (a) Typical interactions between a coagulation serine protease and its substrate during
cleavage; (b) covalent inhibition of thrombin by PPACK.

id "burst." Studies both in vitro and in vivo covalent bond formation in vivo might lead to
e shown that slow binding inhibitors are immunological reactions (through formation
efficacious than fast binding inhibitors of of a hapten) or other undesired side effects
parable potency. Further, the reactive (84a, 90).
ctionality in this class is viewed as a meta- In parallel to the serine trap approach,
c liability and the potential for nonspecific other groups were synthesizing potent throm-
Anticoagulants, Antithrombotics, and Hemostatics

H2N
(12) NAPAP (5) argatroban

(13) napsagatran (14) melagatran H H


(15) ximelagatran -OH -CH2CH3

Figure 6.23. Examples of reversible active site thrombin inhibitors.

bin inhibitors that did not rely on forming a ester (TAME). In the case of argatroban, the
covalent bond at the thrombin active site. Two methyl ester of TAME was replaced by an
early examples, first reported in the early amide, the sulfonyl group was optimized, and
1980s were NAPAP (naphthylsulfonyl-glycyl- a carboxylic acid group was appended to solve
4-AmidinoPhenylAlaninePiperidide,Ki = 6 toxicity problems. X-ray structures for
nM, Fig. 6.23, structure 12) and MD-805, later NAPAP and argatroban bound to the active
named argatroban (Ki = 8 nM) (91). The de- site of thrombin were published in 1991 and
sign of both of these inhibitors evolved from 1992 by two different groups (92). The crystal
an earlier prototype, N-tosyl-arginine methyl structure of argatroban showed that the argi-
3 Physiology, Biochemistry, and Pharmacology

nyl side-chain entered the S1 pocket at an an- thrombosis (HITTS) and is comarketed by
gle different from that of the arginyl chain of GlaxoSmithKline and Texas Biotechnology.
the serine trap inhibitor PPACK and conse- Reviews on argatroban were recently pub-
quently only one of the NHs of the guanidine lished (96).
formed an ionic bond to Asp-189. The tetrahy- Over the years, many other potent revers-
droquinoline inserted into the S4 pocket, with ible active-site thrombin inhibitors were pre-
densities for both methyl isomers making ac- pared, inspired by the D-Phe-Pro-Arg-like
ceptable lipophilic interactions. A section of structures of argatroban and NAPAP. Napsa-
the piperidine ring along with the appended gatran (13)was reported in 1994 and is a very
4-R methyl group inserts tightly into the S2 potent inhibitor of thrombin (Ki = 0.3 nM)
pocket, and the carboxylate points toward the with good selectivity against the fibrinolytic
oxyanion hole, forming a hydrogen bond with enzymes (97); its development was discontin-
Ser-195. Before the X-ray, it was assumed that ued, however. The D-Phe-Pro-Arg mimetic,
the piperidine was occupying the S1' site. The melegatran (Ki = 2 nM; Fig. 6.23, structure
stereochemistries of both piperidine groups, 14),is currently in clinical development by As-
carboxylate (2R) and methyl (4R), were seen traZeneca for patients with DVT and for the
from the X-ray structure to be critical and the prevention of stroke in patients with atrial fi-
other possible stereochemical combinations brillation. Its poor oral bioavailablity (6%)and
were predicted not to be as well tolerated, potency against trypsin (Ki = 4 nM) necessi-
which was in agreement with experimental re- tates that it be dosed parenterally (98). How-
sults. The crystal structure of NAPAP shows a ever, for oral administration, a double prodrug
generally similar binding motif compared to form (ximelagatran, Exanta; 15) is also being
that of argatroban, in terms of placement of developed, wherein the benzamidine moiety is
the major residues in the enzyme S pockets, in modified by hydroxylation and the carboxylate
spite of the fact that the benzarnidine and al- is the ethyl ester. The bioavailabilty of ximel-
kylguanidine for the two inhibitors are at- agatran in humans is moderate (18-24%), al-
tached with different stereochemistries. though it is rapidly absorbed and metabolized
With a Ki value of 8 nM, argatroban not to melagatran (99).
only is a potent thrombin inhibitor, but it is Achieving good oral bioavailability and/or
much less potent against a panel of other co- good plasma half-life within the early classes
agulation and fibrinolytic enzymes (93). Selec- of active-site thrombin inhibitors was frus-
tivity against the fibrinolytic enzymes was trated by the peptidic-like nature of the struc-
seen as especially key, in that potent inhibi- tures and also by the presence of the highly
tion of plasmin or tPA would essentially lead charged guanidine or benzamidine moieties.
to a prothrombotic condition. Argatroban is Further, the requirement for good selectivity
also fairly selective against trypsin (1000- against trypsin was also a frequent problem
fold), although high selectivity against this di- for the development of an oral inhibitor. Over
gestive enzyme may not necessarily be re- the last decade much research has been di-
quired for an intravenously dosed agent. rected toward solving these two problems. To-
(Because of its low oral bioavailability, ar- day, a number of less polar surrogates for the
gatroban is dosed by N infusion.) Argatroban Arg-like side-chain have been identified and
binds rapidly and reversibly to both fibrin- successfully incorporated into nonpeptidic
bound (clot-bound)and soluble thrombin (94). templates that afford very potent active-site
Moreover, it does not induce thrombocytope- thrombin inibitors. Furthermore, by exploit-
nia nor does it interact with the antibody that ing observed structure-activity relationship
causes HIT (95). Argatroban, originally dis- (SARI trends for activities of these thrombin
covered and developed by Mitsubishi, was ap- inhibitors against other enzymes such as tryp-
proved in Japan in 1990 for treatment of arte- sin and the fibrinolytic enzymes, inhibitors
rial thrombosis and, in 1996, for treatment of having very high selectivity for thrombin
acute cerebral thrombosis. In the United could be identified. X-ray crystallography also
States it was approved in 2001 for the treat- aided in the design of more selective inhibitors
ment of patients with HIT and HIT with by revealing differences in the conformations
Anticoagulants, Antithrombotics, and Hemostatics

and interactions of inhibitors bound to throm- of recombinant hirudin variant 1 (rHV-1, de-
bin compared to other enzymes (especially sirudin, 3b) to thrombin is representative of
trypsin). Today, there are numerous examples the class and is shown in Figure 6.24a. The
of nonamidine orally bioavailable thrombin polyanionic C-terminus tightly binds to
inhibitors having excellent selectivity against thrombin exosite 1 [fibrinogen binding do-
trypsin and other serine proteases. As just one main (FBD)], whereas the N-terminus simul-
example, investigators at Merck have opti- taneously occupies the thrombin active-site
mized a series of nonamidine pyridinone tem- region. At the N-terminus, the Val-1 and
plate-based thrombin inhibitors to provide the Tyr-3 side-chains occupy roughly the throm-
pyrazinone L-375,378 (16),having a Kivalue bin S2 and S3 sites, making numerous hydro-
of 0.8 nit4 (100). The X-ray crystal structure phobic contacts, whereas the terminal amino
for this compound shows the aminopyridine group makes hydrogen bonds to His-57 and
occupying S1, with the amino group interact- Ser-214. Additionally, because of the manner
ing, through an ordered water molecule, with of insertion of the N-terminal hirudin peptide
Asp-189 and also interacting with the car- along the active site groove, it forms a short
bony1of Gly-216. The pyridine 6-methyl group parallel set of hydrogen bond contacts to the
makes a productive lipophilic interaction with thrombin backbone (Gly 216 to Gly 218,
Val-213 within S1. The pyrazinone methyl oc- which is opposite to that seen in the antipar-
cupies S2, whereas the phenyl occupies S4. allel interactions of substrates and most inhib-
L-375,378 is selective for thrombin compared itors. The S1 pocket is not occupied by him
to trypsin (2000-fold selectivity) and other din. Much SAR data have been generated for
serine proteases, including tPA and plasmin hirudin involving single and multiple amino
(>100,000-fold)and is 90% orally bioavailable acid substitutions as well as other modifica-
in dogs with a half-life of 231 min; in rhesus tions (105). Desirudin (3b, Fig. 6.3) is mar-
monkeys it is 60% orally bioavailable. keted in Europe for the prevention of DVT in
Thus, it appears that the long-sought goal hip and knee replacement surgery (106).Lepi-
of identifying orally bioavailable and selective rudin (3a, Refludan; U.S. launch 1998) is
active-site thrombin inhibitors has been structurally similar to desirudin but has Leu-
achieved. The subject of small-molecule ac- Thr at the first two N-terminal positions in-
tive-site thrombin inhibitors has been exten- stead of Val-Val (107). Lepirudin is used as a
sively reviewed (90, 101). replacement for heparin in HIT patients.
3.2.3.2 Hirudin and Hirudin-Like Thrombin Even before the crystallographic details of
Inhibitors. More than a century ago, the anti- hirudin's binding to thrombin were known,
coagulating substance hirudin was extracted major structural modifications to hirudin
from the leech Hirudo medicinalis (102). were carried out, resulting in the "hirulogs"
Hirudin is a family of more than 20 related and the "hirugens" (108). The hirugens are
65-66 amino acid peptides containing three peptide fragments of hirudin containing only
disulfide bridges and an 0-sulfated Tyr near the C-terminal FBD and that inhibit thrombin
the carboxylate terminus (103). In 1986 the in the low to submicromolar range. The hiru-
first reports on the preparation of recombi- logs are peptide analogs of hirudin, wherein
nant desulfated hirudins appeared, which al- most of the nonbinding peptide core sequence
lowed the study of single hirudin variants, es- is excised and an active site binding sequence
pecially useful for crystallography purposes. is appended by way of a poly-Gly linker to the
Hirudins lacking the sulfate on the C-terminal FBD, thereby creating essentially a hirugen
Tyr have about 10 times reduced activity, but with an active site binding sequence. Bivaliru-
still potently and specifically inhibit thrombin din (4, hirulog-1; Angiomax) is one such exam-
in the subpicomolar range. The origin of this ple and contains the familiar D-Phe-Pro-Arg
high potency can be explained in the bivalent active-site sequence characteristic of the early
manner in which the hirudins bind to throm- small-molecule thrombin inhibitors (45, 46,
bin, which was first revealed by X-ray crystal- 109). The binding mode of bivalirudin (and
lography of two recombinant hirudin variants other hirulogs) is different from that of him-
reported in the early 1990s (104). The binding din, in that the bivalirudin peptide sequence
3 Physiology, Biochemistry, and Pharmacology

\
S\
Cat triad
I H
N. L-c, S3 52 S1 4 N
c,
Q ,T' D T-Y-V-V-NH3+ D-G-D-F-E-E-I-P-E-E-Y-L-Q-CO~-
G.S-E Active-site groove Exosite 1 (fibrinogen binding groove)
(b) Cat triad

Active-site groove Exosite 1 (fibrinogen binding groove)


(4 0
NH~NH-(CH~~~CO-D-Y-E-P-I-P-E-E-A-C~~-DE-CO~-

0 2 :
Hirudin-like
I

Figure 6.24. (a) Hirudin variant 1 (desirudin)bound to the active site and exosite 1 of thrombin; (b)
binding mode of bivalirudin to thrombin; (c)thrombin inhibitor combining structural elements from
argatroban and the hirudin C-terminus (DF, D-Phe; DE, D-Glu; Cha, cyclohexyl-Ala).

binds continuously along the FBD and active their mechanism of action, each of these
site grooves, with the active site sequence now agents has been shown to be active against
making the usual antiparallel contacts with fibrin-bound thrombin.
the enzyme (108). One consequence of this in- Analogs of bivalirudin incorporating differ-
hibitor structure and its binding mode is that ent active-site binding domains have been syn-
the Arg peptide bond to the P I ' Pro is slowly thesized, with the goal being to stabilize the
cleaved by the enzyme, affording a less potent scissile bond and to increase binding potency
inhibitor. Bivalirudin itself has a Kivalue of at the N-terminus (lola, 105a). One such an-
1.9 nM and upon IV infusion has shown effi- alog (Fig. 6.2413 contains an argatroban-like
cacy similar to that of heparin in preventing active-site binding structure and inhibits
ischemic complication in patients with unsta- thrombin selectively with a Kivalue of 0.17
ble angina who underwent angioplasty. Even pM (110). However, unlike the small-molecule
though both lepirudin and bivalirudin require direct thrombin inhibitors, none of these hiru-
binding to the thrombin exosite 1 as part of din-like inhibitors is likely to have substantial
Anticoagulants, Antithrombotics, and Hemostatics

oral bioavailability, and currently none of established. However, it has been shown that
these newer inhibitors is being evaluated in blocking both receptors provides an additive
the clinic. effect in the inhibition of platelet-mediated
thrombin generation and abciximab achieves
3.2.4 Platelet GPllbAlla Antagonists. After a dose-dependent reduction in thrombin gen-
activation, platelets aggregate by means of eration to a maximum of 45-50% inhibition.
their GPIIb/IIIa receptors, binding to biden- Presumably, the decrease in thrombin gener-
tate fibrinogen, thus allowing formation of a ation is a consequence, at least in part, from
three-dimensional platelet thrombus. Is has the resultant absence of a concentrated plate-
been recognized that, whereas platelet activa- let mass and the attendant dilution of soluble
tion is initiated by a number of stimuli (ADP,
activating stimuli. This reduction in platelet-
thrombin, etc.), fibrinogen binding to the
mediated thrombin generation is believed to
GPIIb/IIIa receptor represents the final com-
mon step to platelet aggregation. Therefore, contribute to the clinical efficacy of abciximab
by targeting the blockade of this interaction, (68, 113).
platelet aggregation should be inhibited, re- The structures of eptifibatide (6) and tiro-
gardless of the source of platelet activation fiban (7) mimic the RGD motif and bind
(111). GPIIb/IIIa is a member of a larger fam- tightly to the platelet GPIIbIIIIa receptor.
ily of integrin receptors and it is also referred Like abciximab, they have been shown in vitro
to as a,,,& (integrin nomenclature). GPIIbI to reduce thrombin generation, although to a
IIIa receptors recognize and bind to the tri- lesser extent. The design for eptifibatide was
peptide sequence RGD (Arg-Gly-Asp) of fi- inspired by a KGD-containing snake venom
brinogen. Fibrinogen has this RGD sequence disintegrin protein, which was known to bind
located at each terminus of its a-chain, thus to the GPIIb/IIIa receptor both potently and
allowing the bidentate interaction that results selectively (114). In the SAR leading to the
in crosslinked platelets (Fig. 6.14). Addition- discovery of this drug, it was found that,
ally, evidence has accumulated that fibrinogen whereas small cyclic peptides incorporating
can bind to GPIIb/IIIa independently of its the KGD sequence were selective, they lacked
RGD sequence through an unrelated dode- the potency of their relatively unselective cy-
capeptide sequence (HHLGGAKQAGDV) lo- clic RGD counterparts. Guanylation of the ly-
cated at each terminus of its y-chain. Al- sine residue on the KGD analogs, resulting in
though it has long been known that RGD a homo-Arg residue, fortuitously provided
peptides (and RGD mimetics; see below) bind compounds that were both potent and selec-
to GPIIbDIIa and effectively inhibit platelet tive. Nonpeptide antagonists such as tirofiban
aggregation, the isolated fibrinogen dode- and many other recent analogs essentially fol-
capeptide can independently bind to the recep- low the design paradigm: (Arg mimetic)-(con-
tor at a location distinct from the RGD binding strained spacer)-(Asp mimetic), such that the
site and can also inhibit platelet aggregation overall length from the basic nitrogen to the
(112). acid group is about 16 A (115).
Abciximab (c7E3, Reopro) is the Fab frag- All three marketed GPIIb/IIIa drugs are
ment of a mouse human chimeric antibody to poorly absorbed by the oral route and are
the GPIIb/IIIa receptor and binds tightly and dosed by continuous IV infusion. Abciximab is
essentially irreversibly, resulting in potent in- approved as an adjunctive therapy with aspi-
hibition of aggregation of activated platelets rin and heparin for percutaneous coronary in-
(111).Interestingly, in spite of this tight bind- terventions (PCI), such as angioplasty, and is
ing, it is thought that abciximab continually being considered as an adjunctive therapy in
redistributes from one platetet to another and other settings of arterial (platelet-rich) throm-
therefore its effect can persist longer than the bosis [e.g., acute myocardial infarction (MI)
8-day lifetime of an individual platelet. Abcix- and ischemic stroke]. Eptifibatide is approved
imab also binds to the platelet vitronectin as an adjunct in PC1 and unstable ischemic
(a;&)receptor, although the clinical signifi- syndromes, whereas tirofiban is approved for
cance of this lack of selectivity has not yet been unstable ischemic syndromes only. Both of
3 Physiology, Biochemistry, and Pharmacology

(17) lamifiban (18) sibrafiban

(19) xemilofiban (20) lotrafiban

(21) roxifiban

Figure 6.26. Several platelet GPIIbflIIa inhibitors investigated in clinical trials by IV (17)and by
oral administration(18-21).

ease oral bioavailability. For


example, sibrafiban (18) contains a hydroxy-
Two other p a r e n t e d GPIIb/IIIa agents lated amidine to reduce basicity and an ester-
, echoing the technique used
7, a humanized Fab fragment directed to prodrug the thrombin inhibitor melaga-
GPIIb/IIIa, but having less affinity tran. Xemilofiban (19),another benzamidine,
at of abciximab for the vitronectin re- is prodrugged as the ethyl ester, whereas the
RGD mimetic lotrafiban (20)
d. The human oral bioavail-
f these orally active agents
an (17, Fig. 6.25), showed a small ben- have not been reported, although xemilofiban
reducing acute coronary syndrome was reported to have a bioavailability of 13%
on oral dosing (119). In contrast to the IV-
ver the past decade, a number of pharma- administered GPIIbDIIa antagonists, these
t demonstrated efficacy in
er and develop orally bioavailable small patients with acute coronary syndromes, and
ptide GPIIb/IIIa antagonists, and most many of them have been associated with an
e effort was directed toward the investi- increase in mortality ( l l l a , 120). The worse
h use of these oral agents
by observations that
of these agents contained highly basic GPIIb/IIIa antagonists can induce the recep-
Anticoagulants, Antithrombotics, and Hemostatics

tor to adopt a ligand-binding conformation similar efficacy for the two agents at prevent-
that transiently persists after dissociation of ing coronary stent thrombosis, the adverse
drug, allowing fibrinogen to bind and, para- event rate was higher for ticlopidine (124).
doxically, platelet aggregation to commence Also, the historical risk of thrombotic throm-
( l l l a , 121). This proaggregation effect may be bocytopenic purpura is lower with use of clo-
general for all GPIIbIIIIa antagonists, but the pidogrel compared to that of ticlopidine.
response may be exaggerated for the oral Only the S-isomer of clopidogrel is active as
agents, given the periods of trough drug levels an antiplatelet agent (125); ticlopidine is
that allow receptor occupancy to fall off. achiral. A third thienopyridine antiplatelet
By contrast, the IV agents are maintained agent, CS-747 (22, Fig. 6.261, a racemate, is
at a continuously high plasma concentration currently undergoing phase I trials (126). As
with uninterrupted and high receptor occu- with clopidogrel and ticlopidine, antiplatelet
pancy. Further, the IV agents have been typi- effects for CS-747 require hepatic conversion
cally administered against the background of to an active metabolite, the structure of which
anticoagulant therapy, which can enhance the has been confirmed and is analogous to the
clinical response to a GPIIbIIIIa antagonist. clopidogrel/ticlopidine active metabolites.
Clinical development of most of these oral Adenosine triphosphate is a competitive
agents has been terminated. Roxifiban (21, antagonist of the action of ADP at the P2Y1,
Fig. 6.251, a more recent oral GPIIbDIIa an- receptor, although it is unacceptable as a ther-
tagonist, appears to differentiate itself from apeutic agent as a result of its weak potency
the earlier oral agents, in that it is bound more and its metabolism to ADP (127). A class of
tightly to the platelet receptors and thus stabilized ATP analog antagonists of P2Y1,
might be able to maintain sufficient receptor exhibit selectivity for this receptor and act di-
occupancy upon oral dosing to achieve the de- rectly, with no metabolic modification re-
sired efficacy (118d). It still remains to be es- quired as for the thienopyridines. Representa-
tablished whether clinical outcomes might im- tive of this class of ATP analogs is cangrelor
prove with oral GPIIbDIIa antagonists having (AR-C69931; 23), which has an IC,, value of
more favorable (tighter) receptor binding 0.4 nM against ADP-induced platelet aggrega- 1
properties and/or having pharmacokinetics al- tion and >1000-fold selectivity for the P2Y1,
lowing higher continuous plasma drug levels receptor compared to the other P2-type re
with less of a trough. ceptors (127). Structurally, the terminal di-
chlorophosphonate group, a phosphate mimic,
3.2.5 Platelet ADP Receptor Antagonists. is stabilized toward hydrolysis. Additionally,
As discussed in section 2.3, the thienotetrahy- modifications to the purine 2 and 6 positions
dropyridine (usually referred to simply as provide cangrelor with increased potency over
thienopyridine) analogs clopidogrel and ticlo- that of ATP.
pidine are inactive per se, requiring hepatic As an IV agent, cangrelor demonstrated
conversion to a ring-opened thiol-active spe- therapeutic efficacy in phase I1clinical studies
cies that irreversibly inhibits the platelet re- in patients with acute coronary syndromes. Its
ceptor P2Y12, presumably by formation of a rapid onset of action and rapid reversal upon
disulfide linkage to a receptor cystein. The cessation of infusion contrasts with the slow
P2Y, receptor is insensitive to thienopyridines onset and reversal of activity of the thienopy-
(67). Clopidogrel and ticlopidine are effica- ridines (128). However, further development
cious antiplatelet agents in humans (12, 54b, of cangrelor has been terminated. Nonphw
122) and, in particular, clopidogrel has shown phate adenosine analog antagonists of P2Y
superiority over aspirin, with comparable have been reported, but these are current
safety, in the prevention of myocardial infarc- less well characterized in terms of their poten
tion and stroke, and when used in combina- tial as antiplatelet drugs (126c, 129).
tion with aspirin has shown a reduction in The first example of a nonnucleosid
ischemic events compared to that of aspirin versible selective P2Y12 antagonist has
alone (123). Although one clinical study com- reported, CT50547 (24). This compoun
paring clopidogrel to ticlopidine demonstrated plays moderate inhibitory potency in a P2Y1,
3 Physiology, Biochemistry, and Pharmacology

H3C/'-NH

- /

HO OH
(22) CS-747 (racemate) (23)cangrelor, AR-C69931

CI

H2O3PO

~ 2 0 ~ ~ 6
(24) CT50547 (25) MRS2279
Figure 6.26. Antiplatelet agents active at the P2Y,, (22-24) and P2Y1(25) receptors.

ligand binding assay (IC,, = 170 nM) phosphate adenosine analog antagonists of
similar level of potency in an ADP-in- the P2Y1 receptor have recently been reported
platelet aggregation assay. It is 1000- (133).
selective for P2Y1, compared to the P2Y1 A question that remains to be answered is
whether antagonism of the P2Y1 receptor
e activation of P2Y1 receptors in plate- alone or dual antagonism of the P2Yl and
contributes to platelet aggregation and P2Y1, receptors might achieve clinical bene-
onists of this receptor may have poten- fits equivalent to or superior to the antago-
as antithrombotic agents (67, 131). Natu- nism of P2Yl, alone.
occurring adenosine bisphosphates (e.g.,
'-bisphosphate) act as weak 3.2.6 Aspirin and Dipyridamole. Aspirin is
etitive antagonists at the P2Y1 receptor the most common antiplatelet drug in use to-
structural modification at the ribose ring day (12, 134). In the platelet, aspirin irrevers-
at the purine 2 and 6 positions have af- ibly inactivates cyclooxygenase-1 (COX-1) by
d competitive inhibitors with enhanced acetylating the hydrorry group of Ser-529 near
cy and selectivity (132). For example, the active site, thereby blocking the binding of
2279 (25) has an IC,, value of 52 n M in a its substrate arachadonic acid. COX-1 in the
antagonism assay that measured inhibi- platelet normally converts arachadonic acid to
of phospholipase C induction elicited by PGH,, a precursor of the potent platelet adi-
omethyl-ADP. This compound also po- vator thromoboxane A, (TxA,). Because plate-
inhibits platelet aggregation and does lets lack a nucleus and do not support protein
the P2Y,, receptor. Non- synthesis, they cannot replenish the acety-
Anticoagulants, Antithrombotics, and Hemostatics

lated COX-1 for the duration of their normal These enzyme or protein preparations act on
lifetime (about 7-10 days). Moreover, because plasminogen either to generate plasmin or to
only about 10% of the platelet pool is replen- create an activated form of plasminogen hav-
ished each day, once-a-day dosing of aspirin is ing plasminlike activity. Plasmin activity acts
able to maintain virtually complete inhibition to dissolve the fibrin component of clots.
of platelet TxA, production. To a lesser extent, There are shortcomings with many of these
aspirin inactivates COX-1 activity in endothe- thrombolytic agents, which include: (1)short
lid cells, leading to a decrease in the synthesis plasma half-life, which may partly reflect the
of the antiplatelet modulator PGI,, although rate of inactivation by the serpin PAI-1, neces-
this effect can be partially overcome by de sitating either continuous infusion or multiple
novo protein biosynthesis. Mucosal COX-1 ac- bolus IV doses; (2) induction of a "paradoxi-
tivity is also inhibited by aspirin, which con- cal" prothrombotic condition, which may lead
tributes to gastric bleeding (Section 2.2.1). As- to a greater tendency to reocclude, or a sys-
pirin also exhibits anti-inflammatory activity temic lytic condition, or both; and (3) immu-
by inhibition of cellular COX-2, although at nogenicity (137a, 138). For example, the first-
doses higher than that needed to achieve its generation thrombolytic, streptokinase, has a
COX-1 mediated antiplatelet effects. Other reasonably acceptable half-life (18-23 min)
COX-1 inhibitors have been investigated, dif- but is immunogenic and prone to induce pro-
fering in their antiplatelet and therapeutic thromboticflytic conditions. Alteplase (natu-
profiles compared to those of aspirin, and a few ral recombinant human tPA), a second-gener-
are marketed in other countries (12, 135). ation agent, is nonimmunogenic and induces
Low dose aspirin is well established at im- less of a prothrombotic/lytic condition than
proving outcomes in patients who have had that of streptokinase, but has a short half-life
thrombotic events or who may be prone to (4-6 min).
them. In patients with acute MI, prior MI, un- The paradoxical prothrombic andlor lytic
stable angina, or stroke, aspirin reduced the condition (see below) induced by several of
long-term risk of recurrences by 25% and in these agents has been associated with the in-
individuals with stable angina, aspirin re- ability of these agents to exhibit "clot selectiv-
duced the risk of MI by 44% (134). ity" (138c,d). The origin of clot selectivity has
Dipyridamole is thought to exert antiplate- its basis in the ability of some plasminogen
let effects in part by inhibiting phosphodies- activators (e.g., tenectaplase) to bind effi-
terase-mediated hydrolysis of the platelet-de- ciently to a unique conformation of plasmino-
activating nucleotides CAMP and cGMP, gen when the plasminogen molecule is itself
although the scope of dipyridamole's mecha- bound at the C-terminal lysine sites of par-
nism or mechansims of action is still not en- tially degraded fibrin. Clot-selective agents do
tirely clear. It appears to synergize with aspi- not bind as readily " to the solution conforma-

rin. In patients with a history of transient tion of plasminogen, which is different from
ischemic attack or ischemic stroke, aspirin its fibrin-bound conformation (139). This clot
and sustained-release dipyridamole decreased selectivity achieves two important physiologi-
risk for stroke by 18 and 16%, respectively, cal results: (1)
. . the PA is localized to the site

whereas aspirin added to sustained-release di- (fibrin + plasminogen), where it will have the
pyridarnole decreased risk by 37% (136). most therapeutic effect; and (2) localization of
the PA to the clot restricts the activator from
3.3 Thrombolytic Agents: Mechanisms
diffusing into the general vasculature, which
and Improvements
can trigger the prothromboticflytic states re-
Over the last decade, thrombolytic therapy ferred to above. In particular, evidence has
has had a significant impact on how acute accumulated that high concentrations of plw
myocardial infarction, and more recently, on minogen activators freely circulating through-
how acute ischemic stroke is treated (137). out the vasculature can trigger activation of
Most thrombolytics, either currently mar- the kallikreinJFXI1pathway that leads to gen-
keted or in trials, are natural or modified eration of FXIa and a resultant prothrombotic
forms of tPA, uPA, or bacterial proteins. state. This may explain, at least in part, why in
Antithrombotic Agents Having Alternative Mechanisms of Action 323

some settings of thrombolytic therapy, partial It has been found that covalently linking poly-
or total reocclusion is observed after initial ethylene glycol to staphlokinase enhances.its
dissolution of the thrombus. Subsequently, af- plasma half-life (140).
ter depletion of procoagulant factors and the The mechanism of the non-clot-selective fi-
a2-antiplasmininhibitor, the circulating plas- brinolytic streptokinase, another nonenzyme
minogen activator continues to freely gener- bacterial protein, is somewhat different from
ate plasmin within the vasculature and may that of staphlok'inase, in that it binds to plas-
induce a systemic lytic state, with possible minogen in a way that conformationally opens
hemorrhagic consequences (138c,d, 140). up and activates the catalytic site, without a
The third-generation agent tenectaplase proteolytic cleaveage to form plasmin.
(TNKase)is a recombinant analog of tPA, en- Other modifications to tPA or bacterial pro-
thrombolytic proteins continue to be studied,
gineered to render it more clot selective, pro-
which may result in potential therapeutic ad-
long its half-life, and make it more resistant to
vantages (141).
PAI-1. The letters TNK in its name indicate
some of the amino acid replacements that
were made as part of this process. For exam- 4 ANTITHROMBOTIC AGENTS HAVING
ple, lysine (K),histidine, and two arginines ALTERNATIVE MECHANISMS OF ACTION
were replaced by four alanines in the catalytic
portion of this enzyme to enhance resistance Treatment of thrombotic conditions using
to PAL1 inhibition. In accord with the inter- currently marketed agents, although largely
pretation of clot selectivity, it was observed effective, have disadvantages. Heparin and
that clinical markers of thrombin generation warfarin anticoagulant activity must be mon-
itored carefully because of safety concerns.
tracked inversely with the extent of clot selec-
Moreover, warfarin has a delayed onset of ac-
tivity for three PAS, with the level of markers
tion. Most antithombotic and antiplatelet
being in the order: streptokinase > alteplase
> tenectaplase. Tenectaplase has a longer agents must be administered either IV or SC,
which is less desirable than oral administra-
half-life than that of alteplase (14-18 versus
tion. Aspirin, although ubiquitously used, is
4-6 min, respectively), allowing less frequent
not highly efficacious when used alone in
dosing. Therefore, because of its longer half-
many settings. To overcome these drawbacks,
and high degree of clot selectivity, tenecta-
a great deal of research is currently directed
lase seems to represent a significant advance
toward the discovery of new treatment op-
a fibrinolytic agent over the older agents.
tions, many of which exploit alternative mech-
Staphlokinase, another third-generation
anisms.
ot-selective PA undergoing trials, is a recom-
inant nonenzyme bacterial protein with a
4.1 Inhibitors of Coagulation Factors
ode of action different from that of the
ne protease PAS.Staphlokinase binds as a The direct inhibition of thrombin represents a
complex to the unique C-terminal fibrin- logical strategy for achieving an efficacious
ding conformation of plasminogen, which and reasonably safe therapeutic anticoagulant
ws small amounts of circulatingplasmin to effect. On the other hand, the inhibition of the
ivate the complexed plasminogen. The re- serine protease coagulation factors that pre-
tant staphlokinase-plasmin complex is pro- cede thrombin in the coagulation pathway,
ded from d-antiplasmin-mediated inacti- FXa, FIXa, or FVIIa, represents an equally vi-
ion while on the surface of fibrin, but is able strategy, and one that may have advan-
dily inactivated if it dissociates into the cir- tages over the inhibition of thrombin alone. By
ation, further accounting for its high clot attenuating the generation of thrombin,
vity (139). The plasma half-life of rather than inhibiting the catalytic activity of
okinase is short, however (6 min); and it thrombin itself, physiological functions medi-
munogenic, inducing neutralizing anti- ated by low levels of thrombin might be
s after 10 days in a majority of patients, spared. Normal hemostasis mediated by
herefore might be restricted to single use. thrombin's action at the PAR-1 receptor, for
Anticoagulants, Antithrombotics, and Hemostatics

Figure 6.27. Potent reversible inhibitors of FXa.

example, could remain intact and therefore in- lective inhibitors of alternate coagulation fac-
hibitors of the earlier coagualtion factors may tors, especially Factor Xa. Similar to the
not only be effective antithrombic agents but search for direct thrombin inhibitors, an em-
may also carry less bleeding risk. Early sup- phasis was placed on the development of
port for this concept was reported using mac- agents having good oral bioavailability to al-
romolecular inhibitors of FXa and TF/FVIIa low convenient chronic dosing.
in animal models of thrombosis. These studies
concluded that, whereas direct thrombin in-
hibitors such as hirudin impaired platelet he- 4.1.1 Direct Active Site Inhibitors of FXa
mostatic function in parallel with their anti- Building on the experience gained from the
thrombotic effects, selective inhibition of FXa successful development of reversible small.
using TAP (tick anticoagulant peptide) or molecule thrombin inhibitors, a number of p
inhibition of TF/FVIIa using active-site-inhib- tent and selective inhibitors of FXa have been
ited FVIIa (FVIIai) could achieve a dose-de- discovered that are efficacious in various mi-
pendent antithrombotic effect with compara- mal models of thrombosis (143). Some have
tively less impairment of hemostatic function also shown good bioavailability and plasma
and hemorrhagic risk (142). Moreover, by in- half-lives upon oral dosing in animals. Fi
hibiting the earlier coagulation factors that 6.27 shows the structures of four represen
are responsible for the generation of throm- tive optimized FXa inhibitors. The most
bin, the "thrombin rebound" effect (section tent of these is the benzamidine CI-1031(26
3.2.3) might be avoided. Starting in the mid- which has a Kivalue for FXa of 0.11 nM
1990s significant research effort was directed which is >1000-fold less potent for throm
toward the discovery and development of se- and trypsin. Surprisingly, in spite of its mult
ns of Action
Antithrombotic Agents Having Alternative Mechanis~ 325

d structure, high plasma levels of thrombolysis. In a baboon model of arterial


1-1031persisting up to 6 h are achieved after thrombosis, a 2-h infusion of rTAP resulted in
in primates (144). a long-lasting (55 h) antithrombotic effect
showed efficacy with favorable (152).
ty (bleeding) profiles in several animal
rombosis, including a tPA fibrino- 4.1.2 lnhibitors o f FIXa. In the intrinsic te-
is model in dogs (145). An X-ray crystal nase complex, Factor IXa activates FX to FXa
cture of CI-1031 shows the benzamidine (Fig. 6.8). Although hereditary deficiencies of
ing a salt bridge with Asp-189 in the S1 FIX result in hemophilia B, studies in animals
et, whereas the distal phenyl along with suggest that agents that block the activity of
appended methyl dihydroimidazolidine FIXa may possess therapeutic utility with an
es the lipophilic S4 pocket defined acceptable safety margin. Factor Ma, which is
Trp-215, Tyr-99, and Phe-174 (146). covalently inhibited at its active site with a
The structure of DPC423 (27) evolved from tripeptide chloromethylketone (DEGR-inacti-
-containing analogs and vated FXIa; FXIai), competes with endoge-
sic benzylamine group that pre- nous FIXa for incorporation into the intrinsic
ably binds in the FXa S1 pocket. DPC423 tenase complex, resulting in diminished con-
= 0.15 nM), selective against version of FX to FXa. FIXai infused into dogs
ombin and trypsin, and shows excellent was as efficacious as heparin in a model of cor-
availabilty (57%)and half-life (7.5 h) when onary artery thrombosis and, moreover, an
t is efficacious in a canine abnormal bleeding response from a surgical
with a good safety pro- wound was present only with heparin, not
e, and has entered clinical trials (147). FIX& FIXai was also effective in a guinea pig
RPR209685 (28) and anthranilic amide in- thrombosis model, with minimal effects on
itor (29) are examples of potent non- normal hemostasis (153). Monoclonal anti-
ged FXa inhibitors. In the case of (29) bodies against FIX/FIXa that inhibit both the
= 11 nM),both computer modeling and activation of FIX and the activity of FIXa have
t h the X-ray crystal structures shown efficacy in models of venous and arte-
ne-containing analogs suggest rial thrombosis in a number of animal species,
henyl group occupies the S1 with no serious bleeding consequences com-
et of FXa and the dimethylaminophenyl pared to heparin. Humanized monoclonal an-
pies S4 (148). RPR209685 (K, = 1.1 n M ) is tibodies to FIX have been dosed to healthy vol-
00-fold selective against trypsin, tPA, unteers (154). No selective small-molecule
in, thrombin, and other serine pro- inhibitors of FIXa have been reported to date,
. It is orally bioavailable in the rat and however.
, respectively) and shows
cy in a dog model of DVT (149). 4.1.3 Inhibitors o f FVlla and TF/FVlla.
id peptide isolated from Four macromolecular inhibitors of TF/FVIIa
nithodoros moubata, is a or FVIIIFVIIa are currently being investi-
selective inhibitor of FXa (150). An X- gated clinically: recombinant TFPI (tissue fac-
of a recombinant form of tor pathway inhibitor), rNAPc2 (recombinant
with FXa has revealed a nematode anticoagulant protein c2), a mono-
motif involving insertion of several N- clonal antibody to FVII/FVIIa, and active-site-
residues of the inhibitor inhibited FVIIa (FVIIai).In their mechanisms
FXa active site and the C-terminus of action, TFPI and rNAPc2 both employ FXa
to a separate domain (1511, reminis- as an initial "scaffold," to bridge to and to in-
t of the hirudin-thrombin motif. A number activate the TF/FVIIa complex. Recombinant
ical in vivo studies have validated its TFPI (tifocogin)is an effective antithrombotic
hrombotic efficacy and relative safety. in several animal models and has been shown
example, rTAP was more effective than in healthy humans to dose-dependently atten-
din when given as an adjunctive treat- uate endotoxin-induced coagulation activa-
ent to dogs undergoing alteplase-induced tion (155). rNAPc2, was shown in patients un-
Anticoagulants, Antithrombotics, and Hemostatics

dergoing elective total knee replacement to be by blocking the TxA, synthase-mediated con-
50% more effective than heparin, with similar version of PGH, to TxA, might achieve an ad-
bleeding profiles. Trials in patients with un- vantageous antiplatelet and vasorelaxant ef-
stable angina undergoing percutaneous trans- fect. Alternatively, or additionally, blocking
luminal coronary angioplasty (PCTA) are the TxA, receptor might also prove therapeu-
scheduled (156). The monoclonal antibody to tically useful. In reality, however, it was found
FVIIIFVIIa (Corsevin M) is being studied in that inhibition of TxA, synthase results in
clinical trials for arterial thrombosis in the build up of the precursor PGH,, which itself
setting of PTCA (157). FVIIa inactivated with activates platelets upon binding to the TxA,
Phe-Phe-Pro chloromethyl ketone (FFP- receptor (135,161). Nevertheless, it was found
FVIIa) competes with endogenous FVIIa for that dual agents that combine both TxA, syn-
binding to TF, forming an enzymatically inac- thase inhibition and TxA, receptor-blocking
tive complex. FFP-FVIIa has shown anti- activities are potent inhibitors of platelet
thrombotic efficacy in several animal models function and interest continues in the design
with a good safety profile. An early clinical and testing of such dual agents (135,161). For
trial employed FFP-FVIIa with heparin in pa- example, terbogrel(301, a potent antagonist of
tients undergoing PCTA and indicated a trend both the thromboxane receptor (IC,, = 11
for efficacy, although with some increase in nM) and thromboxane synthase (IC,, = 4 nM)
minor bleeds (158). Reports of potent, revers- efficiently inhibits collagen-induced platelet
ible small-molecule active-site inhibitors of aggregation (IC,, = 310 nM). Terbogrel is
FVIIa (Ki values of 3-12 nM) have begun to 30% orally bioavailable in rats (162) and is
emerge (159). All of these potent inhibitors currently in Phase I1 clinical trials for throm-
contain a benzamidine group, which presum- botic indications. The nonacidic compound
ably binds to Asp-189 in the S1 pocket at the BM573 (31)prevents platelet aggregation in-
active site of FVIIa. Based on the experience duced by arachidonic acid (IC,,, = 125 nM)
gained with the development of neutral small- and induced by the receptor agonist U466619
molecule inhibitors of thrombin and FXa, se- (IC,, = 240 nM) (163). Pure TxA, receptor
lective FVIIa inhibitors having the potential antagonists having no TxA, synthase activity
for oral bioavailability should, in theory, also [e.g., ifetroban (32) and S-18886 (33)lare also
be feasible. A recent study compared the effi- potent inhibitors of platelet aggregation (164).
cacy and safety of small-molecule inhibitors of In particular, S-18886 inhibited U466619-in-
thrombin, FXa and FVIIa, in a guinea pig duced platelet aggregation with an IC,, value
model and concluded that at equivalent levels of 230 nM and shows antiplatelet and anti-
of antithrombotic effect, the FVIIa inhibitor thrombotic effects when dosed orally in sev-
had the smallest bleeding risk (160). eral animal species (164c-e). Further clinical
trials of dual agents andlor pure TxA, receptor
4.2 Antiplatelet Agents with Alternative antagonists will be needed to more fully assess
Modes of Action whether agents from this class will achieve
significant therapeutic usefulness in humans
4.2.1 Agents That Interfere with the Throm- as antithrombotics.
boxane Receptor and Thromboxane Synthase.
Thromboxane A, (TxA,, Fig. 6.28) activates 4.2.2 Platelet PAR-1 and -4 Receptor An-
platelets upon binding to the platelet TxA, tagonists. Thrombin acts as a powerful plate-
GPCR (Section 3.1). Additionally, TxA, causes let activator through proteolytic-mediated
constriction in vascular tissue. Aspirin indi- agonism of the platelet PAR-1 and PAR-4 re-
rectly inhibits TxA, biosynthesis by blocking ceptors (Section 3.1). As discussed, PAR-1 *
conversion of arachadonic acid to PGH,, the nism provides an immediate activation re-
precursors to TxA,. However, aspirin also sponse at low thrombin levels, whereas PAR-4
blocks biosynthesis of the antiplatelet and va- agonism provides a response at higher throm-
sodilating prostaglandin PGI, in vascular en- bin levels typical of the later stages of clot
dothelium, a potential disadvantage. In the- formation. Peptidic and nonpeptidic PAR1
ory, directly inhibiting the generation of TxA, antagonists structurally derived from the un-
i'
4 Antithrombotic Agents Having Alternative Mechanisms of Action 327

OH
thromboxane A2 (TxA2)

(30)terbogrel (31) BM573

(32) ifetroban (33) S18886

Figure 6.28. Thromboxane A2 and agents that block both the TxA, receptor and TxA, synthase (30
and 31) or the receptor only (32 and 33).

asked tethered receptor ligand (SFLLR) or TRAP and thrombin-stimulated platelet ag-
rived from high throughput screening leads gregation (IC,, values of 0.11 and 0.37 a,
e been described (64a, 115,165). However, respectively) in vitro and furthermore was
compete with the favorable energetics of a shown to be efficacious in a primate model of
thered agonist ligand, a soluble small-mole- arterial thrombosis when dosed by IV infusion
le antagonist is at a theoretical disadvan- (166b,c). Primate platelets, similar to human
. Typically, many of the PAR-1 antago- platelets, have both PAR-1 and PAR-4. This
ts reported to date have shown potent result provides some encouragement that a
ockade of platelet aggregation induced by PAR-1-specific antagonist may have the po-
mall peptide agonists (e.g., SFLLR-NH,; a tential to achieve a therapeutically useful an-
TRAP" = thrombin receptor-activating pep- tithrombotic effect in humans.
de), but are less effective at blocking throm-
-mediated platelet activation. Recently, 4.2.3 Other Platetlet Targets: Serotonin,
wever, druglike antagonists that efficiently PCI,, and PAF Receptors. Serotonin (5-hy-
ock thrombin-induced aggregation have droxytryptamine, 5-HT) binding to platelet
been reported. For example, FR171113 (34, 5-HT, receptors elicits a weak aggregation
fig. 6.29) is efficacious against both SFLLRN- response that is enhanced in the presence of
H, and thrombin-mediated platelet aggrega- collagen at the site of vasculature injury.
tion (IC,, values of 0.15 and 0.29 p M , respec- 5-HT-induced vasoconstriction, mediated by
ly) (166a). Also, the PAR-1 selective binding to endothelial5-HT,, and 5-HT,, re-
agonist RWJ-58259 (35) blocked both ceptor subtypes, contributes to its thrombotic
Anticoagulants, Antithrombotics, and Hemostatics

Figure 6.29. Platelet PAR-1 antagonists.

effect in vivo. Ketanserin (36, Fig. 6.30) and thrombotics (169). Stable, orally bioavailable
sarpogrelate (37) are two well-studied older prostanoids such as iloprost and beraprost
orally active 5-HT antagonists that show anti- (41) have demonstrated antiplatelet effects in
thrombotic effects in vivo (167). Sarpogrelate clinical trials, although their half-lives are
is marketed in Japan for treatment of periph- very short (<1h). Therefore, some current ef-
eral arterial disease. A number of research fort in the field is directed toward improving
groups are continuing to investigate peripher- the pharmacokinetic profile of PGI, agonists.
ally acting serotonin antagonists as potential For example, FR181877 (42), which inhibits
antithrombotics (168). Whereas ketanserin ADP-induced platelet aggregation in vitro
has high affinity for the 5-HT,, receptor and with an IC,, value of 81 nM (IC,, = 4 nM for
inhibits collagen-induced platelet aggrega- beraprost), has a half-life in male rats of 4.3 h
tion, it has low affinity for the 5-HT,, receptor
compared to 0.43 h for beraprost (169a).
subtype. A recent analog, SL65.0472 (38),was
Platelet-activating factor (PAF), a phos-
designed to maintain the 5-HT, blocking ac-
tivity of ketanserin while also potently inhib- pholipid, was discovered in 1971 as a conse-
iting the endothial5-HT,, receptor, offering a quence of its platelet-aggregating properties.
potential advantage (168a). The acetamide In spite of its name, PAF has numerous other
group in SL65.0472 was introduced to limit activities (e.g., proinflammatory and vasodila-
CNS penetration of this compound. SL65.0472 tory) (170). Many small-molecule antagonists
demonstrated equivalence to ketanserin in of PAF having in vitro antiplatelet activity
human platelet aggregation assays, is effica- have been identified, and a few were shown to
cious in the Folts model of coronary artery be efficacious in animal models of thrombosis
thrombosis at an IV dose of 10-30 pglkg, and (171). To date, however, PAF antagonists
has entered clinical trials. Compound (39), an have not performed as effectively in vivo as
analog of sarpogrelate, is a more potent inhib- have other classes of antithrombotics.
itor of platelet aggregation in vitro than either To overcome the deficiencies of antiplatelet
ketanserin or sarpogrelate, but produced gas- agents that target single mechanisms, some
tric irritation in rats (168~). However, the lau- research has sought to combine two different
ryl ester prodrug (40, R-102444) did not pro- antiplatelet activities in one molecule. Agents
duce gastric irritation and, when dosed orally, combining thromboxane receptor antagonism
was more efficacious than sarpogrelate in a rat with thromboxane synthase inhibition have
thrombosis model. been mentioned. Additionally, single agents
PGI, (prostacyclin), produced by endothe- with combined PAF antagonism and throm-
lid cells, deactivates platelets (see Section 3.1) boxane synthase inhibition have been re-
and also acts as a potent vasodilator. Chemi- ported, as have agents combining antagonism
cally stable PGI, mimetics have been prepared for both the thromboxane and 5-HT,, recep
and evaluated for their potential as anti- tors (172). It is still too early to assess whether
Antithrombotic Agents Having Alternative Mechanisnns of Action

H 0
(36)ketanserin (37)sarpogrelate (racemate)

(41) beraprost (42) FR181877

Figure 6.30. Antagonists of the platelet 5HT receptor (36-40); agonists of platelet PGI, receptor
(41.42).

ese newer dual-acting compounds may have ful for enhancing endogenous fibrinolysis or
tential as efficacious antithrombotics. might be used in conjunction with thrombo-
lytic therapy, allowing the use of lower and
Potential Profibrinolytics: Inhibition perhaps safer doses of recombinant tPA or
Factor Xllla, TAFla, PAI-1, other fibrinolytics.
2-Antiplasmin The transglutaminase Factor XIIIa
nts that operate through mechanisms that strengthens fibrin toward plasmin degrada-
en the structure of fibrin or that enhance tion by covalently crosslinking the fibrin
e efficacy of endogenous plasmin or its acti- strands and it also protects fibrin from the ac-
r tPA should behave as profibrinolytics, tion of plasmin by covalently attaching the
may potentially find therapeutic utility in plasmin inhibitor aPantiplasmin to the sur-
ombotic settings. Such agents could be use- face of fibrin (Section 3.1). Therefore, inhibi-
Anticoagulants, Antithrombotics, and Hemostatics

Figure 6.31. Prototype inhibitors of FXIIIa (43), TAFIa (441, and PAI-1 (4648).

tors of FXIIIa have the potential to increase dent (irreversible) inhibitor of FXIIIa and,
the susceptability of clots toward lysis. Tride- based on a binding mode analysis using the
gin, a peptide isolated from the salivary glands known crystal structure of FXIIIa, a mecha-
of the giant Amazon leech is a specific inhibi- nism was proposed involving attack of the ac-
tor of Factor XIIIa, and clots formed in the tive site cysteine-314 on the cyclopropene dou-
presence of this peptide were shown to be ble bond (174). A few other peptide and
more rapidly lysed in vitro (173). A potent nonpeptide inhibitors of FXIIIa have also been
(IC,, = 26 nM)and selective synthetic non- described, although to date, reversible drug-
peptide FXIIIa inhibitor, derived from optimi- like small-molecule inhibitors have not been
zation of a class of FXIIIa inhibitors isolated reported (175).
from a fungus species, contains an unusual The plasma carboxypeptidase TAFIa re
cyclopropeneone structure (43, Fig. 6.31). En- tards the action of plasmin by cleaving C-ter-
zyme kinetics indicate that it is a time-depen- mind lysine residues from partially degraded
5 The Future of Antithrombotic Therapy

fibrin (Section 3.1). A 39 amino acid peptide region containing three arginines (179a). An-
inhibitor of TAFIa has been isolated from po- other research group had earlier reported the
tatoes. In a rabbit jugular vein model of nonacidic diketopiperizine PAI-1 inhibitor
thrombosis, coinfusion of this peptide with (47) (XR5118; IC,, = 3.5 a) and demon-
tPA enhanced thrombolysis or markedly re- strated its antithrombotic effect in a rabbit
duced the amount of tPA needed to achieve thrombolysis model (179b). Systematic modi-
the same amount of lysis (60, 176). A few pro- fication of structure (47) resulted in the car-
totype small-molecule inhibitors of TAFIa boxylate analog (48), one of the more potent
have recently been reported [e.g., the phos- small-molecule PAI-1 inhibitors reported to
phonate-based compound 441. Because TAFI date (IC,, = 0.2 pit4 in a plasmin generation
is activated by thrombin, which is found at the assay, with a similar potency in a functional
site of a thrombus, inhibition of TAFIa might fibrinolysis assay) (179~).It is hypothesized
provide enhanced fibrinolysis with the added that (48) binds at the same PAI-1 site as inhib-
advantage of clot selectivity (see Section 3.3). itor (45) and that the carboxylic acid groups of
PAL1 is the predominant serpin inactiva- both (48) and (48) are interacting with an Arg
tor of tPA and therefore inhibitors of PAI-1 residue within this binding site.
are expected to enhance endogenous fibrinoly- The serpin for plasmin, cy2-antiplasmin
sis. Several in vitro and animal studies sup- (a2AP), has been inhibited by monoclonal an-
port this view. Lysis of human platelet-rich tibodies, resulting in enhancements to fibrino-
clots is accelerated by antibodies against Ivsis.
- Antibodies to a2AP were shown to am-
PAI-1 in uitro and Fab fragments against rat plify the lysis of human plasma clots in the
PAI-1 inhibit venous and arterial thrombosis presence of plasminogen activators (182).Sep-
in uivo (178). A number of small-molecule lead arately, it was also shown in a rabbit jugular
compounds have been reported that allow tPA vein thrombolytic model that specific inhibi-
activity t o be preserved in the presence of tion of fibrin-linked a2AP by anti-a2AP anti-
PAI-1 (179). bodies increased the rate of spontaneous fibri-
The exact mechanism(s) by which these nolysis (183). a2AP antibodies potentially
small molecules are able to prevent the in- suitable for therapeutic use in humans have
activation of tPA by PAI-1 is still being in- been described (184), although clinical trials
vestigated. Antibody studies have revealed have not been carried out to date. Tanninlike
epitopes on PAI-1 distinct from the reactive molecules have been claimed to have inhibi-
center loop that may accommodate the bind- tory activity against cy2AP in early reports
ing of small molecules (179a, 180). In princi- (1851, but small-molecule druglike inhibitors
le, the binding of small molecules to these or of a2AP have not been reported to date.
ther sites might interfere sterically with the
teraction of PAI-1 and tPA, or they might
ert the PAI-1 inhibitory conformation to 5 THE FUTURE OF ANTITHROMBOTIC
rnative conformations that may be either THERAPY
ss reactive ("latent" conformation) or that
allow PAI-1 to serve as a normal sub- Unfractionated heparin is being replaced in
trate for tPA with fast turnover kinetics many of its applications by currently available
alternative p a r e n t e d agents such as the
Two recent examples of PAI-1 inhibitors, LMWHs, fondaparinu, lepirudin, bivaliru-
0th of which contain carboxylic acid groups din, and argatroban (186). These efficacious
(45 and 48, Fig. 6.311, are thought to interfere alternative agents offer advantages in terms
directly with the interaction of PAI-1 and tPA. of more predictable pharmacokinetics and im-
The modestly potent anthranilic acid inhibitor proved safety, reducing or eliminating the
(H029953XX, IC,, = 12 a, derived need for patient monitoring, and reducing or
flufenamic acid (46),is postulated to bind eliminating the risk of heparin-induced
a region of PAI-1, previously identified as a thrombocytopenia. Orally bioavailable direct
tralizing antibody epitope and that con- thrombin and/or FXa inhibitors now under de-
s a hydrophobic cleft flanked by a basic velopment seem poised to become available
Anticoagulants, Antithrombotics, and Hemostatics

over the next several years and will represent TIA transcient ischemic attack
viable alternatives to both the p a r e n t e d an- TM thrombomodulin
ticoagulants and to warfarin, the only cur- UA unstable angina
rently available oral anticoagulant. These UFH unfractionated heparin
agents promise the convenience of an oral VTE venous thromboembolism
drug without the need of intensive patient VWF von Willebrand factor
monitoring, as warfarin requires.
New antiplatelet agents that act by already
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Agen

Contents
1 Introduction, 340
2 Clinical Applications, 342
2.1 The Role of Lipids in the Atherogenic
Process, 342
2.2 Current Drugs on the Market, 342
2.2.1 HMG-CoA Reductase Inhibitors (Sta-
tins), 342
2.2.2 Fibric Acid Derivatives (Fibrates), 346
2.2.3 Bile Acid Sequestrants
(BAS)/Cholesterol Absorption
Inhibitors, 347
2.2.4 Nicotinic Acid Derivatives, 349
2.2.5 Miscellaneous, 350
2.2.5.1 Probucol, 350
2.2.5.2Hormone Replacement Therapy
(HRT), 350
2.2.5.3Estrogen Modulators, 351
2.2.5.4Plant Sterols, 351
2.3 Side Effects, Adverse Effects, Drug
Interactions, and Contraindications, 352
2.3.1HMG-CoA Reductase Inhibitors
(Statins), 352
2.3.1.1Hepatotoxicity, 353
2.3.1.2Myopathy, 353
2.3.1.3 Other Effects, 354
2.3.2 Fibric Acid Derivatives (Fibrates), 354
2.3.2.1Hepatotoxicity, 354
2.3.2.2Myopathy, 354
2.3.3 Bile Acid Sequestrants
(BAS)/CholesterolAbsorption
Inhibitors, 355
2.3.4 Nicotinic Acid Derivatives, 355
2.3.5 Miscellaneous, 355
2.3.5.1 Probucol, 355
2.3.5.2Hormone Replacement Therapy
(HRT),355
2.3.5.3Estrogen Modulators, 355
2.3.5.4Plant Sterols, 355
2.4 Absorption, Distribution, Metabolism, and
Elimination, 355
Antihyperlipidemic Agents

2.4.1 HMG-CoA Reductase Inhibitors 6 New and Future Treatments, 367


(Statins), 355 6.1 New Treatments for Lowering LDLc 2 TG
2.4.2 Fibric Acid Derivatives (Fibrates), 357 Lowering, 368
2.4.3 Bile Acid Sequestrants 6.1.1 Novel HMG-CoA Reductase Inhibitors
(BASYCholesterol Absorption (Statins), 368
Inhibitors, 357 6.1.2 Microsomal Triglyceride Transfer
2.4.4 Nicotinic Acid Derivatives, 357 Protein (MTP) Inhibitors, 368
2.4.5 Miscellaneous, 358 6.1.3 LDL-Receptor (LDLr) Upregulators, 369
2.4.5.1Probucol, 358 6.1.4 Bile Acid Reabsorption InhibitorsDile
2.4.5.2 Plant Sterols, 358 Acid Sequestrants (BAS)/Cholesterol
3 Physiology and Pharmacology, 358 Absorption Inhibitors, 369
3.1 Lipid Transport and Lipoprotein 6.1.5 Acyl-CoA Cholesterol AcylTransferase
Metabolism, 358 (ACAT) Inhibitors, 370
4 History, 362 6.2 New Treatments for Raising
4.1 Identification of the Statin Class of Lipid- HDLc 2 TG Lowering, 370
Lowering Drugs, 362 6.2.1 Peroxisome Proliferator-Activated
4.2 Discovery of Atorvastatin (Lipitor), 362 Receptor (PPAR) Agonists, 370
5 Structure-Activity Relationships, 363 6.2.2 Cholesteryl Ester Transfer Protein
5.1 HMG-CoA Reductase Inhibitors (Statins), (CETP) Inhibitors, 371
363 6.2.3 Liver X-Receptor (LXR)Agonists, 371
5.2 Fibric Acid Derivatives (Fibrates), 364 6.3 New Treatments for Atherosclerosis, 372
5.3 Bile Acid Sequestrants (BAS)/Cholesterol 6.3.1 Chemokines and Cytokines, 372
Absorption Inhibitors, 365 6.3.2 Antioxidants, 372
5.4 Nicotinic Acid Derivatives, 366 6.3.3 Lipoprotein-Associated Phospholipase
5.5 Miscellaneous, 367 A, (Lp-PLA,) Inhibitors, 373
5.5.1 Probucol, 367 6.3.4 New Miscellaneous Treatments, 374
5.5.2 Plant Sterols, 367 7 Retrieval of Related Information, 374

1 INTRODUCTION nary heart disease and other direct manifesta-


tions of atherosclerosis.
Atherosclerosis and the resulting complica- There are a number of nonmodifiable risk
tions including coronary artery disease, pe- factors for CV disease such as age, sex, and
ripheral vascular disease, and cerebrovascular family history of disease, but also a number of
disease are responsible for 41% of all deaths in modifiable risk factors including dyslipidemia,
the United States (la) and remain the leading hypertension, obesity, diabetes mellitus, and
cause of mortality and morbidity in industri- tobacco smoking. It is estimated, by the use of
alized nations. Despite the identification of the current National Cholesterol Education
many of the risk factors for the development of Program (NCEP) guidelines (lb), that more
atherosclerosis and initiation of behavioral than 250 million people worldwide can be clas-
and pharmaceutical intervention to reduce sified as having some form of dyslipidemia.
risk, cardiovascular (CV) disease remains a Dyslipidemia is described in terms of eleva-
significant health-care burden, estimated by tion of lipid (cholesterol and triglyceride) in i
the American Heart Association in 2000 to atherogenic lipoprotein particles LDL (low i1
1
cost some $326 million (la). There are over density lipoprotein), IDL (intermediate den-
650,000 new myocardial infarctions and sity lipoprotein), and VLDL (very low density
450,000 recurrent myocardial infarctions each lipoprotein), or reduction in cholesterol car-
year and about one third of all the people af- ried in protective HDL (high density lipopro-
fected die (la). Although the age-adjusted tein).
death rate from CV disease is falling, the over- Lipids are carried in the blood in lipopro-
all death rate is little changed because of aging tein particles with triglyceride (TG)and cho-
of the population. Of the deaths from CV dis- lesteryl ester carried in the interior and phos-
ease, a large proportion is attributed to coro- pholipid, free cholesterol, and apolipoproteins
1 Introduction

at the surface. These particles can be classified tients with both severe and borderline dyslip-
according to size (density): idemia (3-18).The strongest link with athero-
sclerosis exists for elevated concentrations of
.I. Chylomicrons are postprandial particles LDL-cholesterol (LDLc) and clinical trial data
containing mainly TG with apolipoprotein support the benefits of aggressive LDLc low-
B48, have a density (dl < 0.94 glmL, and ering in appropriate populations (19, 20).
are poorly atherogenic. However, recent epidemiological data have re-
2. VLDL particles are of hepatic origin, carry affirmed that elevated plasma TG and low
mainly TG with some cholesteryl ester, HDL-cholesterol levels are also important risk
have a density range d = 0.94-1.006 glmL, factors for atherosclerotic vascular disease. A
and are weakly atherogenic. major function of HDLc is the transport of
3. IDL particles are formed from the catabo- cholesterol from peripheral tissues to the
I
lism of VLDL, are enriched in cholesteryl liver, a process known as reverse cholesterol
esters, with a density range d = 1.006- transport. Low HDLc concentrations may lead
1.019 g/mL, and are atherogenic. to a failure to export lipid from the vessel wall,
4. LDL particles are rich in cholesteryl ester, leading to atherosclerotic plaque development
-poor in TG, with a density d = 1.019-1.063 and hence increased CV risk.
g/mL, and are highly atherogenic, espe- Several assessments of the importance of
cially small dense LDL (sdLDL). VLDL, elevated TG as an independent risk factor for
IDL, and LDL carry apolipoprotein B100. CHD have also been completed. Most impor-
6. HDL particles are formed from hepatic or tant, multivariate analyses have demon-
intestinal apolipoprotein A1 through a pro- strated that elevated TG concentrations: (1)
cess of lipid transfer from other lipoprotein increase CHD risk independent of HDLc (21,
particles and peripheral tissues; they are 22); (2) increase significantly the number of
very rich in cholesterol, with a density d > cardiovascular events when combined with an
0.063 g1mL. There is a strong, independent, elevated LDLc/HDLc ratio (23, 24); (3)are an
and inverse association between low con- independent risk factor in women aged 50-69
centrations of HDL-cholesterol (HDLc) years (19); and (4) are an independent risk
and coronary heart disease (CHD) risk (2). factor in type 2 diabetes mellitus (DM) pa-
I tients (25).
Although there is debate about the strength Drug therapy can be considered for pa-
i tients who, in spite of adequate dietary ther-
..of association between some forms of dyslipi-
idemia and the risk of developing cardiovascu- apy, regular physical activity, and weight loss,
:tar disease, the evidence exists that the reduc- need further treatment for elevated blood cho-
--
ition in cholesterol and triglyceride in low lesterol levels. Current NCEP guidelines (lb)
;densitylipoprotein classes leads to a reduction are shown in Table 7.1.
kin CHD mortality and morbidity in primary There is also increasing awareness that
,and secondary prevention studies and in pa- current lipid-lowering therapies lack suffi-

iTable 7.1 Current NCEP Guidelines for Treatment


%

r
LDLc (mg/dL)
F
[patient Start Goal
!with CHD or CHD risk equivalentsa 2130 400
l~ithoutCHD and 2 2 risk factorsb 2130 (10-year risk: 10-20%) <I30
.I
I 2160 (10-year risk: <lo%)

hctor for CHD > 20%.


'Risk factors are: (1)male 2 45 yearslfemale 2 55 years; (2)smoker; (3)hypertension; (4)family history of early CAD; (5)
[~IDLC< 40 mgldL.
Antihyperlipidemic Agents

cient efficacy or safety in the treatment of the rupture through a combination of inflamma-
full spectrum of lipid abnormalities. tory cell destruction of the fibrous cap and
physical disruption attributed to blood flow.
2 CLINICAL APPLICATIONS Rupture is followed by formation of a throm-
bus, which obstructs the artery and precipi-
tates acute coronary syndromes.
2.1 The Role of Lipids in the Atherogenic
The reasons for the "instability" of these
Process
plaques are not clear, but they are known to
Specific sites in the vasculature associated have an increased lipid content and decreased
with decreased shear stress or increased tur- fibrous cover (31).The most common plaque
bulence, such as bifurcations and branches, fissures are the junctions of the plaque and the
are favored sites for the development of arte- normal intima (32,33), a site often containing
rial lesions (26). The triggers for the develop- lipid-laden macrophages, which are thought to
ment of atherosclerosis are not known, but a play a role in weakening the plaque (34). Re-
key initial phase in the development of athero- cent work suggests that progressive accumu-
sclerosis is the retention of cholesterol-rich li- lation of lipids may destabilize plaques, thus
poproteins and remnants in the subendothe- leading to thinning, weakening, and ulti-
l i d space (27, 28). mately destruction of the fibrous cap, which is
Lipid deposition is followed by an increase followed by subsequent rupture of the plaque.
in the number of inflammatory cells, including Cholesterol in plasma clearly plays a role
macrophages in the intimal space. These mac- both in the development of "stable" and "un-
rophages become engorged with lipid, forming stable" plaques. Lipid-lowering therapy in hu-
foam cells, visible as nonobstructive fatty mans is thought to play a role, not only in
streaks on the endothelial surface of the aorta preventing the formation and growth of
and coronary arteries. The streaks contain plaques but also in reducing the cholesterol
large accumulations of lipid-filled macro- content of plaques already formed.
phages and smooth muscle cells and fibrous
tissue. The main lipid component is intracel- 2.2 Current Drugs on the Market I

lular cholesteryl ester. The progression of i


these lesions from simple fatty streaks to ad- The four major pharmacological classes of
vanced lesions (29) is a process dependent on drugs routinely used in the treatment of
hemodynamic forces, plasma levels of athero- simple hypercholesterolemia (SH), mixed dys-
genic lipoproteins (30), and other risk factors. lipidemia (MD), and hypertriglyceridemia
Maturing plaques (Starey class IV/V) are (HTG)/lowHDL are: (1)HMG-CoA reductase
areas of intimal thickening, with a fibrous cap inhibitors; (2) fibric acid derivatives; (3) bile
covering a central core made up of intracellu- acid sequestrants/cholesterol absorption in-
lar and extracellular lipid and necrotic cell de- hibitors; and (4) nicotinic acid derivatives (see
bris. These plaques may be angiographically Table 7.2). The drug classes and summary ef-
silent, encased within a thickened arterial fects on lipoprotein classes are summarized in
wall or may project into the lumen and ob- Tables 7.3a and 7.3b.
struct the arterial flow, and are detectable by
angiography. 2.2.1 HMG-CoA Reductase Inhibitors (Sta-
Historically, it was believed that the tins). 3-Hydroxy-3-methylglutarylcoenzyme
plaques gradually encroached into the lumen A (HMG-CoA)reductase inhibitors were first
until flow was either severely restricted or approved by the U.S. Food and Drug Adrninis-
totally occluded. When this occurred in the tration (FDA) in 1987. They have since be-
coronary artery, the patient would present come the most widely prescribed drug class for
clinically with either angina or an acute myo- the treatment of hypercholesterolemia. Mev-
cardial infarction (MI). Recent research has astatin (I),the initial drug of this class, wes
shown, however, the importance of angio- discovered in extracts from Penicillium citri-
graphically silent, small plaques (30). These num by Sankyo (Japan) in 1976 (36). Meva-
smaller plaques are thought to perforate or statin (1)was subsequently shown to inhibit
2 Clinical Applications 343

Table 7.2 Marketed Antihyperlipidemic Agents


Generic Name (structure) Trade Name Originator Dose (mglday)"
.HMG-CoAreductase inhibitors (statins)
Mevacor Merck & Co. Inc 20-80
Lescol Novartis AG 20-80
Pravastatin (3) Pravachol Sankyo/Bristol-Myers 2040
Squibb Co.
Simvastatin (4) Zocor Merck & Co. Inc. 20-80
Atorvastatin (6) Lipitor Warner-Lambert (now 10-80
Pfizer Inc.)
- AG
Bayer 0.2-0.8
Pitavastatin (9) TBD~ Kowa/Nissan Chemical 1 4 (?)'
(previously itavastatin) Industries Ltd/Novartis/
sankyo
e 'ibric
Rosuvastatin (7)
acid derivatives (fibrates)
Crestor Shionogi/AstraZeneca 10-80

Clofibrate (10) Widely generic Widely generic 2 x 1000


Gemfibrozil(11) Lopid P h e r Inc. 2 X 600
Bezafibrate (13) Bezalip Roche 400-600
Micronized fenofibrate (14) TricorILipantil FournierIAbbott 200
Ciprofibrate (16) Ciprol Sterling Drug Ltd. (now 100
Sanofi-Synthelabo)
bile acid sequestrants (BAS)/cholesterol absorption
. inhibitors
Cholestyramine (17) Questran Bristol-Myers Squibb Co. 4000-16,000
Widely generic Widely generic 5000-20,000
Colesevelam (19) Welch01 GelTex 2600-3800
Ezetimibe (20) Zetia Schering Plough Corp. 1-40
Ezetimibe + simvastatin TBD~ Schering Plough Corp.1 10 mg each
Merck & Co. Inc.
licotinic acid derivatives
Niacin (21) Niaspan Kos Pharmaceuticals Inc. 1000-2000
Niacin + lovastatin Advicor Kos 500/10-2000/40
(niacintlovastatin)
kellaneous
Probucol(22) Sinlestal Hoechst Marion Roussel 2 X 500
(now Aventis Pharma)
Tamoxifen (23) Nolvadex Zeneca (now AstraZeneca) 20
Toremifene (24) Fareston Orion Corp. 60
Raloxifene (25) Evista Eli Lilly & Co. 60
"Administered orally unless otherwise noted.
bBayervoluntarily withdrew cerivastatin from all marketsI in August 2001 after increasing reports of adverse effects (35;

"Sanyo announced in November 2001 that its U.S.subsidiary would undertake a second Phase I1 trial of pitavastatin,
sine a lower dose. because of cases of muscle ~ a i associated
n with the elevation of CPK (creatine ~hos~hokinase).
a marker

WG-CoA reductase (37), the rate-limiting tions of lovastatin (2). Lovastatin (2) and sim-
nzyrne in cholesterol biosynthesis (38, 39), vastatin (4) are administered as the inactive
nd to lower serum cholesterol in dogs- .(40)
. lactones. which must be metabolized in vivo to
cynomolgus monkeys (41). A structurally their corresponding open hydroxy-acid forms,
whereas pravastatin (3)is taken as the active
open hydroxy-acid form.
~ealthyvolunteers (42). Both pravastatin (3) The first-generation synthetic statin was
nd simvastatin (4) are chemical modifica- fluvastatin (5),which is also administered as
344 Antihyperlipidemic Agents

Table 7.3a Current Pharmacological Effects of Therapies in the Treatment


of Patients with Mixed Dyslipidemia (characterizedby high LDLc and high TGs)
Effects on Lipoproteins
Drug Class Total Cholesterol LDLc HDLc TG
HMG-CoA reductase inhibitors & 15-45% i 20-60% 2-12% i 1031%
Fibric acid derivatives & 10-25% J, 15-30% 1040% i 20-50%
Bile acid sequestrants/cholesterol
absorption inhibitors 8-30% & 15-20% .T 0-2% 5-10%

(4)

Table 7.3b Current Pharmacological Effects of Therapies in the Treatment


2 Clinical Applications

currently approved FDA maximal doses, ator-


vastatin(6)80 mg is the most efficacious sta-
tin, with LDL reductions of 50 to 60% (441,
followed by simvastatin (4) 80 mg(47%) (441,
cerivastatin (8)0.8 mg (42%) (451,lovastatin
(2) 80 mg (40%) (44),pravastatin (3) 40 mg
(35%) (44),and fluvastatin (5) 40 mg (30%)
(44).Recently published Phase I11 data for ro-
suvastatin (7) suggest that it is better than
atorvastatin (6) at lowering LDL and in the
percentage of patients treated that reach the
NCEP goal of less than 100 mg/dL for LDLc
the active drug substance. The clinical success (46-48). Pitavastatin (9) does not appear to
of the statins has led to newer synthetic mol- offer any advantage of greater LDL lowering
ecules, which are more potent [atorvastatin over the existing statins on the basis of two
(6) and rosuvastatin (7)]or equipotent [ceriv- Phase I11 studies in Japanese patients (49,501,
astatin(8)and pitavastatin (9)]on a milligram so only randomized controlled clinical trials to
per milligram basis compared with fluvastatin assess the long-term effects on CAD would al-
(5). low a differentiation. In addition to statin
Statins are the drugs of choice for reducing monotherapy, there is increasing interest in
LDL cholesterol (LDLc)and the second-gener- combination therapy to obtain greater reduc-
ation statins are moderately effective in reduc- tions in LDLc as well as benefits on other risks
ing TGs at higher doses and when baseline TG factors. These combinations are discussed in
concentrations are >I50 mg/dL (43). At the later sections.
Antihyperlipidemic Agents

shown to moderately lower LDLc and increase


HDLc levels (55-57).
Clofibrate (10) is administered as its inac-
2.2.2 Fibric Acid Derivatives (Fibrates). Fi-
tive ester, which is hydrolyzed in vivo to the
brates have been shown to affect the expres- active drug, clofibric acid (12). Although clofi-
sion of genes implicated in the regulation of
HDL and TG-rich lipoproteins as well as fatty
acid metabolism through the activation of the
peroxisome proliferator-activated receptor a
(PPARa) (51-53). The current fibrates are all
weak PPARa agonists (i.e., require high mi-
cromolar concentrations for receptor activa-
tion), which may explain why high doses are
required for their clinical activity (54). They
were developed as hypolipidemic agents
through optimization of their lipid-lowering
activity in rodents before the discovery of the
PPARs. Clofibrate (10) and gemfibrozil (11)
are two of the older fibrates that have been

brate was shown to improve the lipoprotein


profile and cardiac events in several clinical
trials (58-60), there was a greater all-cause
mortality (59,60). Clofibrate (10)is no longer
recommended as a lipid-lowering agent be-
:
i cause of the increase in overall mortality and Ciprofibrate (16) was first launched in Eu-
. the adverse events that are associated with its rope in 1985 but does not show any advantage
use. over current fibrate treatment (63) at doses
Gemfibrozil ( l l ) , on the other hand, has
demonstrated CV benefit in three studies (57,
61, 62). In particular, the VA-HIT trial (57)
demonstrated a 22% benefit in CV morbidity
and mortality with a 6% increase in HDLc, a
31% decrease in TGs, and no effect on LDLc.
The results of this study stress the importance
of low HDLc as a major risk factor for CHD.
The newer fibrates, bezafibrate (13)and feno-
fibrate (14), also reduce TGs and increase

5100 mgtday. Because of reports that doses of


2200 mg/day have been linked to rhabdomy-
olysis (64) the phase 111 trials in the United
States were suspended (65).
All of the current fibrates require multiple
daily doses except a micronized formulation
of fenofibrate (14), which demonstrates in-
creased absorption and more predictable
plasma levels, allowing dose reductions and
once-daily administration (66). There are cur-
rently three ongoing clinical trials in diabetic
patients with fenofibrate (14), to assess the
benefit on clinical outcomes (67-691, and an
increasing interest in combination therapy
with statins (70-78). The results of these
studies will be used to forge the future market
of these drugs in mono- and combination ther-
apye

HDLc but to a greater extent than clofibrate 2.2.3 Bile Acid Sequestrants (BAS)/Choles-
(10) or gemfibrozil (11) because of increased terol Absorption Inhibitors. The bile acid se-
receptor affinity (53). This may also explain questrants (anion-exchange resins) are non-
the greater extent of LDLc lowering observed systemic drugs, which act by binding bile acids
with bezafibrate (13) and fenofibrate (14). within the intestinal lumen, thus interfering
Like clofibrate, fenofibrate is administered as with their reabsorption and enhancing their
the inactive ester, which is hydrolyzed in vivo fecal excretion (79, 80). This leads to the in-
to the active form, fenofibric acid (15). creased hepatic conversion of cholesterol to
Antihyperlipidemic Agents

bile acid through upregulation of cholesterol colestipol (18) are secondary and tertiary
7a-hydroxylase activity (81). The liver's in- amines and its functional anion exchange ca-
creased requirement for cholesterol is par- pacity varies according to the pH in the intes-
tially met through the hepatic removal of cir- tinal tract (89). Both cholestyramine (17) and
culating LDLc through upregulation of colestipol (18)are effective cholesterol-lower-
hepatic LDL receptors (79, 80). Bile acid se- ing drugs in monotherapy as well as combina-
questrants have a very slight effect on HDLc tion with statins (90-921, fibrates (93-97), ni-
and can lead to TG elevations (79-83). acin (92), or probucol (91, 98, 99); however,
Cholestyramine (17) has been in use for BAS use is limited because of the need of large
30+ years and has been tested extensively. In doses for efficacy as well as their side-effect
both the Lipid Research Clinics Primary Pre- profile and interactions with other drugs. Re-
vention Trial and in the National Heart Lung cently, colesevelam (191, a third-generation
(84,85) and Blood Institute Type I1 Coronary bile acid sequestrant with increased in vitro
Intervention Study (86, 87), cholestyramine- potency (100, 101), has shown similar LDL-
induced reductions of LDLc were associated lowering efficacy at much lower doses, without
with significant reductions in the incidence the side effects associated with the other bile
and progression of CHD, respectively. Cho- acid sequestrants (102,103). The combination
lestyramine (17) is a copolymer of 98% poly- of colesevelam (19) and an HMG-CoA reduc-
styrene and 2% divinylbenzene containing tase inhibitor has been shown to be more ef-
about 4 meq of fixed quaternary ammonium fective in further lowering serum total choles-
groups/gram dry resin. The resin is adminis- terol and LDLc levels beyond that achieved by
tered as the chloride salt but exchanges for either agent alone (104-106).
other anions of higher affinity in the intestinal Ezetimibe (20) is a cholesterol absorption
tract (88). inhibitor that has just completed phase I11tri-
Colestipol(18) is the hydrochloride salt of a als and is in preregistration. It prevents the
copolymer of diethylenetriamine and l-chloro- absorption of cholesterol by inhibiting the
2,3-epoxypropane. The functional groups on transfer of dietary and biliary cholesterol
92 Clinical Applications

A = Primary amines
B = Cross-linked amines
D = Quaternary ammonium alkylated amines
E = Decyalkylated amines
n = Fraction of protonated amines
G = Extended polymeric network

Merck & Co. to develop the fixed combination


HO.
tablet of ezetimibe (20) and simvastatin (4).
This combination has been shown to reduce
LDLc by 52%compared to 35%with simvasta-
tin (4) alone (110).Schering-Plough is also in-
vestigating combinations with the other
statins and the fibrates (111).

2.2.4 Nicotinic Acid Derivatives. The lipid-


lowering effects of nicotinic acid (niacin, 21)
have been known for some time (112). Al-
though the exact mechanism of action of nia-
t
b s s the intestinal wall. The molecular
pechanism by which ezetimibe (20) inhibits
polesterol absorption in the intestine re-
lfains to be elucidated (107-109). Clinical tri-
ke have demonstrated reductions of LDLc bvu
in monotherapy (110, 111). Schering-
ugh recently formed a partnership with
Antihyperlipidernic Agents

cin (21) is unknown, it has been shown to in- cacy and safety and has just received FDA ap-
hibit the mobilization of free fatty acids proval in the United States (142,143).
(FFAs) from adipose tissue, resulting in re-
duced plasma FFA levels and, thus, a de- 2.2.5 Miscellaneous. This section discusses
creased hepatic uptake (113-115). Conse- the drugs and classes of drugs used in the
quently, hepatic TG synthesis is decreased, treatment of dyslipidemia that fall outside the
leading to a reduction in VLDL secretion and four major pharmacological classes discussed
an increase in the intracellular degradation of earlier. Although these compounds exhibit
ApoB (113, 116). As a result of the decreased beneficial lipid-lowering effects, they are re-
VLDL production, the plasma LDL level is garded as second-line drugs for the treatment
also reduced because this is the major product of hypercholesterolemia and they are often
prescribed for other indications.
of VLDL catabolism. The increase in HDL lev-
2.2.5.1 Probucol. Probucol (22) was dis-
els is the result of a decrease in the fractional
covered as a lipid-lowering agent in 1964 from
catabolic rate of ApoAI, the major constituent a screening program of phenolic antioxidants.
of the HDL particle (117, 118).
Niacin (21) has been studied in six major
clinical trials with cardiovascular endpoints
(119). The CV endpoints are reduced in mono-
therapy (120) or in combination (121-126),
and over longer time periods all-cause mortal-
ity is also decreased (119). Niacin (21) is now
available in a slow-release formulation (Nias-
pan; 127-129), designed to decrease the side
effects seen on use of this agent that limit its
utility (130-133; see Section 2.3.4).
The use of niacin (21) and statins in com-
bination has often been avoided because of Its exact mode of action is unclear but it has
concerns of myopathy and liver toxicity on the been shown to reduce both LDLc (8-15%)and
basis of case reports recorded shortly after lo- HDLc (by as much as 40%) (144-146). Studies
vastatin (2) was introduced to clinical practice have shown an increased fecal loss of bile acids
(134,135). Since then, there have been a num- and increased catabolic rate of LDLc (147) as
ber of studies conducted to determine the well as a reduction in LDL synthesis (148).
safety and efficacy of niacin-statin combina- Because of the decrease in HDLc and other
tion therapy (136), including the Arterial Dis-
side effects (see Section 2.3.5. I),probucol(22)
ease Multiple Intervention Trial (ADMIT),
is rarely used in the treatment of hyperlipid-
which demonstrated that it is both feasible
emia and is not marketed in the United States.
and safe to modify multiple atherosclerotic
disease risk factors effectively with intensive Probucol's benefit in restenosis is believed to
combination therapy in patients with periph- be attributed to its strong antioxidant effects,
eral arterial disease (137). More recently, ex- which may prevent endothelial damage and
tended-release niacin (21) has also been eval- LDL oxidation secondary to angioplasty (149).
uated in combination with various statins However, the Probucol Quantitative Regres-
(136,1381, including the HDL-Atherosclerosis sion Swedish Trial (PQRST) failed to show a a
Treatment Study (HATS) evaluating lipid al- clinical benefit of probucol (22) in combina-
tering for patients with coronary disease tion with cholestyrarnine (17) compared to
and low HDLc (139). The findings showed cholestyramine (17) alone or placebo (150).
extended-release niacin (21) with simvastatin 2.2.5.2 Hormone Replacement Therapy
(4) could reduce cardiac events by 48% in dia- (HRT). The use of HRT, estrogen and proges-
betic patients and 65% in nondiabetics (140, terone, in postmenopausal women has in-
141). Nicostatin, a fixed combination of niacin creased dramatically over the last 10 years.
(21) and lovastatin (21, has demonstrated effi- Not only has it shown benefit for the peri-
2 Clinical Applications

menopausal symptoms but also in several


studies, a reduction in cardiovascular events
(151). HRT directly stimulates LDL receptor
activity, leading to reductions in total choles-
terol and LDLc levels, moderate increases in
HDLc levels, and a decrease in HDL and LDL
oxidation (152). HRT in combination with a
statin has also shown to be very effective in
lowering LDLc levels (153).The Heart and Es-
trogenProgestin Replacement Study (HERS),
investigating primary or secondary preven-
tion of HRT, concluded that estrogen plus me-
droxyprogesterone showed no significant ben-
efit in the prevention of coronary artery
disease (CAD) (154). Another recent study
from Duke University found similar results to
those of the HERS trial (155). There are cur-
rently contradictory results from several stud-
ies, which is why the FDA has not approved
HRT treatment for the indications of: ( I ) reg-
ulation of lipids or (2) reduction of CHD. Re-
cently, the American Heart Association issued
a caution on the use of HRT for cardiovascular
disease (156).
2.2.5.3 Estrogen Modulators. Several mech-
anisms are responsible for the cardioprotec-
tive effects of estrogen, including beneficial ef-
fects on the lipoprotein profile and direct
effects on the vascular wall (160). As men-
tioned in the preceding section, the lipid ef-
fects include moderate decreases in LDLc, in-
creases in HDLc, and a decrease in LDL and
HDL oxidation. The effects are modulated
through the binding of estrogen to its nuclear
estrogen receptor (ER) and the regulation of
target gene transcription through the ligand-
receptor complex (161). The discovery of a sec-
ond estrogen receptor subtype (ERP), which
may mediate some of estrogen's cardiovascu-
lar benefits (161-166), has led to increased ER
research to identify selective agonists.
Other estrogen modulators that are used in
postmenopausal women are tamoxifen (23)
and toremifene (24) in the treatment of breast 13% (168). Raloxifene (25) has been shown to
cancer and raloxifene (25) in the prevention of lower fibrinogen, in addition to increasing
osteoporosis. All the drugs have been demon- HDL levels, without raising TGs (169).
strated to reduce total cholesterol and LDLc. 2.2.5.4 Plant Sterols. Plant sterols and
Furthermore, tamoxifen (23) has been as- stanols inhibit the intestinal absorption of
sociated with lower rates of MI, although cholesterol and as a result lower plasma LDLc
higher rates of thromboembolic diseases have concentrations. They occur, in varying de-
been reported (167). Toremifene (24) has been grees, naturally in almost all vegetables. The
associated with increases of ApoAI levels by most abundant of the phytosterols is p-sitos-
Antihyperlipidemic Agents

terol(26) and the fully saturated derivative of stanols have also been shown to reduce serum
p-sitosterol is sitostanol(27). Plant sterols are cholesterol levels in patients on statin therapy
absorbed to a small extent, whereas plant (172). Sterol and stanol esters can be used as
stanols are virtually nonabsorbable. Thus, in- food additives, to allow adequate amounts to
be consumed without affecting food quality or
dietary habits. Low fat stanol or sterol ester-
containing margarines in combination with a
low fat diet have been shown to reduce LDLc
levels in hypercholesterolemic subjects (173,
174).

2.3 Side Effects, Adverse Effects, Drug


Interactions, and Contraindications

2.3.1 HMC-CoA Reductase Inhibitors (Stat-


ins). As a class, HMG-CoA reductase inhibi-
tors have been shown to have a low risk of
severe side effects after chronic exposure in
humans. Most of the known adverse effects
are directly related to their biochemical mech-
anism of action and are the result of potent
and reversible inhibition of an enzyme in-
volved in cellular homeostasis (175). The inci-
dence of adverse effects increases with in-
creasing plasma levels of the active drug;
however, it should be noted that the increase
in risk is not associated with a proportional
increase in cholesterol-loweringefficacy (176-
180). Toxicology studies have shown that the
liver, kidney, muscle, the nonglandular stom-
ach, and lymphatic tissue are potential target
testinal levels of stanols will be prolonged organs and tissues (see Refs. 181-188 and Ta-
compared to that of sterols, which may explain ble 7.4). In rodents acanthosis and hyperkera-
why plant stanols appear to be more effective tosis of the nonglandular stomach was ob-
in decreasing cholesterol absorption (170) and served. These changes were observed only in
reducing serum LDL levels (171). Plant rodents and appear to be linked to the bio-

Table 7.4 Target Organs in Multidose Toxicology Studies with HMG-CoA


Reductase Inhibitors
-- -

Target Organ Lovastatin Cerivastatin Fluvastatin Atorvastatin


Liver J J J J
Gall bladder J - J J
GI tract J J J J
Kidney J J -
Muscle J J J J
Nonglandular stomach J J J J
Lymphatic system J J J - 2

CNS J J - J i
Eyes J J J -
Testes J J J -
Thyroid - - J -
2 Clinical Applications 353

chemical mechanism of this class. The acan- nases have also been reported with most lipid-
thosis and hyperkeratosis side effects require lowering drugs and may be a response to
the direct contact of the fore-stomach squa- changes in lipid metabolism rather than a di-
mous epithelium, with high concentrations of rect effect of lipid-lowering drugs on the liver
inhibitor for long periods. It should be noted (190). Hepatitis is a rare complication of statin
that dogs appear more sensitive than other therapy (0.01-0.02%)and seems to be an idio-
species to statin toxicity probably because of syncratic or cytochrome (CYP) P450-depen-
the fact that the degree of metabolism is less, dent effect (191, 192).
resulting in higher systemic exposure to the 2.3.1.2 Myopathy. Myalgia in patients on
statin therapy occurs with an incidence of
2-5% and is not usually associated with an
ductase prevents the formation of ubiquinone increase in creatine kinase (CK) levels. The
(28) and dolichol (29), which are involved in symptoms disappear upon discontinuing statin
electron transport and glycoprotein synthesis. treatment, or some patients may benefit from
a clinical supplementation with coenzyme Q,,
[ubiquinone (28)1, a mitochondrial electron-
F shuttle transporter whose levels are depleted
1 by statin therapy (193). Myopathy and myosi-
3 tis are rarer (0.5-1%) and characterized by
- muscle pain, weakness, or cramps and CK val-
t ues of at least 10-fold the upper limit of normal
- (176, 177, 179, 181). There is little correlation
- between the degree of CK elevation and the
L- severity of the symptoms (194), although this
r-
Y7
side effect is dose dependent and disappears
e upon discontinuing treatment. The incidence
ll of myopathy is exacerbated by concomitant
- therapy with cyclosporin, gemfibrozil, cho-
~e lestyramine, fenofibrate, niacin, itraconazole,
I- and erythromycin or in the presence of renal
(29) n = 15-20 insufficiency (176, 177, 181, 194-198). These
zt
a- agents interfere with drug metabolism
Although it is to find that a through the cytochrome P450 system, leading
a-
b- to increased plasma concentrations of un-
in S, it changed drug over longer periods of time.
o- Rhabdomyolysis with renal dysfunction is a
very rare complication of statin treatment and
th- is usually idiosyncratic and dose independent
awal of cerivastatin (8) from the market be- (181).It is characterized by the actual break-
- use of muscle damage linked to 31 U.S. down of the muscle membrane, leading to
tin eaths and at least nine more fatalities leakage of the muscle protein myoglobin into
- road, primarily in combination with gemfi- the bloodstream (myoglobinemia). The myo-
ozil (11),has focused attention on the safety globin travels to the kidneys (myoglobinuria),
where it causes the kidney tubules to stop
se- working, thus leading to kidney failure. In ad-
a- dition, the increased potassium levels, re-
s leased from muscle cell breakdown, can lead to
cardiac arrhythmias and death. Recently,
ment should be discontinued (178, 189, Sankyo announced that the launch of pitavas-
. These changes are dose dependent, often tatin (9) would be postponed because of the
sient, and return to normal after the drug necessity to undertake a second phase I1 trial,
discontinued. Small increases in transami- by use of a lower dose. Cases of muscle pain
Antihyperlipidemic Agents

associated with the elevation of CPK (creatine 2.3.2 Fibric Acid Derivatives (Fibrates).
phosphokinase), a marker of myopathy, at the The side-effect profile is similar for all of the
higher doses had been observed (199). Astra- fibrates. The most common side effects are
Zeneca also reported that there were inci- nausea, diarrhea, and indigestion. Other side
dences of rhabdomyolysis in patients treated effects, such as headache, loss of libido, skin
with rosuvastatin (71, but only at the high rash, and drowsiness, occur less frequently.
dose. Toxicological studies have shown that the
The exact mechanism through which the liver, muscle, and kidney are potential target
statins induce skeletal muscle abnormalities organs and tissues (211-213). In general, the
is currently unknown (179, 195, 196). It has fibrates potentiate the effects of oral antico-
been suggested that statins cause intracellu- agulants by displacing these drugs from their
binding sites on plasma proteins, necessitat-
lar ubiquinone deficiency, as mentioned ear-
ing a reduction of the dosage of anticoagulant
lier, which interferes with normal cellular res-
(211,213-215). The fibrates are also contrain-
piration in muscle and results in electron dicated in pregnant or lactating women, or pa-
leakage into the tissue, thus causing oxidative tients with severe liver or renal impairment or
stress and ultimate tissue destruction. These existing gallbladder disease.
changes are reversed by concurrent adminis- 2.3.2.1 Hepatotoxicity. As with the statins,
tration of mevalonic acid in the animal mod- elevations in serum transaminases occur in
els. However, animal studies have failed to 2-13% of the patients (189,190,212,228)tak-
show a relation between tissue ubiquinone ing fibrates, and a level three times the upper
(28)levels and the degree of muscle destruction. reference limit is the point at which treatment
Although the incidence of myopathy and should be discontinued. These changes .+ are of-
rhabdomyolysis has raised concerns over the ten transient and return to normal after the
statins as a class, the benefits of using statins drug is discontinued. In rodents, the activa-
to manage patients' cholesterol far outweigh tion of PPARa by fibrates leads to the induc-
the risks of serious side effects from their use. tion of hepatic peroxisome proliferation,
2.3.1.3 Other Effects. There were concerns which is characterized by an increase in per-
over statin-induced cataracts on the basis of oxisome number andlor peroxisome volume,
toxicology studies in animals but these have and hepatomegaly (51). A greater than three-
now largely disappeared, given that data from fold increase in peroxisome proliferation (PP)
clinical studies involving statins have not re- is associated with the development of hepa-
ported lenticular opacities in patients receiv- tocellar carcinomas in rodents (216-218),
whereas the risk of inducing a hepatocarcino-
ing long-term treatment (177, 178, 200, 201).
genicity associated with a weak PP response
The most common adverse effects are gastro-
(i.e., two- to threefold increase) is unknown.
intestinal, with the occurrence of nausea,
Although all of the fibrates induce this phe-
bloating, diarrhea, or constipation (189, 194, nomenon in rodents, it was believed to be spe-
202). These are usually transient and resolve cies specific (i.e., rodent), given that gemfi-
spontaneously after 2 3 weeks. brozil (11) (219-221) and fenofibrate (14)
Although the statins as a class have been (222-225) have been shown not to induce per-
shown to exhibit favorable hematorheological oxisome proliferative or carcinogenic effects in
effects, atorvastatin (6)has been shown to in- primate and human livers. On the other hand,
crease fibrinogen, a known CV risk factor, in clofibrate (10) (226) has been shown to induce
some patient populations (203-206). Further- PP in human livers, whereas ciprofibrate (16)
more, the higher doses of atorvastatin (6) have (227) has been shown to induce PP in ~ r i -
also demonstrated a lower HDL-raising effect mates. One potential explanation for this dif-
and even HDLc reductions compared to those ference is the very high exposures of both clo-
of the other statins (207-210). The underlying fibrate (10) and ciprofibrate (16)compared to
mechanisms and eventual clinical relevance the other fibrates.
and consequences of these effects still need to 2.3.2.2 Myopathy. The risk of myopathy
be elucidated. with fibrate treatment is increased in patients
2 Clinical Applications

with renal impairment (228-231) because of death in dogs (240), whereas it increased the
the extended systemic exposure of the drug. QT interval in monkeys. It was withdrawn
This side effect is dose dependent and disap- from the US. market because of its potential
pears upon discontinuing treatment. to induce serious ventricular arrhythmias.
2.3.5.2 Hormone Replacement Therapy (HRT).
HRT is associated with an increased relative
2.3.3 Bile Acid Sequestrants (BAS)/Choles- risk of breast and endometrial cancer with
terol Absorption Inhibitors. The use of the bile each year of treatment, as well as a risk of
acid sequestrants is limited by their unpalat- venous and pulmonary thromboembolism
ability, attributed essentially to the large (241-244). Although there is not a strong as-
doses needed for efficacy (3-30 glday). Gastro- sociation of HRT to ovarian cancer, there is a
intestinal side effects are also associated with debatable positive correlation (241). Most of
the low compliance in a large number of pa- the adverse effects of HRT are restricted to
tients. The newer formulations such as tab- current or recent use, and long-term HRT use
lets, caplets, and flavored granules and the de- should be carefully considered on an individ-
velopment of more potent sequestrants have ual basis, taking into account the patient's ex-
been associated with fewer gastrointestinal istent risk factors for breast and endometrial
adverse effects. cancer and for venous/pulmonary thrombo-
The cholesterol absorption inhibitor eze- embolism vs. the potential benefits of treat-
timibe (20) appears to have a very good ad- ment on CV disease and osteoporosis.
verse effect profile, with only limited gastroin- 2.3.5.3 Estrogen Modulators. There is an
testinal side effects. Further large-scale trials increased incidence of hot flashes and leg
will be needed to better define the adverse ef- cramps with all the estrogen modulators, al-
fect profile. though this side effect does not affect drug
compliance. Unlike HRT, tamoxifen (231,
2.3.4 Nicotinic Acid Derivatives. Nicotinic toremifene (24), and raloxifene (25) do not in-
acid (21) is not very well tolerated (132, 133). crease the risk of breast and endometrial can-
Nearly all patients suffer from itching, flush- cer and, in fact, several studies have demon-
ing, and gastrointestinal intolerance, which strated that tamoxifen (23) and raloxifene
usually diminishes with prolonged use. Aspi- (25) are useful in the prevention of breast can-
rin will prevent the flushing, indicating that cer. Currently, a Study of Tamoxifen Against
this side effect is prostaglandin mediated Raloxifene (STAR) is ongoing to compare the
(232). Current guidelines do not recommend two drugs in the prevention of breast cancer
the use of niacin in patients with diabetes be- (245,246).
cause it can exacerbate gout and worsen gly- 2.3.5.4 Plant Sterols. Whereas plant ste-
eemic control (233-236). Recently, results rols are absorbed to a small extent, plant
from the Arterial Disease Multiple Interven- stanols are virtually nonabsorbable. Unless
tion Trial (ADMIT) suggest that lipid-modify- consumed at extraordinarily high levels, prac-
ing doses of niacin can be safely used in pa- tically no side effects have been observed.
tients with diabetes (237).
2.4 Absorption, Distribution, Metabolism,
2.3.5 Miscellaneous and Elimination
2.3.5.1 Probucol. Probucol (22) is gener-
ally well tolerated, with only about 3% of the 2.4.1 HMG-CoA Reductase Inhibitors (Stat-
patients discontinuing treatment because of ins). Two thirds of the total cholesterol found
side effects. The most frequently reported side in the body is of endogenous origin, with the
effect is diarrhea, which may occur in up to major site of cholesterol biosynthesis being
one-third of the treated patients (238). Other the liver. Therefore, to minimize the risk of
less frequently reported gastrointestinal side adverse effects associated with high systemic
effects include flatulence, abdominal pain, exposures, the statins need to show tissue
nausea, and vomiting (239). Probucol(22) in- (liver)-selectiveinhibition of HMG-CoA reduc-
duced ventricular fibrillation and sudden b e , essential for achieving LDLc lowering.
356 Antihyperlipidernic Agents

Table 7.5 Pharmacokinetic Properties of the HMG-CoA Reductase Inhibitors


Plasma Protein Elimination
Statin Half-Life Binding Metabolism Active Metabolites Pathway
Lovastatin (2) 1-2 h >95% Hydrolysis of 83% biliary
inactive 10% renal
lactone and
CYP3A4
Fluvastatin (5) - 2h -99% Hydroxylation No active metabolites 93% biliary 6%
renal
Pravastatin (3) 2-3 h 50% Hydroxylation 75% as parent 70% biliary
compound 20% renal
Simvastatin (4) - 2h >95% Hydrolysis of p-Hydroxy-acid 60% biliary
inactive metabolites
lactone and
CYP3A4
Atorvastatin (6) 14h >98% CYP3A4 ortho- and para- Predominantly
hydroxylated biliary
metabolites
Cerivastatin (8) - 3h >99% CYP3A4 and Demethylation of the 70% biliary
CYP2C8 ether moiety and 30% urinary
hydroxylation of
the isopropyl group
Pitavastatin (9) llh 96% CYP2C9 No active metabolites >98% biliary
(major) >2% urinary
CYP2C8
(minor)
Rosuvastatin (7) 20 h Not CYP2C9 No active metabolites Not reported,
reported (minor) reported most likely
biliarv

Orally administered drugs, once absorbed, All of the statins except pravastatin (3)are
are filtered through the liver through the por- highly protein bound (see Table 7.5), which
tal vein. The drugs are extracted from the por- may limit their use with oral anticoagulant
tal venous blood and concentrated in the he- therapy. Interestingly, the more potent statins
patocyte to an extent that is related to their [i.e., atorvastatin (6)and rosuvastatin (7)]are ?

lipophilicity. associated with long plasma half-lives,indiwt-


The more lipophilic statins [lovastatin (2) ing the necessity of sustained HMG-CoA re
> cerivastatin (8)= simvastatin (4) > fluva- ductase inhibition to obtain the desired lipid-
statin (5) > atorvastatin (6)] exhibit facili- lowering efficacy.
tated passive diffusion through hepatocyte Because cytochrome (CYP) P450 metab
cell membranes, leading to selective accumu- lism is not important in the elimination of flu-
lation in the liver (247,248). Interestingly, the vastatin (51, pravastatin (31,and rosuvastatin
lactones [lovastatin (2) and simvastatin (4)] (7), there is a smaller risk of adverse effeds
selectively accumulate in the liver in anumber resulting from drug interactions. Althou*
of various species studied compared to their fluvastatin (5) is eliminated as hydroxylated
respective acid forms (249-251).The lactones inactive metabolites, pravastatin (3)and rosu-
are then efficiently metabolized into the active vastatin (7) are eliminated as unchanged
open hydroxy-acid form in the liver. On the drug. On the other hand, atorvastatin (6)and
other hand, the hepatoselectivity of the hydro- cerivastatin (8) are extensively metabolized
philic statins [pravastatin (3) and rosuvasta- by CYP3A4 to active metabolites, which ac-
tin (7)l can be attributed to their high affinity count for 70% and 25%, respectively, of the
for a liver-specific transport protein (248,252, total HMG-CoA reductase inhibitory activity
253). observed (254, 255).
Clinical Applications 357

Fibrates

Half-Life Binding Metabolism


Hydrolysis of the ester and minor metabolite
formation conjugated with glucuronide
40% unchanged and 60% numerous metabolites
(conjugated/benzoic acid/phenol/etc.)
50% unchanged, 22% glucuronide conjugates and
22% other polar metabolites (hydroxylothers)
20 h (acid) >95% Hydrolysis of the ester, 50% conjugated and 50%
polar metabolites (phenollbenzhydrol)
70% glucuronide conjugates

2.4.2 Fibric Acid Derivatives (Fibrates). CYP450-mediated metabolism (258). Interest-


ingly, glucuronidation is generally considered
one of the major detoxification processes in
a high degree of protein binding (see xenobiotic metabolism, facilitating biliary
e 7.6). However, the pharmacological re- andlor urinary elimination. It is also regarded
nse (lipid-lowering action) is more accu- as a process that inactivates the drug. In the
case of ezetimibe (20), neither of these gener-
alizations holds true. The glucuronidation of
ezetimibe (30) appears to improve its activity
ly excreted through the kidneys (256).
The shorter plasma half-life associated

es. Dose adjustment is necessary in patients

ereas treatment in patients with severe re-


impairment is precluded.

2.4.3 Bile Acid Sequestrants (BAS)/Choles-

estrants cholestyramine (17), colestipol


in at least two ways: (1)the drug is repeatedly
the intestinal tract, with the extent of delivered back to the site of action (the intes-
tine) through enterohepatic circulation and
to bind bile acids (see Section 2.2.3). The re- (2) glucuronidation appears to increase the
ing solid complex is 100% excreted in feces residence time in the gut (107). In addition,
7) and, because the bile acid sequestrants once ezetimibe is glucuronidated, >95% is ei-
not absorbed, there are neither measur- ther in the intestinal lumen or wall, indicating
plasma levels nor metabolism. that systemic exposure of the glucuronide (30)
he cholesterol absorption inhibitor ezeti- will be very low.

2.4.4 Nicotinic Acid Derivatives. Niacin


marily metabolized through glucuronidation (21) is well absorbed, with >88% of the dose
the intestine and eliminated through biliary recovered in the urine. At the doses used for
retion, with no evidence of significant the treatment of dyslipidemia, niacin (21) is
Antihyperlipidemic Agents

not metabolized to any great extent and the Cholesterol is essential in the synthesis of
elimination is exclusively renal, largely as un- cell membranes, bile acids, and hormones,
changed drug. Niacin (21) is only slightly pro- whereas triglycerides are important to periph-
tein bound (<20%) with a short half-life of eral tissue as a source of energy production.
0.75-1 h. The extended-release formulation, Although the liver is the major site of choles-
Niaspan, allows for sustained blood levels of terol biosynthesis, cholesterol and TG from
nicotinic acid, thus permitting a reduction in the diet can also be absorbed from the intes-
the active dose. tine and transported in the form of chylorni-
crons (Fig. 7.1). The chylomicrons transport
2.4.5 Miscellaneous. Although HRT and cholesterol and TG from the intestine to the
estrogen modulators exhibit beneficial lipid- adipose tissue for storage and the liver for
lowering effects, these drugs are not pre- packaging and resecretion as VLDL or LDL
scribed for the treatment of hypercholesterol- particles. After extensive hydrolysis of TGs,
emia; thus they are not be treated in this the remaining particles, chylomicron rem-
section. nants, are taken up by the liver (260). Pro-
2.4.5.1 Probucol. Probucol (22) is only longed uptake of these TG particles (VLDL or
about 2-8% bioavailable, with a highly vari- chylomicron remnants) by the liver can lead to
able plasma half-life of 12-500 h. Once ab- reduced hepatic production of LDL receptors
sorbed, probucol(22) is 95% incorporated into (LDLr) and to increases in plasma cholesterol
lipoproteins in the blood and can accumulate levels.
in adipose tissue. After stopping treatment, it Hydrolysis of TG-rich particles in the liver
can take up to 6 months to be removed from all leads to release of fatty acids. Fatty acids not
the tissue because of its lipophilicity. Little is used for energy generation by the liver are
known about its metabolism, with only 2% be- converted to TGs for hepatic storage or pack-
ing eliminated in the urine and the remainder aged into VLDL particles along with cho-
in the feces through the bile. lesteryl esters (CE) to be transported to the
2.4.5.2 Plant Sterols. As mentioned earlier peripheral tissue (Fig. 7.2). The VLDL parti-
(Section 2.3.5.41, plant sterols and stanols are cles are hydrolyzed through the lipoprotein
not absorbed to any great extent from the in- lipase (LPL) to form IDL particles. The liver
testinal tract, with their effects being medi- takes up about 60% of the IDL through the
ated by their ability to inhibit the intestinal LDLr and the remainder is hydrolyzed by the
absorption of cholesterol (259). Because the hepatic lipase (HL) to produce LDL particles.
plant sterols and stanols are not absorbed, The major role of LDL is to transport cho-
there are neither measurable plasma levels lesterol to the tissues. When intracellular
nor metabolism. cholesterol is required, cells may synthesize
cholesterol, or acquire exogenous cholesterol
3 PHYSIOLOGY AND PHARMACOLOGY through upregulation of LDLr, resulting in
the increased uptake of LDL. The LDLr is re-
3.1 Lipid Transport and Lipoprotein
sponsible for removing about 60-80% of the
Metabolism
LDL particles (261). Increased intracellular
To understand the development of the differ- cholesterol inhibits HMG-CoA reductase, the !
ent classes of lipid-lowering drugs discussed in rate-limiting enzyme in cholesterol biosynthe I
this review, it is necessary to put the different sis (Fig. 7.31, and decreases the synthesis of 1
therapeutic targets into their physiological LDLr to limit the further uptake of cholesterol
context. The goal of this section is to familar- into the cell (260). LDL can be modified by
ize the reader with the pathways of lipid trans- oxidation and glycation, which leads to de-
port, lipoprotein metabolism, and cholesterol creased recognition by the LDLr, increased
biosynthesis as well as the different tissues residence time in the plasma compartment,
involved. It is not meant to be a comprehen- and increased uptake of modified LDL by scav-
sive review of the area but only a simplified enger receptors on macrophages. This leads to
overview, to give the reader a brief survey of the accumulation of cholesterol, and lipid in
the different drug mechanisms of action. tissue macrophages, which in the arterial sys-
Bile acid sequestrants/cholesterol absorption inhibitors
-1 Plant sterol and stanols

Chylomicron
Intestine remnant

Fibrates-
- LDLr

-
Nicotinic acid
derivatives

rI HMG-CoA reductase
inhibitors II
I <4)V
- \
Fibrates
Nicotinic acid
1
U
Fibrates I derivatives
t Nicotinic acid
derivatives TG @-
LDL IDL
VLDL

Atherosclerotic
plaque

t
Probucol
Peripheral
tissue

Figure 7.1. Interrelationships of the plasma lipoproteins and the sites of action of the lipid-lowering
drugs. HMG-CoA reductase inhibitors upregulate hepatic LDL receptors (LDLr)through their inhi-
bition of cholesterol biosynthesis. Fibrates reduce the hepatic secretion of VLDL and increase li-
poprotein lipase (LPL) and hepatic lipase (HL) responsible for hydrolyzing TG-rich lipoproteins.
Fibrates also increase reverse cholesterol transport (RCT) by increasing circulating HDL levels
through reducing their catabolism by the transfer of cholesteryl esters (CE) from HDL to VLDL by
way of the cholesteryl ester transfer protein (CETP) as well as increasing the number of HDL
particles. The ATP-binding cassette transporter 1 (ABC1)mediates the efflux of free cholesterolfrom
cells to plasma, where it is incorporated into HDL. The hepatic uptake of cholesterol ester from HDL
is then mediated by the scavenger receptor class B Type 1 (SR-Bl).Bile acid sequestrants, cholesterol
absorption inhibitors, and plant sterols and stanols reduce the re-uptake of bile acids andlor choles-
terol through the intestine, decreasing the formation of chylomicrons and the transport of TGs and
CE to the liver. This leads to the upregulation of hepatic LDLr expression. Nicotinic acid derivatives
inhibit the hepatic production of VLDL and increase circulating HDL levels similar to fibrates,
although the mechanism is not fully understood. Probucol is an antioxidant in atheroscleroticlesions
and possibly in LDL particles.
Antihyperlipidemic Agents

Acetyl CoA

clwnr

/ / " Trinlvcerides
Cholesteryl
esters I

VLDL secretion

Figure 7.2. Lipid metabolism in the hepatocyte. Free fatty acids (FFAs)that enter the hepatocyte
are esterified and stored as triglycerides (TGs).The TGs are hydrolyzed as needed by the triglycerol
hydolase (TGH) to FFA and diacylglycerol. The diacylglycerol is further hydrolyzed to produce the
TGs that are used in lipid assembly. Acetyl CoA is used by the hepatocyte either for cholesterol
biosynthesis or TG synthesis and storage, depending on the hepatocytes' needs. Cholesterol is ester-
ified by the acyl-CoAcholesterol acyltransferase (ACAT)to produce cholesterol ester (CE)for use in
lipid assembly or for intracellular storage. The microsomal triglyceride transfer protein (MTP) is
responsible for the lipidation of the nacent apoBlOO with TG and CE to synthesize the VLDL particle,
which is then secreted. If the hepatocyte cholesterol is depleted, the sterol regulatory element-
binding protein (SREBP)/cleavage-activatingprotein (SCAP) is activated, sending a signal to the
nucleus to upregulate the LDL receptor (LDLr) along with other genes implicated in cholesterol
synthesis. Upregulation of the LDLr leads to increased cholesterol uptake from LDL and IDL parti-
cles in the plasma.

tem leads eventually to the establishment of into the lipid core of the HDL particle, thus
atherosclerotic plaques (262). allowing it to increase in size and mature into
HDL is regarded as essential for reverse HDL,. Further addition of CE results in the
cholesterol transport (RCT), the removal of maturation to HDL,. HDL, may: (1)deliver
cholesterol from peripheral tissue and its cholesterol to the liver through interac-
transport back to the liver. Nascent HDL (pre- tions with hepatic HDL receptors and may
p-HDL) is synthesized in the liver and small be converted back to HDL,; (2)exchange lipid
intestine and enters the plasma compartment. with other lipoprotein classes through cho-
When pre-p-HDL particles come in contact lesteryl ester transfer protein (CETP) medi-
with cells rich in cholesterol, there is a trans- ated transfer; or (3) be taken up whole by the
fer of cholesterol to the particle by cell surface liver (264).
proteins [such as the ATP-binding cassette The major class of lipid-lowering drugs
transporter 1 (ABC 111, which are responsible available are inhibitors of an enzyme (i.e.,
for the efflux of cholesterol from cells into the HMG-CoA reductase inhibitors) implicated in
plasma (263). Once transferred to HDL, the the synthesis of cholesterol or inhibitors of the
free cholesterol is esterified by the lecithin- process of cholesterol absorption (i.e., bile acid
cholesterol acyltransferase (LCAT)and the re- sequestrants, cholesterol absorption inhibi-
sulting cholesteryl esters are incorporated tors, plant sterols, and stanols), except for the
Physiology and Pharmacology

Acetoacetyl CoA + Acetyl CoA


4
HMG-CoA synthase
HMG-COA
4 HMG-CoA reductase
Mevalonate
{
Mevalonate kinase
Mevalonate pyrophosphate
4 lsopentyl pyrophosphate synthase
f lsopentyl pyrophosphate

-i
Dolichol { Gernyl pyrophosphate synthase
heme
farnesylation Geranyl pyrophosphate
ubiquinone 1
Farnesyl pyrophosphate synthase
Farnesyl pyrophosphate
{ Squalene synthase
Squalene
4 Squalene epoxidase
2,3-oxidosqualene
4 Oxidosqualene cyclase
Lanosterol

po]
{ Lanosterol demethylase
Desmosterol
1 Dehydroxycholesterol reductase
Cholesterol

o l o H -
HMG-COA -
reductase

S\
\COA CoA
(S)-3-hydroxy-3-methyl- (R)-mevalonic acid
glutaryl-CoA (HMG-CoA)
(31) (32) (33)

Figure 7.3. The cholesterol biosynthesispathway. HMG-CoA reductase is the rate-limitingenzyme


in the pathway and is involved in the transformal:ion of (S)-3-hydroxy-3-methylglutaryl-CoA
(31)
into (R)-mevalonate(33) (see boxed reaction).

rates. The fibrates bind i n t o the PPARa li- get genes (Fig. 7.4). Upregulation o f the apoAI
d-binding domain (LBD), inducing a con- gene leads t o increased apoAI-containing HDL
rmational change o f t h e protein. This en- particles, whereas the downregulation o f apo-
les the complex t o bind t o the DNA in t h e C I I I leads t o increased LPL activity, thus re-
nucleus through a PPAR response element ducing the plasma levels o f TG-rich lipopro-
(PPRE),thus permitting t h e regulation o f tar- teins (51-53).
Antihyperlipidemic Agents

TG-rich lipoproteins
(sdLDL) 4
Figure 7.4. The mechanism of action of the fibrates. In the hepatocyte, the drug binds to the
ligand-binding domain (LBD) of the PPARa receptor. Once bound, the complex binds to the DNA in
the nucleus and transcriptionally regulates target genes in a concerted fashion. ApoAI is upregulated,
leading to an increase in HDL, whereas apoCIII is downregulated. The downregulation of apoCIII,
the natural inhibitor of lipoprotein lipase (LPL),leads to an increased hydrolysis of TG-rich lipopro-
teins, including small dense LDL (sdLDL). Genes implicated in fatty acid poxidation are also up-
regulated.

4 HISTORY 4.2 Discovery of Atorvastatin (Lipitor)

4.1 Identification of the Statin Class of Lipid- An amusing and sensationalized account of
Lowering Drugs the development of atorvastatin appeared
January 24, 2000 in the Dow Jones Business
During the 1970s, epidemiological studies es- News, entitled "Birth of a Blockbuster" (265),
tablished a relation between elevated serum which is summarized below. Since Lipitor's
cholesterol and CHD. This led to increased re- launch in 1997, analysts have predicted that it
search in the field of cholesterol biosynthesis will become the biggest-selling prescription
inhibitors to identify new mechanisms useful
medicine in the world. The recent changes to
in the treatment of elevated serum cholesterol
the NCEP guidelines (discussed in Section 1)
levels. As already stated (Section 2.2.1),
have also doubled the number of Americans
screening of natural product extracts for cho-
lesterol biosynthesis inhibitors led to the iden- that can be considered for statin treatment,
tification of mevastatin (1)from the extracts which makes many analysts believe that Lipi-
of Penicillium citrinum by Sankyo (Japan) in tor may be the first drug to earn $10 billion
1976 (36). Mevastatin (1)was subsequently annually.
shown to inhibit HMG-CoA reductase (37), In the early 1980s, Parke-Davis scientists
the rate-limiting enzyme in cholesterol bio- were several months into the development of
synthesis (38, 39). This one discovery has led an HMGCoA reductase inhibitor, only to
to the development of a $30 billon lipid-lower- learn that Sandoz AG (the former Swiss
ing market, which is expected to continue to drug company that is now part of Novartis)
grow in the future. One molecule that helped had filed a patent that contained their lead
speed up this growth is atorvastatin (6). molecule. Work then switched to a backup
Structure-Activity Relationships

lecule, CI-981, which would later become creased risk of death from non-heart-related
own as atorvastatin (6). illnesses, many doctors in the early 1990s were
Although 8 years of research had already wary of aggressive treatment. The 80 mg
invested in developing the compound, strategy was risky too because, if it turned out
a1 studies had not demonstrated choles- to have unacceptable side effects, it could taint
-reducing superiority over the competi- the drug even at low doses and leave the com-
s. At the time, there was already one statin pany with only a 10 mg tablet to take to the
the market and three others in late-stage market.
an studies, so discussions focused on The LDLc-lowering efficacy demonstrated
by Lipitor in the clinical trials was enough for
hether atorvastatin should progress into hu-
the FDA to put it on priority review. The com-
man clinical trials. Discontinuing the develop- pany enrolled the last patient in its major trial
ment of atorvastatin would have wasted an in October 1995 and filed for approval in June
intense, 2-year effort to come up with a pro- 1996. Six months later, Lipitor was cleared to
cess to manufacture the drug in commercial begin sales.
quantities. The initial molecule was racemic, In the months leading up to approval, the
with only one diastereomer, resulting in the sales of Merck & Co.'s and Bristol-Myers
potent in vitro inhibition of HMG-CoA reduc- Squibb's statins were beginning to soar. Both
tase. This presented an issue for Parke-Davis had recently completed large-scale, long-term
Wientists: 50% of an inactive compound could studies showing use of their drugs reduced
cause unwanted toxicity andlor side effects deaths and heart attacks among high risk pa-
and potentially even jeopardize FDA approval. tients. Warner-Lambert had no data to sup-
The initial large-scale syntheses of the op- port such claims but, thanks to the outcomes
cally pure diastereomer proved difficult be- from the large statin studies, the market and
se of racemization. Scientists then ob- doctors began to react favorably to the statins
served that running the large-scale reactions as a class.
-78°C prevented racemization during the Lipitor, with its low-dose power and com-
action. The final manufacturing process petitive price, hit the market just as doctors
h k 3 weeks from raw materials to end prod- and NCEP guidelines began stressing that
uct and afforded a compound with 100% opti- lower cholesterol levels were healthier.
Merck & Co. and Bristol-Myers Squibb had
The first tests of the drug on 24 employee- spent 10 years educating doctors about statins
volunteers at 10 mg decreased LDLc by 38%. and Pfizer/Warner-Lambert was able to capi-
That was as good as or better than competing talize on Lipitor's superior efficacy. The scien-
mpounds at their recommended maximum tists who developed atorvastatin credit its in-
oses. At 80 mg, LDLc decreased by 58%, creased efficacy to the fact that the drug has a
which was more than any other statin at any much longer half-life than that of its competi-
dose. On the basis of those results, clinical tri- tors.
als were devised that would test 10 mg as a
ing dose and 80 mg for patients with es- 5 STRUCTURE-ACTIVITY RELATIONSHIPS
pecially high cholesterol. Other statins are 5.1 HMC-CoA Reductase Inhibitors (Statins)
prescribed in 20 and 40 mg tablets and Parke-
Davis's strategy would allow them to demon- All of the statins mimic the substrate HMG-
strate LDLc-lowering superiority at even the CoA (31)for the HMG-CoA reductase enzyme.
west dose of atorvastatin. The general structure of the inhibitors con-
Although Parke-Davis officials were confi- tains a central hydrophobic ring system, with
dent that atorvastatin would demonstrate su- a hydroxy-acid (34) or lactone (35; hydroxy-
riority, there was no assurance at the time acid prodrug, which is hydrolyzed to the active
t the market would want a potent low dose open acid form in vivo) side-chain necessary
. Because of several clinical studies in the for anchoring into the active site of the en-
80s linking low cholesterol with an in- zyme.
Antihyperlipidemic Agents

OR
s, CoA
'7Spacer

Hydrophobic
Spacer

Hydrophobic
ring system
ring system

The tight binding of the inhibitors (Table both the HMG-binding pocket and a part of
7.7) into the enzyme blocks access of the sub- the binding surface for the CoA.
strate, HMG-CoA (31),to the active site, al- Interestingly, all of the synthetic statins
though the exact binding mode was unknown contain a kfluorophenyl moiety that distin-
for some 25 years. Furthermore, the elucida- guishes them from the isolated natural prod-
tion of the structure of the catalytic portion of uct analogs of mevastatin (I),which contains
the human HMG-CoA reductase enzyme, co- the substituted decalin-ring system. There is a
crystallized either with HMG-CoA, with HMG stacking between the guanidinium group of
and CoA, or with HMG, CoA, and NADP+ ArggOand the 6fluorophenyl moiety and a
(nicotinamide adenine dinucleotide phos- polar interaction between the arginine m i -
phate), as well as with mevalonate-derived trogen atom and the fluorine atom (268).
products (266, 267), permitted little insight Only atorvastatin (6) and rosuvastatin (7)
into the binding mode of the inhibitors. Re- contain a polar side chain that makes a hydro-
cently, the cocrystallization of several statins gen bond to Ser565through either the carbonyl
bound in the catalytic portion of the human oxygen atom (atorvastatin) or the sulfon-
enzyme has permitted the elucidation of the amide oxygen atom (rosuvastatin). Further-
binding mode of the inhibitors (268). The more, only rosuvastatin (7) forms a polar in-
statins exploit the conformational flexibility of teraction with because of the presence
the enzyme, to create a hydrophobic binding of the sulfonamide moiety. Now that the bind-
pocket near the active site for the hydrophobic ing modes of the different statins can be visu-
ring system of the inhibitor, thus occupying alized in the active site, there is the potential
to use this information to design third-gener-
Table 7.7 Enzyme Activity of Selected ation HMG-CoA reductase inhibitors.
HMG-CoA Reductase Inhibitors I
5.2 Fibric Acid Derivatives (Fibrates)
Potency on Enzyme
Statin IcsO(nM)" The primary structural factor of the fibrates
Pravastatin (3) 44 necessary for receptor recognition is the "fi-
Simvastatin (4) 11 brate head group" (36), which is found in all of
Fluvastatin (5) 28 the synthetic ligands. As mentioned earlier,
Atorvastatin (6) 8 the fibrates were developed as hypolipidemic
Rosuvastatin (7) 5 agents through optimization of their lipid-low-
Cerivastatin (8) 10 ering activity in rodents before the discovery
Pitavastatin (9) 7 of the PPARs. Table 7.8 shows the potency of
"IC5, is the molar concentration that produces 50% of the fibrate drugs on the murine and human
its maximum possible inhibition (see Refs. 248,268,269). PPARa receptors. There is 85% identity at the
5 Structure-Activity Relationships

Table 7.8 Activity of PPARa Agonists in Cell-Based Transactivation Assays


Murine PPARa Receptor Human PPARa!Receptor
hirate EC50 (CJK)" EC50 (aa
Clofibrateb (10) 50 55
>loo >loo
90
18
Ciprofibrate (16) 15 9
"All the data were generated by use of the PPAR-GAIAtransactivation assay with an SPAP reporter, as described in Ref.
810.
bDataare for the active metabolite (i.e., acid).

(38) linoleic acid

to activate the receptor. This has prompted


several groups to search for high affinity nat-
ural ligands among the known eicosanoid me-
tabolites of polyunsaturated fatty acids. The
nucleotide level and 91%identity at the amino lipoxygenase metabolite 8(S)-HETE (39) was
acid level between the murine and human
PPARa ligand-binding domains (LBDs) (53).
These differences may reflect evolutionary ad-
@tationto different dietary ligands and ex-
@n the variations in potencies between the
gpecies. It should be noted that all of the fi-

erous pharmaceutical companies working


to identify more potent compounds with pi- identified as a higher affinity PPARa ligand
ntially greater lipid effects in humans. (272-275), although it is not found in suffi-
Fatty acids have been implicated as natural
ciently high concentrations in the appropriate
ligands-forPPARa by sever2 groups working tissues to be characterized as a natural ligand.
in the area, although all the fatty acids [i.e., Because no single high affinity natural ligand
palmitic acid (37) and linoleic acid (3811iden- has been identified, it has been proposed that
one physiological role of PPARa may be to
sense the total flux of fatty acids in metaboli-
cally active tissue (272-274,276,277).
\/\/\ 5.3 Bile Acid Sequestrants (BAS)/Cholesterol
(37) Palmitic acid Absorption Inhibitors
As mentioned earlier in Section 2.2.3, bile acid
tified to date bind to PPARa with affinities in sequestrants are cationic resins. The more ef-
fective the resin is at selectively binding bile
levels in serum reach these concentrations acids at low doses, the lower the chance of ob-
(271), it is not known whether the free concen- serving the gastrointestinal side effects asso-
trations of fatty acids in cells are high enough ciated with this class of lipid-lowering drugs.
Antihyperlipidemic Age&

The focus of current work in this area is to Studies with radiolabeled [3Hl-(40)in the
increase the loading, that is, the number of bile duct-cannulated rat model indicated rapid
quaternary ammonium groups per gram of appearance of a mixture of metabolites in the
dry resin that can bind bile acids, thus increas- bile (107). This metabolic mixture was also
ing the efficacy of the resin. shown to be a more potent inhibitor than (40)
Research scientists at Schering-Plough had of [14Cl-cholesterolabsorption, which led the
identified the first-generation 2-azetidinone researchers to analyze the putative metabolite
(40, SCH 48461) as a potent cholesterol ab- structure-activity relationship (SAR) (108).
The putative sites of metabolism were identi-
( 4 9 more active than (4R) fied as (1) demethylation of the methoxy
groups, (2) C3-side-chain benzylic oxidation,
and (3) &pendant phenyl oxidation. Finally,
the chemistry program identified ezetimibe
(201, which was designed to block the sites of
Only -OH metabolism and exploit the SAR of the active
and -0-alkyl
active Variety of metabolites.
substituents Interestingly, as mentioned in section
active
2.4.3, ezetimibe (20) is metabolized through
glucuronidation of the C4-phenyl hydroxyl
moiety, which improves the cholesterol ab-
sorption inhibition activity. This explains the
SAR requirement for the substitution of the
C4-phenylgroup.

sorption inhibitor, starting from a chemistry


5.4 Nicotinic Acid Derivatives
program to discover conformationally re-
stricted ACAT inhibitors (278, 279). The key Nicotinic acid (21)is a vitamin of the B family,
structural elements identified for cholesterol but its lipid-lowering action is unrelated to its
absorption inhibition were: (1) the Nl-aryl- role as a vitamin. It is believed that the lipid
substituted 2-azetidinone backbone, (2) a effects result from a decrease in fatty acid re
(4s)-alkoxyarylsubstituent, and (3)a C3-aryl- lease from adipocytes, thereby leading to de-
alkyl substituent (278). It was also shown in creased VLDL production. The molecular tar-
the same paper that a wide variety of para- get of nicotinic acid (21) is unknown; however,
substituents on the Nl-phenyl were permitted its identification would facilitate the develop
but that the C4-phenylrequired a hydroxyl or ment of selective nicotinic acid analogs with
alkoxyl residue. potentially fewer side effects.
Apical

Plasma membrane
Figure 10.13. Structure of the ectodomain of the transferrin receptor. (a) Domain organization of
the transfenin receptor polypeptide chain. The cytoplasmic domain is white; the transmembrane
segment is black; the stalk is gray; and the proteaselike, apical and helical domains are red, green
and yellow, respectively. Numbers indicate domain residues a t domain boundaries.
(b)Ribbon diagram of the transferrin receptor dimer depicted in its likely orientation with respect
to the plasma membrane. One monomer is colored according to domain (standard coloring as
described above), and the other is blue. The stalk region is shown in gray connected to the puta-
tive membrane-spanning helices. Pink spheres indicate the location of SmS+ions in the crystal
structure. Arrows show directions of (small) displacements of the apical domain in noncrystallo-
graphically related molecules. [Reprinted with permission from C. M. Lawrence, S. Ray, M.
Babyonyshev, R. Galluser, D. W. Borhani, and S. C. Harrison, Science, 286, 779-782 (1999).
Copyright 1999 American Association for the Advancement of Science.]
Figure 15.7. DNA-receptor co
DNA binding domain (ribbon).
New and Future Treatments

.5 Miscellaneous (27) is the ethyl side-chain in position C,,.


This suggests that there is a very selective
mechanism of intestinal cholesterol absorp-
estrogen modulators exhibit beneficial
tion based on the structural recognition of
-lowering effects, these drugs were not op-
"cholesterol (41)" and that small structural
zed for this activity. Therefore, they are
modifications interfere with the normal ab-
t treated in this section.
sorption process. This is understandable,
given that p-sitosterol(26) and sitostanol(27)
5.5.1 Probucol. The exact mechanism of
cannot be further processed by the liver and
by which probucol (22) reduces both
peripheral tissues for the synthesis of cell
and HDLc is unclear, making a discus-
membranes and hormones.

cal scavenger action. The antioxidant ef- 6 NEW AND FUTURE TREATMENTS
s of the 2,6-di-tert-butylphenolic moiety
well known and are not further discussed The new therapeutic options available to clini-
cians for treating dyslipidemia in the last de-
cade have enabled effective treatment for
5.5.2 Plant Sterols. These compounds are many patients. Although LDLc is still the ma-
nabsorbable cholesterol analogs that occur jor target for therapy, it is likely that over the
next several years other lipid and nonlipid pa-
structural similarity with cholesterol rameters will become more generally accepted
, the plant sterols are able to inhibit the targets for specific therapeutic interventions.
Major pharmaceutical companies are already
evaluating new therapeutic agents in human
sterol, p-sitosterol (261, and sitostanol clinical trials.
Antihyperlipidemic Agents

6.1 New Treatments for Lowering LDLc -c and phospholipids. MTP inhibitors have been
TG Lowering shown to significantly reduce (>60%) serum
levels of VLDLc, LDLc, and TGs in animal
6.1.1 Novel HMG-CoA Reductase Inhibitors models. The major issue in the prolonged in-
(Statins). Two new competitors in this area, hibition of hepatic MTP is the potential of an
mentioned earlier in Section 2.2.1, are cur- accumulation of TGs in the liver (fatty liver).
rently in late-stage development. Crestor [ro- In addition, BMS discontinued the develop-
suvastatin (7), AstraZenecal is expected to be ment of BMS-201038 (42), claiming mecha-
even more effective than Lipitor [atorvastatin nism of action-based adverse events, in the
(6)]and become a multibillion dollar product form of liver function. However, Bayer is cur-
(248), and Advicor (nicostatin, Kos), a once- rently in phase I11 trials with BAY-139952 (43,
daily combination of Niaspan (21) (extended-
release niacin) and lovastatin (2), lowers LDL
and TGs to a greater extent than lovastatin
alone, and can raise HDL by as much as 40%.
If this product can overcome the safety and
tolerability issues associated with niacin (2)
(see Section 2.3.4) and concerns over myop-
athy/rhabdomyolysis (see Section 2.3.1.21, it
may become a commercial success.
Beyond these, the only other statin of note
is pitavastatin (9). Novartis has recently li-
censed this compound in Europe (where it is in
phase 111)and is in negotiation for U.S. rights.
Recently, it has been reported that both ro- implitapide), whereas Pfizer is in phase I1 tri-
suvastatin (7) and pitivastatin (9) have been als with CP-346086 (structure not published)
associated with rhabdomyolysis at the higher and Janssen is in phase I with R-103757 (44).
doses evaluated, which wilI potentially delay their Interestingly, animal data and results from
development and complicate regulatory approval. the completed clinical trials also suggest that
the intestinal inhibition of MTP results in de-
6.1.2 Microsomal Triglyceride Transfer Pro- creased fat absorption and weight loss associ-
tein (MTP) Inhibitors. MTP, which is found in ated with an antiobesity effect. The future of
the liver and intestine, plays a pivotal role in this class of compounds will depend on the
the assembly and secretion of TG-rich lipopro- ability of these drugs to resolve the potential
teins (VLDL and chylomicrons), and also cat- liver toxicity issues associated with MTP inhi-
alyzes the transport of TGs, cholesterol esters, bition.
6 New and Future Treatments

rN
N\ NA
/

S
C
qyov'
N 1 \ LA
-CkNd
0 O
(44) R-103757

6.1.3 LDL-Receptor (LDLr) Upregulators. The active in this field. Recently, scientists at Glaxo-
up-regulation of LDL receptors has potential SmithKline described a new class of com-
as a novel means of lowering serum LDL. pounds that reduce blood levels of cholesterol
These compounds could be significant compe- in an animal model by upregulating the LDLr
tition in the dyslipidemia segment of the mar- through a mechanism different from that of
ket arising from the large unmet need in this the statins (280). Two series of molecules, the
area. Pfizer, Tularik, Lilly, and Aventis are all steroidlike analogs represented by GW707
(45) and the nonsteroidal molecules repre-
sented by GW532 (461, were identified with
nanomolar activities.

6.1.4 Bile Acid Reabsorption lnhibitors/Bile


Acid Sequestrants (BAS)/Cholesterol Absorp-
tion Inhibitors. Although these three classes of
molecules can be used as monotherapy, their
greatest potential resides in combination ther-
apy. Aventis is currently in phase I trials with
" 0 the bile acid reabsorption inhibitor HMR-1453
(structure not published) for the treatment of
atherosclerosis. BMS is currently in Phase I1
I with the bile acid sequestrant DMP-504
(structure not published), which is reported to
(45) GW707 be more potent than cholestyramine in reduc-
ing serum cholesterol levels but appears to

H p c l

H G N 0

p-N / (46) GW532


0
Antihyperlipidemic Agents

have the same side effect profile, which is a phase I11 trials, whereas Sankyo (CS 505,
disadvantage compared to GelTex's second- structure not published) and bioMerieux-
generation bile acid sequestrant GT-102279 Pierre Fabre (F12511, 48, or eflucimibe) are
(structure not published), also in Phase I1 tri- both reported in the phase I stage. There are
als. Ezetimibe (20) is the only cholesterol ab- currently many other pharmaceutical compa-
sorption inhibitor currently in clinical trials nies reported to be working in this field.
(see section 2.2.3) and has been shown to re-
duce LDLc between 10 and 19% in mono- 6.2 New Treatments for Raising
therapy. Interestingly, the reduction of LDLc in +
HDLc TC Lowering
combination of ezetimibe (20) with a statin ke.,
simvastatin (4) or atorvastatin (6)] is additive. 6.2.1 Peroxisome Proliferator-Activated Re-
ceptor (PPAR) Agonists. Competitors are gen-
6.1.5 Acyl-CoA Cholesterol AcylTransferase erally PPARy or mixed PPARaly agonists, fo-
(ACAT) Inhibitors. ACAT is a ubiquitous en- cused primarily on diabetes [Dr. Reddyl
zyme responsible for esterifying excess intra- NovoNordisk (DRF-2725,49;phase 111), Astra-
cellular cholesterol. The cholesterol ester is
then transferred to lipoprotein particles to be
stored in their core and, subsequently, depos-
ited into forming atherosclerotic lesions. The
activity of ACAT is enhanced by the presence
of intracellular cholesterol; however, whether
inhibition of ACAT will prevent atherosclero-
sis is not yet clear. Furthermore, inhibition of
hepatic ACAT decreases the secretion of apoB-
containing lipoproteins (VLDL) by the liver.
The combination of an ACAT inhibitor and
another lipid-lowering agent, particularly a
statin, could have added benefit on CV mortal-
ity and morbidity. Pfizer is leading the field
with Avasimibe (CI-1011, 471, currently in Zeneca (AZ-242, 50; phase II), BMS (BMS-
298585, 51; phase II), Merck (KRP-297, 52;
phase 11), and LigandLilly (LY519818, struc-
ture not published; phase I)]. There is also a
series of PPARa agonists from Kyorin (531,
the first of which is in preclinical development
for atherosclerosis, whereas GlaxoSmithKline
has also reported two PPAR agonists in phase
I trials for dyslipidemia [GW 590735 (stucture
not published) and GW 501516 (5411as well as
Dr. Reddy's DRF-4832 (structure not pub
lished), which is to start phase I trials for dys-
lipidemia later this year.
6 New and Future Treatments

CP-529414 (structure not published), a 70%


increase in HDLc, indicating these compounds
may potentially have a large impact on the
dyslipidemia market. Avant Immunothera-
peutics is currently in phase I1 trials with
CETi-1 (structure not published), a peptide
(53) Kyorin vaccine against CETP to reduce risk factors
for atherosclerosis, and in November 2001 at
6.2.2 Cholesteryl Ester Transfer Protein the American Heart Association 2001 Scien-
(CETP) Inhibitors. CETP is a plasma glycopro- tific Sessions, Japan Tobacco presented the
tein that mediates the transfer of cholesteryl phase I1 data, where oral administration of
ester from HDL to VLDL, IDL, and LDL. Com- 900 mg of JTT-705 (55)once daily for 4 weeks
pounds that inhibit CETP are expected to in- led to a 34% increase in HDLc levels and a 7%
crease plasma HDL cholesterol levels and im- decrease in LDLc.
prove the HDLILDL cholesterol ratio. They
could be used as monotherapy, or more 6.2.3 Liver X-Receptor (U(R) Agonists. I.XRa
likely in combination with statins. Pfizer re- is a nuclear receptor implicated in lipid ho-
cently reported excellent phase I1 results of meostasis. LXRa can modify the expression of
Antihyperlipidemic Agents

gating new approaches to treat directly the


atherosclerotic plaque resulting in plaque sta-
bilization and/or regression.

6.3.1 Chemokines and Cytokines. Chemo-


kines and cytokines may act directly at the
atherosclerotic plaque, interfering with the in-
flammatory process thought to destabilize the
plaque. All the research activities seem to be
in the preclinical stage, although many large
companies seem to be involved in this area,
including Merck, Novartis, Aventis, AstraZen-
eca, and GlaxoSmithKline.
genes for lipogenic enzymes through regula-
tion of the sterol regulatory-element binding 6.3.2 Antioxidants. Oxidative modification
Protein l c (SREBP-lc) expression (281a). On of LDL has been accepted as an important
the basis of current animal model data, LXRa event in the development of atherosclerosis.
agonists will afford large increases in HDL lev- Therefore, antioxidants have been expected to
els, leading to increased reverse cholesterol have potential as antiatherogenic agents.
transport (RCT) (281b). Several pharmaceuti- However, clinical trials of antioxidants have
cal companies are working in this area but given rise to controversy regarding their real
none of the molecules is beyond the preclinical clinical benefit. Although vitamin E has been
stage. reported to reduce the risk of coronary heart
disease, two large-scale trials failed to demon-
6.3 New Treatments for Atherosclerosis
strate the effect of a-tocopherol (561,the most
In the past, the treatment of CV disease was active species of vitamin E, on cardiovascular
addressed by modifying the major risk factors disease or cerebrovascular mortality (282).
(i.e., LDLc, HDLc, TG, etc.) associated with This failure of a-tocopherol (56) is probably
the progression of atherosclerosis. Recently, attributable to the fact that it cannot reach
pharmaceutical companies have been investi- the core of LDL particles. On the other hand,
6 New and Future Treatments 373

probucol (22) showed antiatherogenic effects versing the progression of coronary artery dis-
in animal models but had the untoward effect ease. Phase I11studies are expected to begin in
of lowering HDL levels. 2003.
Several of these are beyond the preclinical Experimental data have shown that
stage, including AGI-1067 (571, from Athero- BO-653 (58), currently in phase I1 studies, is a
Genetics (licensed to Schering-Plough, in superior antioxidant to either a-tocopherol
phase 11), which is a structural analog of pro- (57) or probucol (22) (282). It can penetrate
bucol (22); a compound [BO-653 (58)l from into the core of LDL particles, does not lower
HDL levels, and shows antiatherogenic and
antirestenosis effects in animal models. How-
ever, studies are still needed to determine
whether BO-653 (57) has therapeutic utility
in humans.
The nonpeptidic compound AC-3056 (struc-
ture not published), in-licensed by Amylin
Pharmaceuticals, is being developed for the
prevention of atherosclerosis and restenosis
after angioplasty procedures and metabolic
disorders relating to cardiovascular disease.
AC-3056 has been characterized in vitro and
in animal models as having three different
Chugai, also in phase 11;and a compound (AC- modes of action-targeting steps in the athero-
3056) from Aventis that has just completed sclerosis cascade: (1)lowering of serum LDL
phase I. cholesterol but not HDL cholesterol; (2) inhi-
AGI-1067 (57) is a VCAM-1 (vascular cell bition of lipoprotein oxidation; and (3) inhibi-
adhesion molecule 1) gene expression inhibi- tion of cytokine-induced expression of cell ad-
tor under development by AtheroGenics for hesion molecules in vascular cells (283).
the potential prevention of atherosclerosis
(hypercholesterolemia)and restenosis. VCAM-1 6.3.3 Lipoprotein-Associated Phospholipase
is the surface protein to which various types of A, (Lp-PLA,) Inhibitors. Lipoprotein-associ-
leukocyte attach themselves, forming the ated phospholipase A, (Lp-PLA,), an enzyme
starting point of new plaques. By inhibiting associated with low density lipoprotein, would
the expression of VCAM-1, AGI-1067 (57) has appear to be a novel target for therapy to pre-
the potential to prevent atherosclerosis at the vent heart attacks on the basis of a study pub-
very earliest stage. In November 2001 further lished by scientists from GlaxoSmithKline
data, presented at the American Heart Associ- and Glasgow University (284). In the study, in
ation 2001 Scientific Sessions, showed that addition to being a potential drug target, the
AGI-1067 (57) met its primary endpoint in enzyme could be a new risk factor for cardio-
preventing restenosis in the phase I1 studies vascular disease and as such could serve as a
and showed a direct antiatherosclerotic effect marker, independent of LDL, to predict the
on coronary blood vessels, consistent with re- occurrence of heart attacks. Lp-PLA, is found
Antihyperlipidemic Agent!

in the bloodstream, bound to LDL. During have on overall cardiovascular morbidity and
LDL oxidation, Lp-PLA, breaks down the fats mortality is not known. Nonetheless, the abil.
in LDL, producing substances that attract in- ity to reverse lipid accumulation in atheroscle-
flammatory cells, which in turn engulf LDL, rosis with this therapy is claimed to provide
eventually contributing to the formation of substantial benefits compared to those of ex-
atherosclerotic plaques. SB-480848 (structure isting therapies.
not published), which targets Lp-PLA,, is cur- Preliminary findings from Esperion Ther-
rently in phase I clinical trials (285) and tar- apeutics' phase IIa clinical study of ETC-588,
gets a different rationale from cholesterol re- or LUV (large unilamellar vesicles), for the
treatment of ACS indicated that the product
duction in the prevention of heart attack. This
met the primary endpoint of demonstrating
would therefore benefit people at risk of a
safety and tolerability in patients with known
heart attack, but who do not have increased vascular disease. The study was a double-
cholesterol levels. blind, randomized, placebo-controlled, multi-
ple-dose trial designed to determine the opti-
6.3.4 New Miscellaneous Treatments. Es- mal dosing schedule and effect of ETC-588 in
perion Therapeutics and the University of Mi- 34 patients with stable atherosclerosis and
lan are developing ETC-216, apolipoprotein HDL of 45 mg/dL or less. Patients received one
AI Milano (also known as apoAI Milano or of three dose strengths (50,100, or 200 mgkg)
AIM), a recombinant variant of normal apoli- or placebo every 4 or 7 days. Patients receiving
poprotein AI, the major protein component of the 100 and 200 mgkg doses each received
HDL, which is thought to protect against car- seven doses for either 4 or 6 weeks, whereas
diovascular disease by efficiently removing those on 50 mglkg received 14 doses for either
cholesterol and other lipids from tissues in- 8 or 13 weeks. All dose levels and regimens
cluding the arterial wall and transporting were found to be safe and well tolerated, and
them to the liver for elimination. A multiple- an optimal dosing schedule of once every 7
dose, multicenter phase I1clinical trial has ini- days was defined for future use. Evidence
tiated with ETC-216 in patients with acute of dose-related cholesterol mobilization was
coronary syndromes (ACS). The trial will as- noted. Evaluation is still under way of brachial
sess the efficacy of ETC-216 in regressing cor- artery ultrasound measurements and changes
onary atherosclerosis by measuring changes in inflammatory markers. ETC-588 is made
in plaque size of one targeted coronary artery, of naturally occurring lipids that circulate
measured by atheroma volume through the through the arteries and is claimed to remove
use of intravascular ultrasound. The double- accumulated cholesterol and other lipids from
blind, randomized, placebo-controlled study cells, including those in the arterial wall. It is
will enroll 50 patients with ACS who are designed to augment HDL function for the
scheduled to undergo coronary angiography acute or subacute treatment of ischemia.
and/or angioplasty. ETC-216 offers an attrac- ETC-588 has demonstrated a high capacity to
tive mechanism for the treatment of athero- transport cholesterol from peripheral tissues
sclerosis because it aims to reverse the lipid to the liver, improve endothelial function, and
accumulation already present in atherosclero- regress atherosclerosis in preclinical models.
sis, as well as preventing further accumulation.
There are lipid-lowering agents currently
available that decrease serum cholesterol lev- 7 RETRIEVAL OF RELATED INFORMATION
els and stop the progression of atherosclerosis,
but no therapies currently exist that selec- Related information can be retrieved through
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are currently no human studies available on use of combinations of key words or phrases,
this compound, the effect that this drug will plus specification of a single year to narrow
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CHAPTER EIGHT

Oxygen Delivery by Allosteric


Effectors of Hemoglobin, Blood
Substitutes, and plasma
Expanders
BARBARA CAMPANINI
STEFANO BRUNO
SAMANTA ~ O N I
ANDREA MOZZARELLI
Department of Biochemistry and Molecular Biology
National Institute for the Physics of Matter
University of Parma
Parma, Italy

Contents
1 Introduction, 386
2 Allosteric Effectors of Hemoglobin, 388
2.1 History, 388
2.2 Clinical Use of Right Shifters, 388
2.2.1 Organic Phosphates, 389
2.2.2 Bezafibrate Derivatives, 389
2.2.2.1 Efaproxiral, 391
2.2.3 Aromatic Aldehydes, 394
3 Blood Substitutes: Modified Hemoglobins and
Perfluorochemicals, 396
3.1 History of Blood Substitutes, 396
3.1.1 Development of Modified Hemoglobins,
396
3.1.2 Perfluorochemicals as an Alternative to
Hemoglobin, 398
3.2 Clinical Use of Modified Hemoglobins and
Perfluorochemicals, 398
3.3 Hemoglobin-Based Blood Substitutes on
Clinical Trial, 399
3.3.1 General Side Effects, 399
3.3.1.1 Hypertension, 401
3.3.1.2 Nephrotoxicity, 403
Burger's Medicinal Chemistry and Drug Discovery 3.3.1.3 Gastrointestinal Effects, 404
Sixth Edition, Volume 3: CardiovascularAgents and 3.3.1.4Hemoglobin Oxidation:
Endocrines Oxidative Toxicity and
Edited by Donald J. Abraham Reperfusion Injury, 404
ISBN 0-471-37029-0 O 2003 John Wiley & Sons,Inc. 3.3.1.5 Antigenicity, 405
385
Allosteric Effe iemoglobin, Blood Substitutes, and Plasma Expa

3.3.2 Crosslinked Hemoglobins, 406 3.4.1 Current Drugs, 421


3.3.2.1 HernAssist (DCLHb), 406 3.4.1.1 First-Generation Fluoro
3.3.3 Recombinant Hemoglobins, 408 Based Blood Substitutes, 421
3.3.3.1 Optro (rHbl.l), 408 3.4.1.2 SecondGeneration
3.3.4 Polymerized Hemoglobins, 412 Perfluorochemicals, 422
3.3.4.1Hemopure (HBOC-201),413 3.4.2 General Side Effects, 423
3.3.4.2PolyHeme (Poly SFH-P), 414 3.4.3 Pharmacokinetics, 424
3.3.4.3Hernolink, 415 3.4.4 Physiology and Pharmacology, 425
3.3.4.4Recent Developments, 417 3.4.4.1 Hemodynamics, 427
3.3.5 Conjugated Hemoglobins, 417 3.4.5 Structure-Activity Relationship, 427
3.3.5.1PEG-Hb, 417 3.4.6 Emulsion Stability, 427
3.3.5.2PHP (Pyridoxalated 3.4.7 Recent Developments, 428
Hemoglobin-Polyoxyethylene 4 Plasma Volume Expanders, 429
Conjugate), 419 4.1 Clinical Use, 429
3.3.6 Recent Developments of Modified 4.1.1 Current Drugs on the Market, 429
Hemoglobins, 420 4.1.2 Side Effects, 430
3.3.6.1Encapsulated Hemoglobins, 420 4.1.3 Pharmacokinetics, 432
3.3.6.2Albumin-Heme, 421 4.2 Physiology and Pharmacology, 432
3.4 Perfluorochemicals, 421 5 Web Site Addresses, 433

1 INTRODUCTION lar, increased levels of 2,3-diphosphoglycer


are responsible for the adaptation to low
Oxygen supply is vital for human life. In the gen pressures at high altitudes and to low
lung, oxygen binds to hemoglobin, a protein moglobin contents in anemia. The sigmoidal
contained in red blood cells and, in the circu- shape of the binding curve indicates that o
lation, it is delivered to the peripheral tissues, gen binding increases oxygen affinity and
where hemoglobin loads carbon dioxide (1). decrease of the oxygen saturation favors the
The oxygen content of the air at sea level is unloading of more oxygen. This behavior is an
20.95 %, which corresponds to a partial pres- indication of hemoglobin positive cooperativ-
sure of oxygen of about 160 Torr. In the lung, ity.
the oxygen pressure is about 100 Tom and in Several models have been proposed to ex-
the mixed venous circulation is about 40 Torr. plain the allosteric properties of hemoglobin:
The corresponding oxygen fractional satura- the Monod-Wyman-Changeux model (MWC)
tion (i.e., the concentration of oxygenated he- (4), the Koshland-Nemethy-Filmer model (4
moglobin over the total hemoglobin concen- the Perutz's stereochemical mechanism (6),
tration) is 0.97 and 0.75. Thus, only a small and the Ackers's Symmetry rule (7). A modi-
fraction of the oxygen bound to hemoglobin is fied version of the MWC model, which includea
delivered to the tissues. The affinity of hemo- Perutz's stereochemical mechanism, appears
globin for oxygen is expressed as p50, the oxy- to explain most of the functional properties of
gen pressure at which 50% of hemes are satu- hemoglobin (8). The fundamental crystallo-
rated (Fig. 8.1) (2, 3). Under physiological graphic study, carried out over more than 50
conditions, pH 7.4 and 3TC, the p50 of human years by the Nobel laureate Max Perutz, shed
blood is 26 Torr. Molecules that bind to hemo- light on hemoglobin structure and function (6,
globin and shift the oxygen binding curve ei- 9). Hemoglobin is a tetrameric molecule com-
ther to the left or to the right are called allo- posed of two a- and P-subunits, arranged in a
steric effectors. Left shifters increase oxygen tetrahedral geometry (Fig. 8.2). Each subunit
affinity and right shifters decrease oxygen af- contains a heme, a tetrapyrrole ring coordi-
finity. Physiological right shifters are protons, nating an iron ion in the center. The iron is
chloride ions, carbonate, and the organic phos- also coordinated to a histidine in the heme
phate 2,3-diphosphoglycerate. An increased binding pocket of hemoglobin. Oxygen, carbon
concentration of these compounds favors the monoxide, and nitric oxide competitively bind
unloading of oxygen to the tissues. In particu- to the ferrous iron, whereas carbon dioxide
-
-
-
-
-
-
-
-
-
I I

1 00
Oxygen pressure (torr)
Figure 8.1. Oxygen binding curves of hemoglobin.
Whole blood (wh)under physiological conditions ex-
hibits a p50 of 26 Torr (1)."Stripped" hemoglobin
(st) (i.e., hemoglobin in the absence of allosteric ef-
fectors)exhibits a p50 of 5 Tom at 37"C,pH 7.2 (19).

binds to the amine termini. 2,3-Diphospho-


glycerate binds to positively charged residues
of the p-chains in the central cavity, whereas
chloride ions bind both in the central cavity
and at a-chain residues. The deoxyhemoglobin
structure is called T (tense) because several
salt bridges constrain the molecule, decreas-
ing by 20- to 300-fold the oxygen affinity with
respect to the oxyhemoglobin structure, called
R (relaxed) (3). The binding of oxygen triggers Figure 8.2. Structure of hemoglobin in the R state
a series of tertiary and quaternary conforma- (a) and in the T state (b). In the T to R transition,
tional changes, leading to the breakage of salt one c@ dimer rotates by about 15", with respect to
bridges and other bonds and to the release of the other, and the central cavity narrows.
protons and both organic and inorganic anions
(6, 9). This description of hemoglobin struc- state and as iron nitrosyl complex in the T
ture, dynamics, and function is necessarily state (13, 14). The release of NO from hemo-
simplified and mainly aimed at providing key globin and the reaction with glutathione to
elements of hemoglobin complexity. More de- form S-nitrosoglutathione have been sug-
tailed descriptions are reported in the above- gested to be relevant steps in complex mecha-
quoted books (1-3) and papers (4-9) and ref- nisms that regulate NO activities. It is known
erences therein. that, under physiological conditions, Hb con-
A still controversial function of hemoglobin centration is about 1000-fold higher than that
is nitric oxide (NO) transport. NO is involved of NO (11). Hemoglobin is also involved in NO
in the control of vasoactivity, blood vessels di- oxidation, considered the major pathway for
lation, platelet aggregation, and brain-regu- NO catabolism (15) and oxidase and peroxi-
latedrespiration (10). NO enters the red blood dase activities (16).
cells through mechanisms still under investi- Within the above-outlined frame of hemo-
gation (11,12) and predominantly binds to he- globin structure and function, three research
moglobin as S-nitrosothiol at p-Cys93 in the R projects have been developed in the last 30
388 Oxygen Delivery by Allosteric Effectors of Hemoglobin, Blood Substitutes, and Plasma Expanders

years. The first investigation is aimed at de- the erythrocyte membrane (19). Attempts to
signing new allosteric effectors of hemoglobin. engineer red blood cells to make them perme-
Compounds that increase the oxygen affmity able to IHP were carried out (20).
can be used for the treatment of diseases as In the early 1980s two antilipidemic
sickle-cell anemia, and those that decrease the agents, clofibrate and bezafibrate, were shown
oxygen affinity can be used for the treatment to lower the affinity of hemoglobin for oxygen
of ischemia, hypoxia, and to improve the effi- as the organic phosphates (19,21). These com-
ciency of radio- and chemotherapy. The sec- pounds were reported to reversibly cross the
ond project, strongly stimulated in the 1980s erythrocyte membrane, thus opening the way
by the risk of HIV contamination in trans- to their use as therapeutic agents. However,
fused blood, is focused on blood substitutes the more promising of the two molecules,
either by designing new types of oxygen carri- bezafibrate, exhibited a dissociation constant
ers or by constructing genetically and/or to hemoglobin that was still too low to make it
chemically modified hemoglobins able to oper- suitable as a drug. Moreover, it was shown
ate in the plasma outside the red blood cells. A that bezafibrate interacts strongly with serum
third line of research is aimed at formulating albumin, reducing its in uiuo activity. Never-
solutions able to replace the blood, maintain- theless, a class of structurally related com-
ing the volume and the oncotic pressure of pounds was developed with the aim of increas-
blood fluids in severe hemorrhagic events. Up ing their affinity for hemoglobin and reducing
to now, no allosteric effectors or blood substi- the competition with albumin (see Ref. 22 for a
tutes have been approved for human use in the review). From these studies, in 1992, a com-
United States, a clear indication of the diffi- pound initially called RSR13 emerged as a
culties in mimicking the multifaceted roles of promising drug candidate. Its approved non-
hemoglobin. proprietary name is efaproxiral.
In the same period, a completely new class
of right shifters of the oxygen-binding curve
2 ALLOSTERIC EFFECTORS OF
was discovered. It was known that aromatic
HEMOGLOBIN
monoaldehydes, as vanillin and 12C79 (23),
were able to shift the binding curve to the left,
2.1 History
thus increasing the overall affinity of hemo-
In 1967 (17, 18) the endogenous compound globin for oxygen. This effect might be of clin-
2,3-diphosphoglycerate (2,3-DPG) was discov- ical interest in the therapy of sickle-cell ane-
ered to be a powerful allosteric effector of he- mia. The polymerization of the hemoglobin
moglobin. 2,3-DPG exhibits a physiological mutant associated with this pathology occurs
concentration in human erythrocytes of only when a critical concentration of deoxyhe-
4.6 mM, high enough to form a 1:l complex moglobin is reached upon unloading of oxygen
with hemoglobin. 2,3-DPG is responsible for in the peripheral tissues. A shift of the bin*
the relatively low oxygen affinity of whole curve to the left increases the concentrationof
blood compared to that of purified hemoglo- oxyhemoglobin, thus reducing the tissue dam-
bin. By removing 2,3-DPG, the p50 drops from ages caused by the polymerization. In the
26 Torr to approximately 12 Torr. Other nat- course of investigating this class of compounds
'
ural and synthetic allosteric effectors, struc- in search of new antisickling agents, surpris-
turally related to 2,3-DPG and known as or- ingly, some aromatic aldehydes were discov-
ganic phosphates, were discovered over the ered to lower the oxygen affinity (24). Al- ,
years. Among them, inositol hexaphosphate though they have not yet been developed as
(IHP) and its structural analog inositol hexa- drugs, they could be used for the same
sulfate (IHS) have been extensively studied. It cations as efaproxiral.
was shown that they all share a common
2.2 Clinical Use of Right Shifters
mechanism of action and a common binding
site with 2,3-DPG (1). Unfortunately, this The modulation of oxygen delivery to periph-
class of compounds revealed to be unsuitable eral tissues through the direct and reversible
as drugs because they do not efficiently cross interaction of drugs with hemoglobin has long
2 Allosteric Effectors of Hemoglobin 389

been recognized as a possible treatment for


several pathological states. Besides antisick-
ling agents, which will be treated elsewhere,
drugs interacting with hemoglobin of poten-
tial clinical use are essentially those that in-
i duce a rightward shift of the oxygen dissocia-
tion curve. This approach is beneficial in all
conditions that require a transient increase in
oxygen delivery in the tissues, either to over-
come an insufficient amount to healthy organs
(ischemia) or to sensitize solid tumors to ra-
diotherapy and chemotherapy. Up to now, no
drug of this class is on the market, mainly be-
cause of the poor pharmacokinetic properties
of the molecules tested so far. However, efa-
proxiral is currently being tested in advanced
clinical trials.

2.2.1 Organic Phosphates. The interaction


of the endogenous allosteric effector 2,3-DPG
(la)with hemoglobin is well known. 2,3-DPG
binds noncovalently in the cleft between the
two p-subunits of deoxyhemoglobin, forming
several ionic bonds with positively charged
residues and counterbalancing the excess of Figure 8.3. Binding sites of 2,3-DPG (a) and
positive charges in the central cavity (Fig. RSR13 (b) to hemoglobin. Ligands are shown in
8.3a) (25). By preferentially binding to T-state space-filling mode and hemes in ball-and-stick
hemoglobin, 2,3-DPG stabilizes the deoxy mode.
state with respect to the R state. It was also
shown that 2,3-DPG affects the intrinsic affin-
ity of T-state hemoglobin, even in the absence
of a switch to the R state (3). Inositol
hexaphosphate (lb),also known as phytic
acid, binds to the same site (26).

2.2.2 Bezafibrate Derivatives. The binding


: site of the bezafibrate derivatives is different
from that of the organic phosphates. Bezafi-
brate (2a)and its analogs bind to twofold sym-
metry-related sites in the central water cavity
of deoxyhemoglobin,approximately 20 A from
the binding site of 2,3-DPG (21,27) (Fig. 8.3b).
Each molecule is engaged in several interac-
tions, mostly hydrophobic contacts and water-
mediated hydrogen bonds with both p-sub-
units and one a-subunit. Efaproxiral and its
structural analogs hinder the transition from
T to R by preventing the narrowing of the cen-
t tral water cavity. Because the binding sites of
: efaproxiral and 2,3-DPG are different, they
act synergically and not in competition.
390 Oxygen Delivery by Allosteric Effectors of Hemoglobin, Blood Substitutes, and Plasma Expanders

The bezafibrate derivatives developed so pure affinity contribution, it does not affect
far have the general structure (2b)(28). the T to R equilibrium, and, therefore, the Hill
coefficient is the same as the control curve. It
was shown that all the bezafibrate derivatives
induce, to a different extent, a change both in
the affinity and the allosteric equilibrium.
In some cases, an approximated linear cor-
relation was found between the affinity of the
allosteric effectors for hemoglobin and the in-
duced variation of p50 (28). However, some of
the strongest allosteric effectors show a re-
markable variability in effectiveness (change
of p5O), in spite of similar binding constants to
hemoglobin. Abraham and coworkers (28) in-
troduced an intrinsic affinity coefficient and
proposed a molecular mechanism. The intrin-
As a rule, the shortening of the four-atom sic affinity might be influenced by the capacity
bridge of bezafibrate to a three-atom bridge of the effectors to interact with key residues
increases the potency of these compounds as and, particularly, with aLys99.
allosteric effectors of hemoglobin. Depending Of the five structural classes listed in Table
on the X-Y-Z link, the bezafibrate derivatives 8.1, only two, A and C, both characterized by
tested as allosteric effectors can be grouped an amido group, exhibit high potency. The
into five structural classes (28). The substitu- first bezdbrate derivatives were reported by
ents of the more interesting molecules are re- Lalezari and coworkers (3032) and they all
ported in Table 8.1. The R, groups are hydro- share a phenylureido-substituted phenoxy-
phobic moieties and the X-Y-Z system is the isobutylic structure. They differ in the num-
bridge between the two substituted phenyl ber and position of the chlorine substituents
rings. on the phenyl ring (2b).The most potent mol-
The shift of pi50 induced by these com- ecule of the series, L345, lowers the oxygen
pounds results from two contributions (29). affinity of human erythrocytes 30-fold when it
The so-called allosteric factor arises from the is present at a concentration of 1 mM.It also
perturbation of the equilibrium between the strongly decreases the cooperativity. These
T- and R-state hemoglobin. This perturbation compounds show a partial competition with
is reflected in a low value of the Hill coeffi- chloride ions but act synergically with
cient, which approaches unity for potent effec- 2,3-DPG. X-ray diffraction studies have r e
tors when hemoglobin remains in the low af- vealed that they do not bind just to the bezd-
finity T state even at high oxygen partial brate site, but also, with lower affinity, to two
pressures. The so-called affinity factor origi- symmetrically related sites near Argl04P. Un-
nates from a variation of the intrinsic affinity fortunately, all the members of this class lose
of both T and R states. If an effector exhibits a their activity in the presence of physiological

Table 8.1 Allosteric Effectors of Hemoglobin That Decrease the Oxygen Affinity
Series Link Relevant Compounds
Series A ("RSR series") -NH--CO--CH2- RSR4 (R3,R, = C1)
RSR13 (R3,R, = Me)
Series B ("MM series") -C&NH--CH,- MM25 (R, = C1)
Series C ("Lseries") -NH--C&NH- L35 (R3,R6 = Cl)
L345 (R3,1&,R, = C1)
Series D -CH,-NH40-
Series E -CH2--CO--NH-
2 Allosteric Effectors of Hemoglobin

concentrations of albumin, even if palmitic whole blood in vivo and the oxygen pressure in
acid or tripalmitin is added. the tissues, measured directly by inserting a
A more successful group of bezafibrate de- microelectrode in the muscle. Experiments on
rivatives, named RSR series, was obtained by healthy rats (36) showed that the administra-
replacing one amide nitrogen of the urea with tion of efaproxiral also results in a decreased
amethylene group (27,33).The reversal of the cardiac output and in an increased vascular
arnide bond results in the much weaker MM resistance. These changes in hemodynamics
series. Although the RSR series is equally or are unlikely to be caused by a direct interac-
even less potent in vitro than the urea deriva- tion of efaproxiral with the vascular tissue.
tives, it is the least affected by the presence of Rather, they are caused by a regulatory ad-
albumin, probably because of its less polar na- justment in response to the increased delivery
ture. The bezafibrate derivative (2-[4-[[3,5- of oxygen to the tissues, probably mediated by
dimethylanilino]methyl]phenoxy]-2-methyl- oxygen-sensing systems, which are not yet
propionic acid) (3),efaproxiral, proved to be well characterized. This hypothesis is confirmed
the least affected and, therefore, the most by the observation that the structurally unre-
promising drug candidate. lated compound inositol hexaphosphate pro-
duces similar changes in hemodynamics. Be-
cause efaproxiral is also used as an enhancer
of the effects of radiotherapy, its pharmacolog-
ical activity was proposed to arise from an ad-
ditional, unknown mechanism. The hypothe-
sis originates from experiments in which
normally oxygenated and hypoxic EMT-6 mu-
rine mammarv " carcinoma cultured cells were
exposed to radiation in the absence and pres-
ence of efaproxiral and some of its less effec-
The substitution of the gem-dimethyl tive structural analogs (37). Efaproxiral
groups of efaproxiral with different alkyl proved to greatly enhance the cytotoxicity of
groups, as well as the substitution of the ether radiation on cultured cells. The effect is
oxygen atom with a methylene group, resulted greater in cells kept under hypoxic conditions,
in a decreased aMinity for hemoglobin (34). but it is also present in normally oxygenated
Unlike the urea analogs, this class seems not cells. Such an effect is obviously hemoglobin
to bind to a secondary site. independent and demands alternative expla-
2.2.2.1 Efaproxiral. Efaproxiral is the only nations. The fibric acid class, to which efa-
drug belonging to the compounds that right proxiral belongs, is known to perform its
shift the oxygen-binding curve, which is cur- antilipidemic activity by inhibiting the biosyn-
rently being investigated in clinical trials. It is thesis of cholesterol at a not well-defined level
produced by Allos Therapeutics (Denver, CO, in the steps preceding the synthesis of meval-
USA). It is usually administered as sodium onate, and altering the synthesis of com-
salt. pounds, such as geranyl-PP, farnesyl-PP, and
2.2.2.7.1 Physiology and Pharmacology. The geranylgeranyl-PP. Because these compounds
primary pharmacological effect of efaproxiral are involved in signal transduction pathways,
is a decreased affinity of hemoglobin for oxy- they might hinder DNA repair mechanisms,
gen, which implies a steeper gradient of hemo- thus making the neoplastic cells more sensi-
globin oxygenation between the lung and tis- tive to DNA-damaging therapies.
sues (Fig. 8.4). Therefore, a higher fraction of 2.2.2.1.2 Potential Clinical Use of Efaprox-
oxygen is delivered to the cells, thus increas- ira I
ing both its free concentration and the frac- 2.2.2.1.2.1 Efa~roxiral
, * as Radiation En-
tion bound to myoglobin. Early experiments hancer. The most promising application of
on healthy mice (35) showed that the admin- efaproxiral is as an enhancer of the effective-
istration of efaproxiral leads to a significant ness of radiation therapy in the treatment of
and reproducible increase in both the p50 of solid neoplasia. The oxygen-dependent sensi-
392 Oxygen Delivery by Allosteric Effectors of Hemoglobin, Blood Substitutes, and Plasma Expanders

cultured mammal cells with ionizing radiation


at different oxygen pressures (38). A low par-
tial pressure was shown to increase the resis-
tance of the cells. In viuo, by directly measur-
ing the oxygen pressure in solid tumor, it was
possible to correlate low values of oxygen with
poor response to radiotherapy, at least for
squamous cell carcinoma metastases (40), car-
cinoma of head and neck (41), and cancer of
the uterine cervix (42,43).
Hyperbaric oxygen is not always effective
as enhancer of radiotherapy, possibly because
hemoglobin is almost completely loaded at air
oxygen pressure; thus higher pressures do in-
crease dissolved oxygen only. An alternative
approach to reduce intratumoral hypoxia is
Oxygen pressure (torr) based on the liberation of part of the oxygen
Figure 8.4. Oxygen binding curve of hemoglobin that remains bound to hemoglobin at periph-
under physiological conditions (solid line) and in the eral oxygen pressures. Efaproxiral was tested
presence of a right shifting agent (dashed line). A for safety in a phase I clinical trial as radioen-
10-Torr right shift of the p50 results in about a two- hancement agent in 19 patients with newly
fold increase in the unloaded oxygen in the periph- diagnosed glioblastoma multiform brain can-
eral tissues. cer treated with standard cranial radiation
therapy (44). Phase I1 and I11 evaluations are
tivity of tumors to radiotherapy is attributed currently underway.
to an indirect mechanism of cytotoxicity. The A general advantage of this type of chemo-
ionizing radiation produces damages in the and radioenhancers is that they do not have to
DNA chain by forming free radicals, mostly reach the neoplastic cells for efficacy. There-
derived from oxygen. The efficacy of radiation fore, their activity does not depend on the me-
is therefore strongly reduced by intratumoral tabolism of the specific involved tissue.
hypoxia, which is one of the causes for the fail- 2.2.2.1.2.2 Efaproxiral in the Treatment of
ure of therapy, particularly in advanced tu- Ischemia. Another possible application of efa-
mors. To achieve the same toxic effect in hy- proxiral currently under investigation is as ox-
poxic conditions as in conditions of normal ygen-delivery agent in the treatment of isch-
oxygenation, 2.5- to 3-fold the standard dose of emia. Ischemia is an imbalance between
radiation should be applied (38). Hypoxia was oxygen supply and demand in peripheral tis-
shown to play an important role in multistep sues, which can undergo permanent damage if
carcinogenesis because it provides a selective the metabolic needs are not met for a suffi-
pressure for the proliferation of neoplastic ciently long time. It may be caused by trauma,
cells with a low apoptotic potential (39). -
neoplasia, or cardiovascular events. Ischemia
Therefore, drugs capable of inducing an in- is particularly dangerous when either the car-
creased intratumoral oxygen pressure are po- diac tissue or the central nervous system is
tentially useful not only in enhancing the ra- affected. In the former case. the whole cardio-
diation therapy but also in the general vascular function can be impaired, resulting
treatment of tumors, slowing down the pro- in possible secondary tissue damages. In the
gression toward malignancy. latter case, the clinical consequences can be
Intratumoral hypoxia may be caused either very different, depending on the involved area
by an increased consumption of oxygen due to of the central nervous system.
actively proliferating neoplastic cells or by a Efaproxiral proved effective in reducing
poor or inefficient vascolarization of the tu- the effects of prolonged ischemia in a number
mor. The effect of a poor perfusion on radio- of animal models. It was shown to reverse the
therapy can be mimicked in uitro by treating hypoxia-induced cerebral vasodilatation in
2 Allosteric Effectors of Hemoglobin

rats (45). Preischemic administration of efa- this study, it was shown that the rightward
proxiral to cats subjected to 5 h of permanent shift of the oxygen binding curve in vivo is
middle cerebral artery occlusion resulted in a dose dependent. A dose between 75 and 100
significant reduction in the size of the in- m a g was found to increase the whole blood
farcted region (46). However, the administra- p50 by approximately 10 Torr. As for animal
tion of efaproxiral proved ineffective in more models, no significant hemodynamic effects
severe ischemia (47). Preischemic administra- were noticed. A small number of patients
tion of efaproxiral was effective in the treat- showed a transient increase in serum creati-
ment of transient focal cerebral ischemia in nine. Clinical trials are currently assessing the
rats only if it was combined with the adminis- benefits of efaproxiral in patients with unsta-
tration of the N-methyl-D-aspartatereceptor ble angina and are planned for the treatment
tagonist dizocilpine (48). Efaproxiral proved of myocardial infarction and stroke.
ffective in improving the recovery of the con- 2.2.2.1.Z.3 Efaproxiral as Performance En-
actile function of stunned myocardium in -
hancer. The observation that efa~roxiralin-
ogs, a model of ischemic heart disease (49). It creases the aerobic capacity of skeletal mus-
as also effective in improving the myocardial cles in animal models raises the concern of an
echanical and metabolic recovery after car- illicit use as a performance-enhancing sub-
opulmonary bypass, using a dog model of stance. This application is motivated by the
urgically induced myocardial ischemia (50). acceleration of the oxidative muscular metab-
his application may be of significant clinical olism that results from a higher availability of
portance, given that the risk of ischemia oxygen in the tissues. In view of this use, a gas
g cardiac surgery is diminished but not chromatography/mass spectrometry method
mated by using hypothermic cardioplegia. for detection of efaproxiral in urine samples
oreover, patients with chronic medical con- has been developed (53).
tions, such as coronary artery disease, diabe- 2.2.2.1.3 Adverse Effects. Because clinical
s, and hypertension, have a significant risk trials are still ongoing, no definitive indication
f experiencing complications associated with of side effects is known. However, some conse-
a, even in noncardiac interventions. quences of the decreased oxygen affinity of he-
e effect of the administration of efaprox- moglobin can be foreseen. One predictable ad-
on metabolism during ischemic events was verse effect that may result from the lowering
vestigated by monitoring with 31Pnuclear of oxygen affinity is the reduced loading of he-
gnetic resonance the levels of the so-called moglobin in the lungs, which may cause hyp-
-energy phosphates, particularly creatine oxia in the tissues. For this reason, the
phosphate and adenosine triphosphate. Dur- amount of efaproxiral that can be safely ad-
ingischemia, because of the impairment of the ministered should not overly reduce the frac-
oxidative metabolism, the level of the high- tional saturation of hemoglobin at atmo-
energy phosphates, phosphocreatine and spheric oxygen pressure. A dose of 100 mglkg
adenosine triphosphate, decreases and the was reported to cause a significant decrease of
concentration of inorganic phosphates simul- the arterial oxygen saturation in a limited
increases. The resulting overall number of patients (52). In mice (54) a dose of
ic impairment may cause permanent 300 m a g resulted in a desensitization of
ages in the tissues. The levels of high-en- Fsall fibrosarcoma to radiation therapy, likely
gy phosphates were determined before, dur- because of the poor oxygenation of hemoglo-
g, and after causing myocardial ischemia in bin in the lungs. This side effect could be over-
gs (51). It was shown that efaproxiral re- come by administering supplemental oxygen.
s the decline of high-energy phosphates if In rat models it was shown that the increase in
inistered before ischemia and accelerates oxygen pressure in breathed air can compen-
return to normal values if administered sate for the reduced oxygen affinity (55).
The high oxygen concentration in the tis-
linical data are also available for efaprox- sues may increase the formation of oxygen-
al. It was administered to patients undergo- derived free-radical species, which are in-
general anesthesia and surgery (52). In volved in the pathogenesis of the ischemia-
Oxygen Delivery by Allosteric Effectors of Hemoglobin, Blood Substitutes, and Plasma Expanders

reperhsion injury (56). Oxygen reacts with the therapeutic dose range. A partial hepatic
hydroxyl radicals and is also used by several glucoronidation before renal clearance might
oxide synthases to produce nitric oxide, form- take place.
ing peroxynitrite in the presence of superoxide
radicals. These species may further damage 2.2.3 Aromatic Aldehydes. Monoaldehyde
the cells. The rat acute subdural hematoma, a allosteric effectors affect the oxygen affinity of
model of human head injury, was used to as- hemoglobin in opposite ways. Some of them
sess the effect of efaproxiral on the production (23) induce a left shift of the oxygen binding
of free radicals, the levels of which were al- curve and, therefore, are potentially useful for
ready known to increase during ischemia. It the treatment of sickle-cell anemia. The most
promising compounds are 5-(2-formyl-3-hy-
was demonstrated that the administration of
droxyphenoxy) pentanoic acid, referred to in
efaproxiral does not increase the formation of
the literature as 12C79 (4a), and vanillin (4b).
free radicals (57).
The aldehydic compounds Bformylsalicylic
Given that acute renal failure is believed to acid (5a),2-(benzy1oxy)-5-formylbenzoicacid
arise from hypoxic conditions in the outer me- (5b), and 2-(pheny1ethyloxy)-5-formylbenzoic
dulla, a rat model was used to assess the sup-
posedly beneficial effect of efaproxiral (58).
However, the opposite effect was observed, as-
sociated with an increase of the levels of serum
creatinine and a worsening of the overall con-
a
H~~~~~~~ Q
b CHO

0CH3
ditions. Pretreatment with furosemide, which OH
reduces sodium transport and, consequently,
diminishes the rate of oxygen consumption, (4)
resulted in a less severe dysfunction. This be-
havior, consequent to efaproxiral administra- acid (5c)were designed to increase the affinity
tion, if confirmed for humans affected by renal for oxygen, but, unexpectedly, showed the op-
dysfunction, may represent an important side posite effect (24).
effect of the drug. Other adverse effects, such
a COOH b COOH
as nausea, headache, and neurologic symp-
toms, were reported in clinical trials. o H c e o H o H c G o 4
2.2.2.1.4 Pharmacokinetics. Preliminary
pharmacokinetic data were obtained from
phase I clinical trials on patients undergoing c COOH
radiation therapy (44, 52, 59). The adminis-
tration of a dose of 100 mgkg i.v. resulted in a J = - qo e cHo
variation of approximately 10 Torr of the p50
a t peak plasma concentration of efaproxiral. (5)
This dose is considered to be safe and effective,
even when administered daily for several To identify the structural differences that
weeks. Higher doses, although not toxic, may are responsible for this effect, X-ray diffrac-
not be significantly more effective in raising tion studies and molecular modeling tech-
the tumor oxygenation, as shown for rat mod- niques were used. By solving the structure of
els of fibrosarcoma (54). hemoglobin complexed with different aro-
The plasmatic half-life of efaproxiral ranges matic aldehydes of both functional classes, it
between 3.5 and 5 hand the whole blood p50 is was discovered that they all form a Schiff base
linearly related to the plasmatic concentration with the terminal amino groups of the two
of the drug. In patients treated up to 6 weeks, symmetry-related avall. The N-termini of the
no drug accumulation was detected. Efaprox- p-subunits are not equally affected, although
iral is mostly eliminated by glomerular filtra- partial occupancy was observed for some of
tion with a mechanism that saturates within the complexes. The only exception is vanillin,
2 Allosteric Effectors of Hemoglobin

o sites, one near aCysl04, tested compounds. Therefore, the overall ef-
PGlnl03 in the central cavity fect on the oxygen affinity of hemoglobin un-
the other on the surface near PHis116 and der physiological conditions may depend on
A possible explanation for the different and very small contributions. In the
urprising opposite effects brought about by case of the left-shifting compounds, the higher
tructurally related compounds that bind to affinity is attributed both to the rupture of the
he same residue was proposed by Abraham ionic bond between aVall and cuArgl41and to
4). In deoxyhemoglobin, the inhibition of the binding of 2,3-DPG and
a&gl41 (and a2Vall and chloride ions, the endogenous allosteric effec-
g141) interact through a water molecule. tors. In the case of the right-shifting com-
carboxylate group that characterizes the
pounds, the formation of a strong intersub-
fting aromatic aldehydes may replace
unit interaction prevails and leads to a
water-mediated bond by forming an ionic
ridge with the positively charged guani- stabilization of the T state.
urn group of Argl41 of the other a-sub- Because the side-chain of the monoaldehydes
this bond is stronger than the was observed to point toward Lys99 of the oppo-
d one, the result is an increase site a-subunit, it was foreseen that the addition
the stability of the T state. In the R state, of a terminal group capable of forming a bond
Val1 and Argl4l of the opposite a-subunits with it might result in a further stabilization of
are too far apart to form any direct interac- the T state. Two groups were tested (62),an al-
tion, and do not allow the formation of a bridge dehyde moiety, which could form a Schiff base
between them. This is the reason that, al- with Lys99, and a carboxylic moiety, which
khough the aldehydes bind to aVall both in could form an ionic bond with the same residue
ates, they stabilize only the T in the protonated form. For each class of com-
te. Secondary interactions with other resi- pounds, the distance between the two reading
r specific compounds may jus- centers was modulated by varying the length of
the differences in the strength of these the link that connects the phenyl rings (6). De-
They bind parallel to the pending on the linker between the phenyl rings,
substituents in the para these compoundsare classifled as bis(2-carboxy-
oward Lys99 of the other 4-formy1phenoxy)-alkanes (6a),(2-carboxy-4-
formy1phenoxy)-(4-carboxyphenoxy)-alkanes
The left-shifting aldehydes either do not (6b), and bis(2-carboxy-4-formy1phenoxy)-xy-
acidic group or the group is present lenes (6c). In (6c),the R group can be a meta
not correctly oriented to form an interac-
-CH,(C6H4)CH2-, an ortho --CH2(C,H4)-
th cuArgl41. The compound 12C79, for
CH2-, or a pam -CH2(C6H4)CH2-, +Hz-
, binds hemoglobin parallel to the CH=CHCH,-.
axis, as the right-shifting aldehydes,
he side chain points in the opposite direc- These compounds were shown to bind he-
binding to the a,Vall, these com- moglobin as predicted and the addition of a
ds disrupt the water-mediated ionic bond new bond resulted in a much higher potency
aVall and PArgl41, but they do with respect to the monoaldehydes tested by
replace it with a stronger bridging Abraham and coworkers (24). Among the com-
pounds capable of binding aLys99, the bisal-
ng of both left-shifting and right- dehyde allosteric effectors are stronger than
g aldehydes has been shown to reduce the monoaldehyde bisacids. This is expected,
loride effect, possibly by narrowing the given that the former binds covalently to the
ss to the central water cavity, where chlo- residue. The increase of p50 depends strongly
ions probably bind in a nonspecific way. on the length of the molecule. The shorter the
e effectors interfere with the bridging chain, the higher the shift of p50.
g of the endogenous allosteric effector This is also expected, given that a less flexible
DPG, the effect of which is abolished or chain is likely to produce a greater constraint
nished to a different extent for most of the on the T state.
396 Oxygen Delivery by Allosteric Effectors of Hemoglobin, Blood Substitutes, and Plasma Expande B [

a COOH binds 2,3-DPG, present at equivalent concen- effec


trations, to form a stoichiometric complex. As (69).
O H C ~ O - ( C H 2 ) . - O ~ C H O a result, the oxygen affinity is low (p50 = hem,
26 Tom) and modulated by allosteric effectors. sequ
On the contrary, the concentration of dimeric in t k
hemoglobin molecules increases in dilute he- pro1
moglobin solutions. The dimer binds 2,3-DP(3 fac
very weakly. As a result, free hemoglobin ex:- ma
hibits an increase in oxygen affinity and loseS c a ~
/ cooperative oxygen binding. lar
HOOC Perfluorochemicals are inert, synthetic, en;
C
COOH linear, or cyclic fluorocarbon compounch, acc
/
which act as high-solubility solvent for oxygel
and do not display any cooperative properties
/ The amount of dissolved gas increases linearl;r bii
HOOC with increasing oxygen pressure and the tota1 in,
amount of gas carried in the circulation is pro- PC
portional to the concentration of fluorocar- se
bons in solution. m
3 BLOOD SUBSTITUTES: MODIFIED C1
HEMOGLOBINS AND 3.1 History of Blood Substitutes tl
PERFLUOROCHEMICALS W
3.1 .I Development of Modified Hemoglo- cl
The transfusion with whole blood is still the bins. The problem of finding an "artificial"
most used therapy in emergencies, surgery, substitute to whole blood to be used in trans-
and pathologies involving blood loss and insuf- fusions is not a new aspect in pharmacological
ficient oxygen delivery to organs and tissues. and biochemical research. Since the end of the
The use of whole blood in therapeutics is al- 19th century, some researchers thought that
ways associated with practical and sanitary free hemoglobin in solution could be admi&
problems, such as the need for careful cross- tered as blood substitute to treat severe ane
matching and blood typing, limitations in the mia (63). In 1916 Sellards was the first to ad-
availability from healthy donors, the short minister hemoglobin solutions to humans and
shelf life of whole blood, and the concern about to study their adverse effects (64). This study,
contamination by infectious agents, such as focused on the renal clearance of hemoglobin
hepatitis virus and HIV. The above-men- solutions, was the first to report a serious re
tioned problems have pushed research toward nal toxicity induced by hemoglobin adminis
molecules with biochemical and physical prop- tration. It is only in 1937 that Amberson no
erties as close as possible to those of hemoglo- ticed a hypertensive effect as a consequence a
bin in red blood cells. Up to now two categories intravenous administration of hemoglobi!
of blood substitutes have been developed: (65). At that time, hemoglobin was quite fa
modified hemoglobins and perfluorochemi- from being pure. Potential sources of toxicit
cals. were red cell membrane debris contaminant
Modified hemoglobins are prepared from that induce nephrotoxicity and hemolysis. I
human, bovine, or recombinant hemoglobin, 1970 Rabiner developed a protocol for the p~
by chemical or genetic methods. Modifications rification of hemoglobin from cell membrane
of hemoglobin are needed to stabilize the tet- (stroma-free hemoglobin, SFHb) (66). In th
ramericform and to increase its p50 to im- 1970s Moss and De Venuto tested SFHb o
prove oxygen delivery to tissues. In fact, the animals (67, 68). These preliminary tests di
tetramer-dimer equilibrium of hemoglobin is not reveal any severe toxicity and phase I clir
shifted in red blood cells toward the tet- ical trials were allowed. Clinical trials ind
rameric form because of the high hemoglobin cated that even stroma-free hemoglobin wa
concentration (about 5 mM). The tetramer highly toxic in humans, mainly because of th
3 Blood Substitutes: Modified Hemoglobins and Perfluo~

effect on kidney and cardiovascular system tion, was used to crosslink hemoglobin be-
(69).This study points out that the toxicity of tween P-chains (74) and, then, between
hemoglobin solutions is not exclusively a con- a-chains (75). These studies led to the devel-
sequence of cell membrane impurities present opment of DBBF-Hb, or aa-Hb, by the U.S.
in the preparation but also of the biochemical Army and DCLHb or HemAssist by Baxter.
properties of hemoglobin free in solution. In Baxter and the Army collaborated on the
fact, hemoglobin out of red blood cells is project since 1985, but the negative results of
mainly dimeric and is, thus, filtered by kidney, clinical trials led the U.S. Army, at the begin-
causing nephrotoxicity. Furthermore, acellu- ning of the 1990s,and Baxter, in 1998, to drop
ar hemoglobin shows an increased NO scav- the project. In 1969 the effect of pyridoxal
enging activity, which is the main mechanism phosphate (PLP) binding to hemoglobin was
accounting for the hypertension, frequently reported (76). PLP binds at the 2,3-DPG site,
observed upon free hemoglobin administration. thus increasing the p50 to a value closer to
A project aimed at polymerizing hemoglo- that of hemoglobin in red blood cells. In 1971
in to lower its colloid oncotic pressure and to Chang, for the first time, used glutaraldehyde
crease its molecular weight was first pro- to crosslink hemoglobin and catalase (77).
osed (70) and further developed by other re- Two companies later exploited this procedure,
earch groups (see Section 3.3.4). In 1964 a Northfield Inc. with Polyheme, and Biopure
ethod for crosslinking hemoglobin mole- Corporation with Hemopure. Both products
les to be used as an artificial membrane for are glutaraldehyde polymerized hemoglobins.
he preparation of "artificial red blood cells" The Northfield product is human pyridox-
as developed (70). In this reaction, sebacyl dated hemoglobin, whereas the Biopure prod-
hloride forms an amidic bond with amino uct is bovine hemoglobin. Because of the
groups on the surface of the protein, as shown higher p50 of bovine hemoglobin with respect
in Reaction 1. In an attempt to decrease the to the human protein, the oxygen affinity of
Polyheme is higher than that of Hemopure.
The good rheological properties of polymer-
ized hemoglobin, its high molecular weight,
+ Hb-NH2 and its modest hypertensive effect explain the
0 positive results in clinical trials and the ap-
(8.1) proval of Hemopure for human use in South
0 H
Africa.
N A-,-q(N\ Hb Since the 1970s many studies on hemoglo-
H 0 bin-based blood substitutes also focused on
the preparation of conjugated hemoglobin
ize of the artificial cells, Chang obtained po- aimed at reducing its potential antigenicity
erized hemoglobin, containing both intra- and prolonging its vascular retention. Dex-
intermolecular linkages. The hemoglobin tran (781, polyethylene glycol (79), and
obtained with this procedure is a stable tet- polyoxyethylene (80) are the most used poly-
ramer with an increased molecular weight mers for the modification of hemoglobin
compared to that of unmodified hemoglobin. surface.
In 1968 Bunn was the first to develop an in- The problem of hemoglobin dimerization in
tramolecular crosslinking procedure aimed at solution was later approached by exploiting
stabilizing the tetrameric form of the protein recombinant DNA techniques. In 1984 human
without polymerizing it (71). A bifunctional /3 globin was expressed in Escherichia coli as a
agent, bis(N-maleimidomethyl)ether, that fusion protein with the coding region of bacte-
crosslinked the two p-chains of each dimer, riophage lambda repressor protein (81). This
was used. Afterward, another crosslinking expression system was found to be unsuitable
agent, acetylsalicylic acid, was used to stabi- for the production of both the a-and p-chains.
lize the hemoglobin tetramer (72,73). A deriv- Further improvement of the expression sys-
ative of aspirin, bis(dibromosalicyl)fumarate, tem in bacteria was attained using a fully syn-
which is more reactive in the acylation reac- thetic gene with codons optimized for E. coli
398 Oxygen Delivery by Allosteric Effectors of Hemoglobin, Blood Substitutes, and Plasma Expanders

that led to the overexpression of myoglobin because its efficacy in reducing myocardial
(82). In 1990 at Somatogen, the same ap- ischemia and angina and in maintaining ven-
proach was used to express fully functional tricular function was demonstrated (88). The
human hemoglobin in E. coli (83). long body retention time of one of its compo-
Even though the above-mentioned modi- nents, the adverse effects attributed to the
fied hemoglobins have been improved, leading surfactant, associated with a low fluorocarbon
to better performances and to the marketing content that conferred only low oxygen-carry-
of some of them as blood substitutes for veter- ing capacity, limited the potential range of its
inary use, they are far from exhibiting the applications. Moreover, a limited stability at
same physiological, biochemical, and pharma- room temperature, requiring the product to be
cological behavior of hemoglobin within red shipped and stored in the frozen state and to
blood cells. Microencapsulation of hemoglobin be formulated as two annex solutions that had
(70) and polymerization with catalase and su- to be mixed before use, probably accelerated
peroxide dismutase (77, 84) are advanced ap- the decline of Fluosol. Manufacturing and
proaches to the issue of blood substitutes. marketing of the product were discontinued in
Their development might provide a blood sub- 1994 when improvements in angioplasty tech-
stitute with improved half-life, less toxic ef- nology made it unnecessary for its approved
fects resulting from reperfusion injury (see indication (89). Nevertheless, the approval of
section 3.3.1.4), and low level of methemoglo- Fluosol proved that artificial oxygen carriers
bin formation and NO scavenging. could be used as alternatives to blood transfu-
sion and it represented the reference point for
3.1.2 Perfluorochemicals as an Alternative successive development of second-generation
to Hemoglobin. The first demonstration that PFCs.
perfluorochemicals (PFC) can sustain life was
reported in 1966 when Clark and Gollan (85) 3.2 Clinical Use of Modified Hemoglobins
showed that mice fully immersed in oxygen- and Perfluorochemicals
ated PFC could breathe in the liquid and that
the amount of oxygen dissolved was sufficient The clinical applications of the two categories of
to support the respiratory function. In 1967 blood substitutes are discussed together in this
Sloviter and Kamimoto (86) observed that the section, whereas other topics are presented sep
activity of a rat brain could be maintained for arately, because of deep differences in their
several hours when perfused with emulsified physical and pharmacological characteristics.
perfluorocarbon. Successively, experiments Blood substitutes can be grouped into two
carried out by Geyer demonstrated that, de- main categories on the basis of their potential
spite the replacement oftheir blood with PFCs clinical use:
emulsion to a hematocrit less than 1%, "blood-
less" rats survived and grew without apparent 1. As alternatives to whole blood, oxygen de-
abnormalities (87). Fluorocarbons used in livery to tissues being their main applica-
these experiments exhibited an organ reten- tion. Moreover, blood substitutes could be
tion time too long to be feasible in human ad- used for volume maintenance in surgery
ministration. Other perfluorochemicals were and trauma with blood loss, treatment of
tested to obtain compounds with more favor- ischemia (stroke, sickle-cell crisis) and re-
able excretion rates to be used as blood substi- fractory anemia. Blood substitutes can also
tutes. In the early 1980s studies on perfluoro- be used in organ preservation before
decalin led to Fluosol. Although its ability to transplantation.
deliver oxygen was demonstrated in clinical 2. As a product with different applications
studies on anemic patients, Fluosol did not re- with respect to blood substitute. For exam-
ceive FDA regulatory approval for treatment ple, Optro [recombinant di-alpha human
of large-volume blood loss because of its short hemoglobin (9011and HBOC-201 [glutaral-
intravascular persistence. In 1989 FDA ap- dehyde polymerized hemoglobin (91)l can
proved Fluosol for use in conjunction with per- stimulate erythropoiesis and be potentially
cutaneous transluminal coronary angioplasty useful in the treatment of severe anemia.
3 Blood Substitutes: Modified Hemoglobins and Perfluorochemicals 399

Another application for blood substitutes is perfluorochemicals and, perhaps, recombi-


envisaged in the treatment of cancer, in as- nant hemoglobin might be envisaged as the
sociation with chemotherapy and radio- only blood supplies administrable to pa-
therapy. As already discussed, solid tumors tients who cannot receive donor blood be-
are more susceptible to radiotherapy and cause of religious beliefs.
chemotherapy if the oxygen delivery to the
sick tissues is improved (38). Both modified
hemoglobins (92) and perfluorochemicals
(93) can be potentially useful for this appli-
3.3 Hemoglobin-Based Blood Substitutes on
cation. Clinical trials using hemoglobin
Clinical Trial
linked to PEG (174) were carried out, but
research halted while on phase I clinical In April 2001 Hemopure, a glutaraldehyde-po-
-
trials. One of the main disadvantages of he- lymerized hemoglobin, received the South Af-
moglobin solutions as blood substitutes is rica Medicines Control Council's approval for
the hypertensive effect, which is discussed its use as a blood substitute to treat acute ane-
in Section 3.3.1.3. On the other hand, in mia in adult surgery patients. The same prod-
case of hypovolemia or systemic inflamma- uct is under marketing approval in the United
tory response syndrome, this effect could States and in Europe. In 1998 Oxyglobin [glu-
be beneficial and accelerate recovery (94). taraldehyde polymerized hemoglobin (HBOC-
For this reason, pyridoxalated hemoglobin 301)l was the first blood substitute to be ap-
entered phase I1 clinical trials in the treat- proved for the market by the U.S. Food and
ment of septic shock (95). HemAssist, a Drug Administration and the European Com-
crosslinked tetrameric hemoglobin (961, mission, in the treatment of anemia in dogs.
was administered during clinical trials to The product is now commercially available in
treat hypotension induced by hemodialysis the United States. Other modified hemoglo-
and was found to stabilize the pressure bin-based blood substitutes are on clinical tri-
without significant adverse effects. Fur- als, some of them entering phase 111. Clinical
thermore, modified hemoglobin solutions trials on three hemoglobin-based blood substi-
could be more convenient than whole blood tutes have been halted: (1)HemAssist by Bax-
in emergency cases in which blood typing ter on May 1998, because of increased mortal-
would be an excessively time-consuming ity in the test groups with respect to control
procedure. Perfluorochemicals may be groups in the therapy of trauma (103) and
used for the treatment of acute respiratory stroke (104); (2)Optro by SomatogenIBaxter
failure by liquid ventilation because they in 1998, after Baxter's acquisition of Somato-
are thought to help the reopening of col- gen, which was developing the product; and
lapsed alveoli (97-99). Liquivent from Alli- (3)PEG-Hb by Enzon, duringphase Ib clinical
-
ance Pharmaceutical Co. contains perfluo- trials. In Table 8.2 the complete list of hemo-
rocityl bromide and is currently in phase globin-based blood substitutes is reported.
MI1 clinical trials (100). The low molecular The route of administration is intravenous
weight fluorocarbons, which are gaseous at infusion. Given that the majority of products
body temperature, such as perfluoropen- are not yet available on the market, the ad-
tane, Echogen from Sonus Pharmaceuti- ministered dose and the infusion rate depend
cals, and perfluorohexane, Imavist, for- on the settings during clinical trials. The "max
merly known as Imagent, from Alliance administered dose" refers to the highest dose
Pharmaceutical Co., can be used as con- administered to patients during clinical trials
trast agents for the assessment of heart without causing severe adverse effects. When-
function and detection of perfusion deficits ever in literature the dose was expressed as
by ultrasound imaging (101). Liquid and mglkg the "max administered dose" was cal-
emulsified perfluorochemicals are cur- culated for an average 70-kg subject.
rently being evaluated as oxygen-carrying
culture media supplements for eukaryotic 3.3.1 General Side Effects. Side or adverse
and prokaryotic cells (102). In addition, effects that have been observed upon adminis-
Table 8.2 List of Hemoglobin-Based Blood Substitutes, Including Only the Products That Were in Clinical Trialsa
Clinical Max
Proprietary Nonproprietary Hemoglobin Chemical Trial Administered
Name Name w e Class Originator Phase Dose (g) Reference
o-Raffinose Human Crosslinked Hemosol I
polymerized polymerized I1
crosslinked hemoglobin
hemoglobin
HBOC-201 Bovine Polymerized Biopure I
(glutaraldehyde hemoglobin I1
polymerized I11
hemoglobin)
Poly-SHF-P Human Polymerized Northfield I
(glutaraldehyde
polymerized
pyridoxalated
Q
0
0
hemoglobin)
None PHP Human Conjugated Apex I 7 95
(pyridoxalated hemoglobin Biosciences ID1 7
hemoglobin- I1 - 180
polyoxyethylene
conjugate)

Hemoglobin-based blood substitutes for which clinical trials were halted or suspended
OptroT" rHbl.1 Recombinant Crosslinked BaxterISomatogen I 22.4
hemoglobin I1 50-100
None PEG-Hb (PEG Bovine conjugated Enzon
modified hemoglobin
hemoglobin)
HemAssist'" DCLHb (diaspirin Human Crosslinked Baxter I 1.75-7 145, 146,
crosslinked hemoglobin I1 3.5-75 148
hemoglobin) I11 75
"Other products that may have clinical or biological interest but have never been tested on human subjects are reported in Section 3.3.6.
Blood Substitutes: Modified Hemoglobins and Perfluolrochemicals

-based blood substitutes erin, both sources of NO, are effective in re-
n intensity and clinical importance, de- ducing the hypertension induced by diaspi-
g on the type of product, its molecular rin-crosslinked hemoglobin (106).
, the chemical modification of the mol- It was shown that it is possible to reduce
he viscosity of the solution, and many the rate of reaction of NO with hemoglobin by
other factors. In this section, the mechanisms introducing mutations in the heme pocket,
responsible for some of the most important or thereby obtaining hemoglobins with a de-
frequently observed adverse effects are de- creased vasoactivity (109).However, hemoglo-
scribed mainly from a biochemical and physi- bins with mutations in the heme pocket are
ologic point of view. Specific adverse effects, likely to exhibit altered oxygen affinity and
their relative importance, and their influence their use as blood substitutes is still an open
on the safety profile of the blood substitute are question. If the formation of S-nitrosohemo-
reported in separate sections dedicated to in- globin is confirmed to play a role in the hemo-
dynamic properties of hemoglobin, a new
n. The administration possibility for the design of recombinant mol-
obin induces an increase in ecules with a selective modulation of NO scav-
e mean arterial pressure (MAP), accompa- enging activity will open (110). In the past
ed by a decrease in heart rate, as a conse- much effort was put in the development of
uence of systemic vasoconstriction in sev- polymeric hemoglobins that could not extrav-
ral vascular beds (105-107 and references asate and that could hopefully give milder hy-
ority of physiological and pertensive effects. Some of these products
thological scenarios this is considered an demonstrated to be effective in reducing he-
verse effect because it reduces tissue per- moglobin-induced hypertension. However, no
sion, a deleterious event in subjects suffer- definitive conclusions can be drawn about the
ing from blood loss or hemodilution. The relationship existing among extravasation,
cause of hemoglobin-induced vasoconstric- molecular weight, and hypertensive effect (see
tion is still an open question in blood substi- Ref. 111 and references therein for an exhaus-
tute research and remains one of the most tive critical review on blood substitutes issue).
serious drawbacks to the use of hemoglobin Recently, many investigators pointed out
solutions as blood substitutes. Currently, that hemoglobin molecular weight might have
the best-characterized and widely accepted a marginal role in determining hypertensive
mechanism of vasoconstriction induced by effects, at least for two reasons. First, hemo-
acellular hemoglobin is nitric oxide (NO) globin free in solution is not subjected to the
scavenging. NO is a naturally occurring mol- hydrodynamic separation exerted by blood
ecule, released by endothelial cells both in flow, which accounts for the formation of a
the interstitial space and in the lumen. In "red blood cells free zone" in blood vessels un-
the interstitial space NO activates guanylate der flow conditions both in vivo and in vitro
eyclase on the smooth muscle cells, causing (11).As a result hemoglobin free in solution
vasorelaxation (Fig. 8.5A). Given that the can have a scavenging potential several times
affinity of hemoglobin for NO is about higher than that of hemoglobin inside red
200,000-fold higher than that for oxygen, blood cells. Furthermore, a study carried out
acellular hemoglobin can efficiently scav- on rHbl.1 (recombinant di-alpha human he-
enge plasmatic NO (see also section moglobin) and its glutaraldehyde-modified
3.3.3.1.4). Furthermore, both dimeric and forms has questioned the understanding of
tetrameric hemoglobin can extravasate by the exact mechanism of hemoglobin extrava-
passing through the interendothelial gap sation (112). In fact, polymerization with glu-
) (log), thus binding NO taraldehyde is likely to prevent extravasation
of action, causing vaso- not because of the increase in the molecular
rtension (Fig. 8.50. weight of hemoglobin, but as a consequence of
cyanornethemoglobin, a decrease in the endocytotic transport of the
ich does not bind NO, does not induce protein attributed to glutaraldehyde decora-
soconstriction. L-Arginine and nitroglyc- tion. This finding opens new insight in the de-
Oxygen Delivery by Allosteric Effectors of Hemoglobin, Blood Substitutes, and Plasma Expanders

(4

.".---.,..-.-<..*--2-.?"
cell

Hb(FeH) ------+ Hb(Fe*-NO)


(Nitrosylhemoglobin)

Figure 8.5. Action of NO on blood vessels and NO scavenging by hemoglobin. (A) Mechanism of
NO-dependent smooth muscle cells relaxation. NO (@I is produced by endothelial cells and released
both into the interstitial space and the lumen of the vessel. In the interstitial space of smooth muscle
cells NO activates soluble guanylate cyclase and causes vasorelaxation. (B)Schematic representation
of a blood vessel. Hemoglobin inside the red blood cells cannot extravasate (a). Dimeric and tet-
rameric acellular hemoglobin (b, c) extravasates, passing either through the endothelial intercellular
junctions or intracellularly by endocytotic mechanisms. (d) Both cellular and acellular hemoglobin
bind NO. Scavenging of NO in the interstitial space results in vasoconstriction. (C) Reaction of
deoxygenated and oxygenated hemoglobin with NO. NO binds to deoxyHb, preferentially forming
nitrosylHb, and to oxyHb, preferentially forming S-nitrosoHb. In addition, NO oxidizes oxyHb to
produce rnetHb and nitrate.
3 Blood Substitutes: Modified Hemoglobins and Perfluora

sign of hemoglobin-based blood substitutes Usually, blood substitutes were formulated


and refocuses the role of the polymerization in as low viscosity solutions, based on the as-
the prevention of vasoconstriction. Further- sumption that high viscosity can reduce blood
more, some immunohistochemical studies on flow through the capillary beds and lead to a
abdominal aorta of rats show that Dex- decrease in cardiac output. Blood viscosity is
BTC-Hb (dextran lo-benzene-tetracarboxy- responsible for the shear forces that are ex-
late hemoglobin) is found in the endothelial erted on capillary walls. The lower the viscos-
cells within 30 min after the administration. ity, the lower the shear force exerted. The ef-
Dex-BTC-Hb is a high molecular weight poly- fect of shear forces is likely to be transduced
into chemical mediators of the vascular tone,
mer formed by stable tetrameric hemoglobin
as, for example, NO and prostacyclins (118).
molecules that cannot extravasate (113).
When shear forces are too weak, the produc-
Probably, the vasopressor effect of hemoglo- tion of NO decreases with induction of vaso-
bin is mediated by other mechanisms, includ- constriction. This finding is supported by the
ing an increase in endothelin-I production demonstration that the administration of low
(106) and a direct effect of hemoglobin on viscosity hemoglobin solutions is correlated
smooth muscle cells. In fact, it was reported with low tissue oxygenation (119).
that hemoglobin can enhance the responses of 3.3.1.2 Nephrotoxicity. Renal toxicity was
smooth muscle cells to noradrenaline (107) one of the first adverse effects observed after
and yohimbin can reduce the hypertension fol- infusion of unmodified hemoglobin solutions
lowing diaspirin crosslinked hemoglobin ad- (64). Even when a higher degree of purifica-
ministration (114). In rats the effect mediated tion of hemoglobin was attained, the adminis-
by adrenoreceptors is not under central ner- tration of unmodified hemoglobin was fol-
vous system control, nor it is mediated by the lowed by nephrotoxicity. In 1991 among
release of catecholamines from the adrenal various recommendations for toxicology tests
of blood substitutes, the importance of careful
More recently, studies aimed at unveiling monitoring of nephrotoxicity through both se-
between blood substitutes ad- rum creatinine levels tests and evaluation of
hypertension have focused renal function was stressed (120).
ly underestimated factors: ox- Following hemolysis or destruction of aged
n the precapillary arterioles red blood cells, hemoglobin released in the
d solution viscosity (for a review see Ref. plasma is picked up by haptoglobin, a carrier
15). In the past, research on blood substi- protein that can bind as much as 150 mg/dL of
tes led to the production of high p50-modi- hemoglobin. If free hemoglobin in solution ex-
ed hemoglobins with the purpose of enhanc- ceeds this binding capacity, it dissociates into
ry to the tissues and thereby a$ dimers, and is filtered in the glomerules
accelerating the recovery. Works at the begin- and partially reabsorbed by proximal tubules.
ning of the 1980s (116) demonstrated that Following renal filtration, two possible mech-
vasoconstrictionand vasorelaxation of precap- anisms of toxicity can be envisaged: precipita-
illary arterioles depend on oxygen concentra- tion of hemoglobin in the kidneys and de-
tion. Vasorelaxation occurs at low local oxygen crease in renal function attributed to local
concentrations, whereas high local oxygen vasoconstriction induced by NO scavenging.
concentrations lead to vasoconstriction. The Recently, it has been reported that persistent
control of blood flow by oxygen demand repre- renal damage occurred in rats treated with
sents a strategy to protect tissues from oxida- high (>0.96 g) doses of stroma-free hemoglo-
tive damage and to ensure, at the same time, a bin (121). The cause of renal failure was
physiological oxygen supply. In this light, low mainly necrosis accompanied by a prolifera-
afbity hemoglobin administration is not al- tion of smooth endoplasmatic reticulum in the
ways correlated with enhanced perfusion. proximal tubules.
Conversely, lower p50 and lower hemoglobin It is worth mentioning that a recent study
concentrations might lead to better blood sub- on the interactions of ultrapure hemoglobin
with renal epithelial cells (122) demonstrated
404 Oxygen Delivery by Allosteric Effectors of Hemoglobin, Blood Substitutes, and Plasma Expanders

that the severe renal toxicity observed in early blood substitutes, although it is not well estab-
reports on hemoglobin solutions administra- lished whether a relationship exists between
tion was mainly attributable to the presence of low NO binding and reduced gastrointestinal
red cell membranes debris and endotoxin con- effects.
taminants, and that administration of highly 3.3.1.4 Hemoglobin Oxidation: Oxidative
purified hemoglobin solutions causes toxicity Toxicity and Reperfusion Injury. Hemoglobin
only at high doses. Progress in blood substi- is naturally susceptible to autooxidation, a re-
tutes research has led to the production of sta- action that leads to the formation of methemo-
ble tetrameric hemoglobins and highly poly- globin and superoxide radicals, as shown in
merized hemoglobins with only small renal Reaction 2. Methemoglobin does not bind ox-
toxic effects.
3.3.1.3 Gastrointestinal Effects. Hemoglo-
bin-based blood substitutes administration is
often accompanied by nausea, vomiting, or
ygen. Furthermore, oxidized hemes have a
other gastrointestinal (GI) side effects related
lower affinity for hemoglobin and, after being
to GI dysmotility.
released into circulation, they can trigger cy-
Gastrointestinal motility depends on blood
totoxic reactions (16) and contribute to the
perfusion and is regulated by a complex inter-
formation of free radicals (131). Hemoglobin is
play of transmitters and mediators acting on
also endowed with pseudoenzymatic activi-
nerve fibers. NO is a nonadrenergic, noncho-
ties, such as peroxidase activity. The reaction
linergic inhibitory transmitter (123) that con-
of methemoglobin with hydrogen peroxide
trols the peristaltic activity and the sphinc-
leads to the formation of ferryl-hemoglobin, a
ters' tonus. Depletion of NO can reduce the
higher oxidation state of hemoglobin (Reac-
rate of gastric emptying (124) and attenuate
tion 3). This species, because of its high redox
sphincter relaxation (125).On the other hand,
a reduced perfusion can alter the electric and
contractile activities of gastrointestinal mus-
cle cells (126,127). As stated in Section 3.3.1.1,
many hemoglobin-based blood substitutes act
as NO scavengers, leading to smooth muscle
cell contraction. Hemoglobin administration potential, can in turn react with different sub-
can lead to reduced gastrointestinal motility strates, causing free-radical formation and ox-
in two ways: through NO scavenging on the idative damage (16), particularly lipid peroxi-
smooth muscle cells of the GI tract and, indi- dation, protein crosslinking, and degradation
rectly, through vasoconstriction of the vessels of carbohydrates (see Ref. 132 and references
perfusing the GI apparatus. Although it is well therein). In the red blood cells, enzymes such
established that hemoglobin-induced gastro- as methemoglobin reductase, catalase, and su-
intestinal dysmotility is mainly caused by NO peroxide dismutase reduce methemoglobin
scavenging (128, 129), it is not clear whether back to its active form, thus eliminating au-
some unknown effects of hemoglobin on dif- tooxidation products, such as superoxide rad-
ferent regulatory mechanisms of the gastric icals and hydrogen peroxide. The absence of
motility are also involved. Furthermore, it was these enzymatic mechanisms in the plasma
found that both NO donors and NO synthesis compromises both the efficacy of hemoglobin
inhibitors can inhibit gastric motility, likely as oxygen carrier and its safety. This situation
because of the action of NO on different mod- is even worse when hypoxic tissues are reper-
ulators of GI activity (130).Some hemoglobins fused. In fact, hypoxia activates xanthine
are reported to have negligible effects on GI oxidase and leads to the accumulation of hypo-
motility, as, for example, PEG hemoglobin xanthine. In the presence of oxygen, hypoxan-
(128) and rHb3011, a new generation recom- thine is oxidized to xanthine with the release
binant hemoglobin (129). Both hemoglobins of superoxide (Reaction 4). Superoxide and ox-
are known to have a lower affinity for NO com- ygen radicals generated in the reaction with
pared with that of other hemoglobin-based hemoglobin are potentially toxic for cells and
3 Blood Substitutes: Modified Hemoglobins and Perfluor

the redox potential of modified hemoglobin is


sufficiently high to allow electron transfer.
The redox potentials of modified hemoglobins
vary greatly, depending on the type and de-
gree of modification. Hemoglobin crosslinked
with superoxide dismutase and catalase has
been proposed as a feasible way to reduce rad-
icals and superoxide production and to de-
crease the autooxidation rate of hemoglobin
(see Ref. 137 and references therein). A new
hemoglobin, crosslinked with o-ATP and
o-adenosine and conjugated with glutathione,
seems to exhibit a significantly reduced
prooxidant potential, and anti-inflammatory
properties (138).
SynZyme Technologies LLC (La Jolla, CA,
USA) is developing a new hemoglobin-based
blood substitute, HemoZyrne, endowed with
superoxide dismutase-like activity that might
be useful in preventing and treating reperfu-
sion injury. HemoZyme is a polynitroxylated
hemoglobin that also possesses anti-inflam-
matory and vasodilator activities. The pro-
posed clinical use of HemoZyme is in ischemia
tissues (133). Damages to tissues and organs and in hemorrhage to prevent reperfusion in-
following reperfusion are referred to as reper- jury and postreperfusion multiple organ fail-
ure (139, 140).
Only few studies (134) have been carried 3.3.1.5 Antigenicity. Because hemoglobin
out on the oxidative processes taking place is considered only a weak antigen (1331, it is
upon the administration of blood substitutes unlikely that a single administration of hemo-
and on their consequences on the safety profile globin can elicit an immune response. How-
of hemoglobin-based products. It has been re- ever, the administration of heterologous he-
rted that crosslinked hemoglobins show an moglobin (e.g., bovine hemoglobin in humans)
or repeated administrations, as would be the
case in transfusion medicine, might poten-
anged or increased peroxidative activity tially activate an undesired immune response.
6). The increased concentration of pancre- It was proved that the infusion in rats of au-
ic and liver enzymes, frequently observed tologous hemoglobin, both stroma-free and
polymerized, never results in high levels of an-
tibody production or in anaphylactic reac-
tions, whereas infusion of polymerized heter-
er investigations are required to assess the ologous hemoglobin in immunized rats
produces anaphylactic reactions (133). The
problem was further addressed by studying
ed blood substitutes (136). The redox po- the immune response to repeated administra-
ntial of hemoglobin-based blood substitutes tions of polymerized hemoglobin in hyperim-
munized rabbits (141). These results show
eir safety profile. In fact, reducing agents, that the immune system is activated upon re-
ch as reduced glutathione, are present in peated administrations of hemoglobin, but the
e plasma, and they can be effective in reduc- immune response might not be stimulated fol-
g the oxidized states of hemoglobin only if lowing a few transfusions. Repeated infusions
406 Oxygen Delivery by Allosteric Effectors of Hemoglobin, Blood Substitutes, and Plasma Expanders

of human polymerized hemoglobin in rhesus tion in the presence of tripolyphosphate leads


monkeys further confirmed the absence of se- to the stabilization of the T form of hemoglo-
vere immune response or hypersensitivity bin, thus increasing its p50 to a value of about
(142). 30 Torr. The result of the reaction is a stabi-
Polymerization seems to enhance hemoglo- lized hemoglobin tetramer, crosslinked be-
bin immunogenicity, whereas modifications tween Lys99 residues of the a-chains. This
with polyethylene glycol (PEG) or encapsula- modified hemoglobin can be heat sterilized,
tion are likely to mask the protein from the through a patented treatment (144),to inacti-
immune system (143). vate viruses and to separate crosslinked mole-
cules from noncrosslinked dimers. The latter
3.3.2 Crosslinked Hemoglobins are less stable and denature upon heat treat-
3.3.2.1 HemAssist (DCLHb). HernAssist is ment. DCLHb is one of the most widely stud-
the commercial name for a crosslinked deriv- ied hemoglobin-based blood substitutes. Clin-
ative of human hemoglobin developed by Bax- ical trials on this product stopped in 1998 (103,
ter between 1985 and 1998, and in collabora- 104) because of the higher mortality observed
tion with the Letterman Army Institute of in the treated groups compared to that of the
Research for a few years. DCLHb (Diaspirin control groups.
Crosslinked Hemoglobin) is the generic name 3.3.2.1.1 Clinical Use of DCLHb. He&-
of the product. It is formulated in a balanced sist was tested with a great variety of clinical
physiological electrolyte solution at a concen- settings during clinical trials, spanning from
tration of 10 g/dL (144). The mean oncotic stroke to surgery, hypovolemic and hemor-
pressure of the formulation is 43 mmHg. The rhagic shock, and hemodyalisis (144).
p50 of DCLHb ranges from 32 to 33 Torr 3.3.2.1.1.1 Adverse Effects. The most fre-
(145). HemAssist is stable for 1 year at -20°C quently observed adverse effects following
and for 1 month at 4°C. DCLHb administration were mild abdominal
The crosslinking agent, bis(3,5-dibromo- pain and increased values in MAP (145, 146).
salicyl)fumarate, is a derivative of aspirin. These are reported to be around 15-20 mmHg
This molecule (7) was -proved to be one of the and mainly affect the diastolic pressure (145).
most effective acylating agents of hemoglobin The increase in MAP is usually dose depen-
(74). dent (961, frequently accompanied by a de-
crease in heart rate or cardiac output, and is
transient in nature (147). In some studies, the
increase in MAP was reported only after the
first infusion and no further increases in sys-
temic and pulmonary arterial pressures were
noticed upon subsequent administrations
(148). In rats the increase in MAP is not dose
dependent at doses higher than 62.5 mg/kg
(149), thus suggesting a saturation mecha-
nism responsible for the hypertension. Stud-
The product obtained from the reaction of ies on abdominal aortic aneurysm repair and
bis(3,5-dibromosalicy1)fumarate with hemo- cardiac surgeries reported a higher systemic
globin depends on the oxygenation state of vascular resistance (SVR) in treated subjects
the protein: oxyhemoglobin is crosslinked be- than that in controls (147, 148). In a phase I1
tween the p-subunits, whereas deoxyhemoglo- study of hemodyalisis patients an improved
bin in the presence of 2,3-DPG, IHP, or other hemodynamic stability without evidence of re-
2,3-DPG analogs is crosslinked between the nal impairment was associated with HemAs-
a-subunits (75). sist administration (96).
HemAssist is produced from outdated hu- Levels of lactate dehydrogenase, creatine
man blood. It is deoxygenated and crosslinked kinase and its myocardial isoenzyme, and liver-
in the presence of a 2,3-DPG analog, tri- functionality markers vary depending on the
polyphosphate (144). The crosslinking reac- experimental setting, but, even in the cases in
3 Blood Substitutes: Modified Hemoglobins and Perflu01

which an increase was reported, it was not with an increased oxygen delivery and perfu-
nsidered a safety concern. sion of ischemic tissues. Preclinical studies on
In phase I1 clinical trials of orthopedic and rats showed that the increase in MAP is fol-
dominal surgery significantly higher levels lowed by an enhanced perfusion of vital or-
globin (metHb) in the treated gans (153,154).
oup were observed, without affecting pa- Administration of DCLHb is followed both
ients' outcome. The whole blood methemo- by a reduction of the cardiac output to the
els were found to be higher than skeletal muscles and an increase of cardiac
a methemoglobin, suggesting a output to the gastrointestinal system. This ef-
-induced conversion of patients' own fect is likely attributable to a redistribution of
o metHb (150). Other reported the blood flow following vasoconstriction (153,
were the increase in amylase 154). The oxygen delivery in the presence of
evels without any signs of pancreatitis, hemo- HemAssist is improved with respect to that of
globinuria, hematuria, hyperbilirubinemia, the whole blood because of both the increased
and abnormal hepatic function. oxygen diffusion from the cell-free hemoglo-
3.3.2.1.1.2 Pharmacokinetics. Data from bin and the lower viscosity of the solution
phase I clinical trials (145)show a dose-related (144).
plasma concentration of HemAssist with a The interpretation of preclinical and clini-
peakat about 30 min after the administration. cal studies seems to be quite controversial on
Studies on rats (151) demonstrated that both the perfusion and oxygen-delivery prop-
olized in the circulation erties of HemAssist or DCLHb. Some authors
through urine and feces as low suggested that the low viscosity of the formu-
lation could result in peripheral vasoconstric-
The t,,, for elimination is dose dependent tion and reduced perfusion to hypoxic tissues
in human subjects (145) and ranged from 2.5 h (110).
fora 25-50 m&g dose to 3.3 h for the highest 3.3.2.1.3 Structure-Function Relationships.
e of 100 m&g. According to Bis(3,5-dibromosalicyl)fumarate reacts with
clinical studies, the elimination route of Lys residues in the hemoglobin 2,3-DPG bind-
LHb is species specific and can be described ing pocket to form a stabilized tetramer. He-
a two-compartment model. Preclinical moglobin can be crosslinked through either its
dies showed that DCLHb has a widespread p- or a-subunits, depending on crosslinking
ue distribution, a consequence of extrava- conditions. Oxyhemoglobin is preferentially
tion of the protein from the bloodstream crosslinked between P,Lys82 and p2Lys82
(74), whereas deoxyhemoglobin in the pres-
Pharmacokinetics studied on patients un- ence of organic phosphates is preferentially
rgoing elective orthopedic and abdominal crosslinked between alLys99 and a2Lys99
surgery (152), treated with an average dose of (75) (Fig. 8.6). In oxyhemoglobin, the two
936 mglkg, showed that the elimination func- Lys99 are located in a cluster of polar residues
clearance was that is inaccessible to the crosslinker. As a
the harmonic mean half-life consequence, the crosslinking takes place pri-
10.5 h, and the volume of distribution was marily at Lys82 residues in the p-chains. Upon
ndings seem to be quite dif- deoxygenation, the site becomes accessible
rent from those on healthy volunteers, likely and the reaction between Lys99 residues in
d loss and dilution effects. the a-chains takes place. The formation of
3.3.2.1.2 Pharmacology. HemAssist is re- p-crosslinked hemoglobin represents the ma-
rted to exhibit good oxygen-delivery proper- jor side reaction of deoxyhemoglobin cross-
better perfusion of some linking (75), which is diminished in the pres-
tissues as a consequence of blood ence of 2,3-DPG or IHP.
edistribution (144). Generally, the hy- The specificity of the crosslinking reactions
rtension generated by the administration of is very high (74) and this is surprising, given
emoglobin-based blood substitutes is consid- the presence of several Lys residues on the
erse effect if it is not associated surface of hemoglobin, which, for example, re-
~-

408 Oxygen Delivery by Allosteric Effectors of Hemoglobin, Blood Substitutes, and Plasma Expanders

upon oxygenation). The cooperativity is con-


served (n= 2.6 compared with a value of 2.9 in
HbA) and the p50 increases from 6.6 to
13.9 Torr, attributed to a decrease in KR and
& and an increase in L (75).All these changes
are modest, demonstrating that the perturba-
tion produced by the crosslinking agent on the
structure of hemoglobin is small. The main
property that is affected by the crosslinker is
the alkaline Bohr effect, which significantly
decreases by about 50%. Investigators sug-
gested that this is likely caused by a glutamic
residue, PGlulOl, that becomes an acidic Bohr
group (75).

3.3.3 Recombinant Hemoglobins


3.3.3.1 Optro (rHbl.1). Optro is the com-
Figure 8.6. Three-dimensional structure of deoxy- mon name that designates a-fused recombi-
hemoglobin (PDB code 2HHB, human deoxyhemo- nant Presbyterian hemoglobin (rHbl.1).
globin). aLys99 residues are shown in black space- Optro is the only recombinant hemoglobin
filling mode. PLys82 residues are shown in gray that entered clinical trials, although experi-
space-filling mode.
mentation ended in 1998 when Baxter ac-
quired Somatogen. At the same time, a new
act with other molecules (e.g., glutaraldehyde) research line opened, focusing on the produc-
to give polymeric hemoglobins. It is known tion of recombinant hemoglobin with a re-
that diesters are more effective acylating re- duced rate of reaction with NO. Because clin-
agents of hemoglobin than their monoester ical trials on this new product have not yet
counterparts and that the bromine substitu- begun, this section deals only with Optro,
ents further increase their efficacy. This is at- whereas information about the potential ap-
tributed to the strengthening of apolar inter- plications of low NO binding recombinant he-
actions by benzene rings and bromine atoms. moglobins is presented in Section 3.3.3.1.4.
Furthermore, the two negative charges of car- rHbl.1 was cloned at Somatogen in collab-
boxylic groups may increase the affinity of oration with the Laboratory of Molecular Bi-
bis(3,5-dibromosalicyl)fumarate for the posi- ology in Cambridge (156). The molecule has
tively charged 2,3-DPG binding pocket (74). two main features: a low oxygen affinity in the
Crosslinking between P-subunits has a pos- absence of 2.3-DPG and a stable tetrameric
itive effect in preventing the formation of he- structure. The reduced oxygen affinity is
moglobin S fibers. In fact, the crosslinking mainly attributed to the pAsnl08 to Lys mu-
causes a displacement of helix F and EF corner tation. The hemoglobin carrying this muta-
with consequent burial of two hydrophobic tion is commonly indicated as Presbyterian
residues, Phe85 and Leu88, in the P-subunits and it is a naturally occurring form, first re-
that are involved in the interaction with Val6 ported in 1978 (157). The stable tetrarneric
in hemoglobin S. As a result, the two residues structure is obtained by the genetic fusion of
are less susceptible to interactions with Val6 the two a-chains with a glycine residue. More
(155). The quaternary structure is not per- details about cloning and structure-function
turbed by the crosslinker and hemoglobin co- relationships are provided in Section 3.3.3.1.3.
operativity is preserved. The oxygen affinity is Optro is formulated at a concentration of
only marginally increased as a consequence of 5 g/dL. The resulting solution has an oncotic
the stabilization of the R form of the protein. pressure of 12 mmHg and exhibits a p50 of
Crosslinking between the a-subunits does 33 Torr (158).
not prevent the T to R transition (the distance 3.3.3.1.1 Clinical Use of rHb7.7. The pro-
between Lys99 residues increases by only 1 A posed applications of Optro are elective sur-
3 Blood Substitutes: Modified Hemoglobins and Perfluorocht

gery and stimulation of erythropoiesis. It was and decreased perfusion of liver, spleen, and
demonstrated that bone marrow depression kidney (161). Other reported adverse effects,
following administration of AZT to normal both during phase I and phase I1clinical trials,
and to murine model of AIDS mice is allevi- are increased amylase and lipase levels with-
ated by the administration of rHb1.1 alone or out signs of pancreatitis (158, 162), and fever
in combination with erythropoietin (159). (158). The former effect is likely another con-
Furthermore, it was proposed that rHbl.1- sequence of NO scavenging with inhibition of
induced renal vasoconstriction leads to the the relaxation of the sphincter of Oddi. In fact,
production of erythropoietin (go), thereby it was reported that, in opossum, rHbl.1 can
stimulating erythropoiesis, and that the ad- alter sphincter of Oddi motor activity by bind-
ministration of rHbl.1 together with eryth- ing NO (163). Fever is likely the consequence
ropoietin can increase the production of red of a contamination of the hemoglobin solution
od cells (133). with bacterial endotoxin. In fact, hemoglobin
3.3.3.1.1.1 Adverse Effects. Gastrointesti- purified from bacterial cells lysate is never
nal discomfort is an adverse effect frequently 100% pure and, sometimes, is contaminated
found upon administration of many hemoglo- by endotoxin, often below the detection limit
bin-based blood substitutes and was reported of the commonly used limulus amebocyte as-
for the first time during phase I clinical trials say. Because high doses of rHbl.1 may be nec-
with Optro. For this reason, a phase I clinical essary for this blood substitute to be effective,
study was designed to assess the role of aber- endotoxin contamination has to be carefully
rant regulation of the motor function of the evaluated.
esophagus in the GI symptoms reported for 3.3.3.1.1.2 Pharmacokinetics. Pharmaco-
rHbl.l(l60). rHbl.1 was reported to alter the kinetic parameters of rHbl.1 were measured
esophageal motor function in humans, inter- during a phase I clinical trial designed to as-
fering with swallow-induced relaxation of the sess the renal toxicity of the product (158).
low esophageal sphincter, determining an ab- rHbl.1 was administered intravenously over
normal retrograde peristalsis and increasing 0.8-1.9 h and the doses administered varied
the velocity of the normal esophageal peristal- from about 1 to 25 g. Because the largest dose
sis. These effects were not accompanied by any administered in this setting is far lower than
signs of vasoconstriction or hypertension. The that administered in a surgical or emergency
authors suggested that the adverse GI effects setting, pharmacokinetics is expected to vary
induced by rHbl.1 administration were a con- to some extent with dose scalingup. For a 25-g
sequence of an alteration in the fine-tuning of dose of rHbl.1 the plasma concentration is
excitatory and inhibitory neurotransmitters about 6 mg/mL and the typical volume of dis-
of the enteric nervous system. It was proposed tribution is 53 mL/kg. In rats rHbl.1 metabo-
at rHbl.1, like many other modified hemo- lism takes place in the liver, as for HbA.
obins, is an NO scavenger. Its specific action rHbl.1 elimination is reported to be a satura-
the GI system without any changes in he- ble process and the half-life is dependent on
modynamics might be explained by the prefer- plasma concentration ranging from 2.4 h for a
ntial access of rHbl.1 to the GI neuromuscu- 0.5 mgImL dose to 12 h for a 5 mg/mL dose.
ar apparatus through the vascular system Clearance is well described by a one-compart-
rfusing the myenteric ganglia. ment model. The largest amount of rHbl.1
Gastrointestinal side effects were also re- found in the kidney is about 0.04% of the total
rted in another phase I study on 48 healthy administered dose, given that a-a fusion pre-
volunteers. In this case, an increase in MAP, vents dimerization and renal filtration.
ting 2 h after rHbl.1 infusion, was ob- 3.3.3.1.2 Physiology and Pharmacology.
wed. The increase in MAP was typically as- Blood substitutes are generally administered
ciated with a decrease in heart rate (158). In to replace blood lost during surgery or trau-
eclinical studies in rats it was observed that matic events. Hemorrhage is often accompa-
increase in MAP and systemic vascular re- nied by vasoconstriction and reduced blood
stance was associated with a redistribution flow through capillaries. One of the main goals
blood flow with enhanced perfusion of heart of a good blood substitute is to increase perfu-
410 Oxygen Delivery by Allosteric Effectors of Hemoglobin, Blood Substitutes, and Plasma Expanders

Table 8.3 Functional Data of Hb&, Recombinant Hbl.1, and the Natural Mutant
Hb Presbyteriana
Hemoglobin Form P,, (Tom) Bohr Coefficient Hill Coefficient (n,,)

"Data were obtained in a solution containing 50 mM Hepes buffer, 100 mM NaC1, pH 7.4, at 25°C (156).
*Data refer to the mutant Vall to Met in both a and fi chains.

sion and oxygen delivery to vital organs. Oxy- ygen affinity of pAsnl08Gly mutant in solu-
genation of tissues is a delicate balance be- tion and the unmodified affinity - of the T state
tween cardiac output, plasma volume, of this mutant in the crystal suggest a desta-
peripheral vascular tone, hemoglobin concen- bilization of the oxygenated state relative to
tration, oxygen unload, and plasma viscosity. the deoxy state as the origin of the reduced
31P-NMRstudies on rats demonstrate directly oxygen affinity (166).
in vivo that rHbl.1 is capable of sustaining The role of the glycine bridge in the equi-
metabolic function in the tissues following to- librium between unbound and ligated forms is
tal blood replacement (164). not fully understood. The glycine bridge can
3.3.3.7.3 Structure-Function Relationships. be quite easily accommodated in the deoxy
rHbl.1 was expressed in E. coli using a con- form of the protein, but, upon oxygenation,
struct under the control of a single TAC pro- the distance between the two a-chains [-3.3 A
moter. The construct contained a single (167)l and the torsion angles of glycine are
operon encoding for two a-chains and one incompatible with the transition from T to R.
p-chain. The two a-chains are linked by a sin- In fact, the presence of the glycine forces the
gle codon for a glycine residue, whereas the protein to adopt a new ligated quaternary
P-chains contained the mutation AsnlO8 to structure, the B form (167). This form has a
Lys that is naturally found in Presbyterian tertiary structure that is almost superimpos-
hemoglobin (156). Furthermore, both a- and able with tertiary structures of other ligated
P-chains contained the substitution Vall to hemoglobins (i.e., R, Y, and R2), whereas the
Met to improve the expression of the protein quaternary structure has a different organiza-
in the bacterial host. The crystal structure tion. The net effect of the genetic crosslinking
confirmed the presence of the Gly bridge in the is still not fully understood. In fact, the func-
deoxy form of the molecule (156). A more re- tional data are compatible with an increase in
cent work on deoxy rHbl.1 at higher resolu- the oxygen affinity, whereas the structural
tion showed that crosslinking does not perturb data suggest that the glycine bridge stabilizes
either the quaternary or tertiary structures of the T state or eventually the ligand-bound B
rHbl.1 (165). A preliminary characterization state (167). Finally, the presence of N-termi-
of the functional properties of rHbl.1 was in nal methionines and the glycine bridge, which
agreement with the structural findings. Even stabilize the protonated deoxy form of the pro-
though the Gly bridge slightly constrains both tein, is responsible for the decrease in the
the C- and the N-terminal of the a-chains, the Bohr effect (167).
cooperativity of hemoglobin is almost fully re- 3.3.3.1.4 Recent Developments. After the
tained (Table 8.3), whereas oxygen affinity is suspension of clinical trials on rHbl.1 in 1998,
drastically reduced (Table 8.3), likely attribut- Baxter focused on recombinant hemoglobins
able to the presence of Am108 to Lys muta- with a reduced reaction rate with NO. Usu-
tion. This residue is at the alpl interface and, ally, the rate of reaction of NO with hemoglo-
hence, only marginally involved in the confor- bin is reported for the deoxy state. The reac-
mational changes associated with the T to R tion rate constant is about 107 M-1 s-1 (951,
transition. The substitution of Am108 with supporting the idea that hemoglobin could act
Lys might lead to the formation of a hydrogen as an excellent NO scavenger in vivo. In pre-
bond between Lysl08 and T~I-35. The low ox- capillary arterioles, hemoglobin is mainly in
3 Blood Substitutes: Modified Hemoglobins and Perfluorochernicals 411

Table 8.4 Pharmacological and Biochemical Data of Recombinant Hemoglobins with Low
NO-Induced Oxidation Rate Constantsa
Pressure Percentage Emptying k ' (NO-Induced Oxidation
c
, Hemoglobin Increase (%Ib of t h e Stomach P5, (mmHgId of Hb)' ( j ~ i k - ~ l s - ~ )

-
"Data for rHbl.1 are reported for comparison.
bPercentage
- of the mean arterial pressure increase following- administration ;determines mean increase
of 25.8mmHg and has the highest NO-induced oxidation rate.
'Data refer to a 750 mgkg dose of hemoglobin. The negative control was an increasing dose of human serum albumin
(HSA);750 mgkg HSA administration was associated with a gastric emptying of about 60%.
dDatawere obtained in a solution containing 50 mM Hepes buffer, 100 mM NaCl, pH 7.4 at 37°C.
'Data were obtained in a solution containing 100 m M sodium phosphate, pH 7.4 at 20°C.
&f. 109.

the R state and, therefore, the reaction of NO NO scavenging is responsible not only for
with oxyhemoglobin might have a greater bi- the pressor effect of acellular hemoglobin ad-
oloeical relevance than that of the reaction ministration but also for many other adverse
with the deoxy form. NO reacts with oxyhemo- effects of hemoglobin-based blood substitutes
globin, forming both s-nitrosol hemoglobin as, for example, gastrointestinal dysmotility
and methemoglobin and nitrate, through a bi- and increased plasma levels of pancreatic en-
molecular process that is slightly faster than zymes. In a preclinical study on rats (129), a
the reaction of NO with deoxyhemoglobin recombinant hemoglobin (rHb3011) with re-
(168). Preliminary studies with sperm whale duced rate of NO scavenging and decreased
myoglobin and human hemoglobin showed oxygen affinity (Table 8.4) showed a reduced
that substitution of some residues in the distal gastrointestinal dysmotility, measured as per- -

heme pocket, Leu(B10) and Val(E11), can re- centage emptying of the stomach, compared
duce the reaction rate of NO with oxyhemoglo- with that of rHbl.1. Presently, Baxter is com-
bin resulting from steric hindrance (168). In a mitted to the development of the above-men-
-
subseauent work (109). a series of mutant re- tioned hemoglobins with reduced NO scaveng-
combinant di-a-hemoglobins was produced by ing rate.
- - residues Leu(BlO), Val(E11), and
changing 3.3.3.1.5 Things to Come. The production
Leu(G8) in the distal heme pocket to larger of recombinant hemoglobins can be achieved
hydrophobic residues (Phe, Leu, Trp, and Ile). in different expression systems, ranging from
As a result, changes in both oxygen affinity E. coli to yeast, insect cells, plants, and trans-
and rate of NO oxidation were obtained. A genic mammals. Large-scale production and
good correlation was found between rate of purification of recombinant hemoglobin to be
NO scavenging and the pressor effect exerted used as a blood substitute have to cope with
on conscious rats, whereas no correlation ex- problems such as high expression yields, easy
isted between oxygen affinity or autooxidation and cheap purification, and successful elimi-
rate and pressor effect (Table 8.4). This study nation of contaminant endotoxins. In spite of
demonstrated for the first time in a quantita- Somatogen's efforts in the scaling up of the
tive way that NO scavenging is directly come- process, leading to excellent results in terms of
ed with NO-dependent oxidation rate of expression yield and purity of the final prod-
myhemoglobin and that the rate of NO scav- uct, it is likely that a bacterial expression sys-
,.enging, and not the affinity of NO for hemo- tem will not be the ideal solution for a large-
globin, correlates with the pressure responses scale production of recombinant hemoglobin
observed upon- administration of recombinant (169). In fact, the low yield of pure proteins
hemoglobin. expressed in bacteria, even using highly opti-
412 Oxygen Delivery by Allosteric Effectors of Hemoglobin, Blood Substitutes, and Plasma Expanders

mized expression vectors, cannot meet the in- ber of macromolecules in the solution, the
creasing needs of transfusion therapy. Fur- higher being the number, the higher the exerted
thermore, given that the administered dose of oncotic pressure. Capillary walls are impeme-
a blood substitute easily reaches tens of able to plasma proteins. The concentration of
grams, the product has to be extremely pure, plasma proteins is about 7 g/dL and a solution
the risk associated with endotoxin-induced with a concentration higher than 7 g/dL is re-
side effects being proportional to the amount ferred to as hyperoncotic. The infusion of a hy-
of recombinant product administered. peroncotic solution results in a withdrawal of
Two alternatives for the production of pure fluids inside the capillaries followed by an in-
and endotoxin-free hemoglobin are envisaged crease in the arterial pressure. Even though cel-
in transgenic animals and in vitro differentia- lular hemoglobin has a concentration of about
tion of embryonic cells to hematopoietic stem 14 g/dL, it exerts no oncotic pressure because
cells. For example, human hemoglobin can be the protein is confined inside the erythrocytes.
expressed in transgenic mice (170) or in trans- Hemoglobin polymerization was attempted to
genic swine (171, 172), the latter being the optimize the balance between an efficient oxy-
more feasible way because of the higher yield gen transport and a physiological oncotic pres-
of hemoglobin per animal. Recombinant he- sure. Therefore, it is possible to administer he-
moglobin purified from transgenic swine moglobin solutions with a concentration of
shows some heterogeneity attributed to the hemoglobin up to 13 g/dL, which exert an on-
formation of hybrid molecules, as a conse- cotic pressure of about 25 mmHg, similar to that
quence of contamination with swine hemoglo- normally found in human plasma. Further-
bin. Human and swine hemoglobins exhibit more, polymerization increases the half-life of
high sequence, structure, and function homol- hemoglobin up to about 24 h.
ogies, making feasible the complete substitu- Three types of polymerized hemoglobins
tion of swine hemoglo'di w i t h the human pro- have been tested on human subjects so far:
to improve the homogeneity of the w R ~ ~ Q PBU ~ u~Corp
' bY ~~e Y. (Cambridge,MA,
product (173). Recently, it has been reported USA), PolyHeme by Northfield Laboratories
that human embryonic stem cells can differenti- Inc. (Evanston, IL, USA), and Hemolink by
ate, under certain growth conditions, to hema- Hemosol Inc. (Toronto, Canada). Both Hemo-
topoietic lineages that can, among others, gen- Pure and PolyHeme are glutaraldehyde-poly-
erate erythroid cells that are identical to those merized hemoglobins, whereas Hernolink is
naturally produced in human adult subjects an 0-raffinose-polymerizedhemoglobin.
(174).An attractive alternative strategy for ~b Glutaraldehyde crosslinks hemoglobin,
production is the preparation of transgenic to- both inter- and intramolecularly, predomi-
bacco containing human a! and p genes (175). nantly through a Schiff base bond with E
amino groups of lysines. The Schiff base can be
3.3.4 Polymerized Hemoglobins. Polymer- further reduced to obtain an aminic bond (Re-
ization of hemoglobin was initially aimed at action 5). The polymerization conditions (re-
increasing the molecular weight of the mole-
cule to prevent renal filtration. When scaveng-
ing of NO by hemoglobin was found to be one
of the major causes of the frequently observed
hypertensive effect associated with hemoglo-
bin-based blood substitutes administration,
hemoglobin polymerization was exploited for
preventing extravasation of the protein and reduction
------ Hb, Hb
for reducing its vasoconstrictive effect. N" " " N'
Solutions of polymerized hemoglobin exhibit H H
distinct physical properties and physiological
and pharmacological effects. In fact, the oncotic action time, glutaraldehydelprotein ratio) can
pressure exerted by a solute through an imper- be controlled to obtain the desired molecular
meable membrane is proportional to the num- weight distribution.
3 Blood Substitutes: Modified Hemoglobins and Perfluoroche

According to recent studies (112,176), glu- diac, hepatic, vascular, and gastrointestinal
taraldehyde reacts with many of the lysine surgery, and in trauma and ischemic events.
residues located on the surface of hemoglobin 3.3.4.1.1.1 Adverse Effects. During phase I
and with the N-termini of both a- and and I1 clinical trials no significant adverse ef-
fects were noticed. The coagulation profile did
eased oxygen affinity consequent to the sta- not change upon administration of 45 g of
ilization of the deoxy form of hemoglobin. HBOC-201 (179, 181). In some cases, leuko-
e constraints introduced by polymerization cytes and reticulocytes counts were higher in
the treated groups than those in control
T to R. Furthermore, faster oxidation groups (182). An increase of IgG antibodies to
dation rates are observed with glut- HBOC-201 was observed during phase I1 clin-
-polymerized hemoglobin. This fmd- ical trials,at day 7 after administration. The
consequence of the higher expo- increase in antibodies titer lasted about 1
e pocket to the solvent, induced week. According to the authors, this finding
puts into question the feasibility and safety of
3.3.4.1 Hemopure (HBOC-201). Hemopure repeated administrations of HBOC-201 (182).
polymerized bovine hemoglobin formulated In some clinical trials mild gastrointestinal ef-
modified Ringer's lactate at a concentration fects were reported but they did not require
any treatment (178). Markers of liver, pancre-
c pressure of 18 mmHg, atic, and renal function did not show any in-
osmolarity of 300 mOsm/kg, a viscosity of crease above physiological values in any clini-
Torr (177). Hemopure cal trial.
rature for more than 1 Hemodynamic effects of HBOC-201 admin-
istration vary in different clinical trial set-
The purification procedure leads to a con- tings. A study on 13 abdominal aortic surgery
olecular weight distri- patients demonstrated that oxygen delivery
main fraction with a was decreased in the treatment group as a con-
ng between 128 and sequence of reduced cardiac output (183).This
0 kD and a minor fraction of octamers of finding is in contrast with previously reported
out 128 kD,with no traces of dimeric or tet- studies on animals (184). Oxygen delivery to
tissues was calculated from cardiac output in
Recently, the concern about a potential human study, whereas in animal studies the
ntamination of bovine blood products and oxygen tension in the skeletal muscle was
Ref. 180 for a re- measured directly by a microelectrode histo-
on this topic) has led to the optimization graphic technique. Decreased cardiac output
e sterilization procedure that, according can be a direct and positive consequence of an
wers the content increased tissue oxygenation. Furthermore,
of prions by 5 log units (177). other studies demonstrated that HBOC-201
Biopure is shipping about 2000 units of the has no negative effects on systemic blood pres-
duct at no cost to some hospitals in South sure (177) or heart rate, both during anesthe-
d the product is expected to be freely sia and in postoperation recovery. These ef-
fects, when observed, were only transient and
related to only mild hemodynamic perturba-
tions (177, 178).
3.3.4.1.1.2Pharmacokinetics. Pharmaco-
kinetic data were collected duringphase I clin-
ical trials on healthy subjects aimed at evalu-
ating the physiology and pharmacokinetics of
HBOC-201(178)and at monitoring the hema-
tological effects of HBOC-201 both on male
donation settings, in orthopedic, car- and female subjects (179).
414 Oxygen Delivery by Allosteric Effectors of Hemoglobin, Blood Substitutes, and Plasma Expanders

It was found that HBOC-201 metabolism is efficacy of alkylating agents used in chemo-
well described by a one-compartment model therapy (188).
and does not depend on gender. The elimina- In 2000 Biopure announced preliminary re-
tion is a first-order process, involving the re- sults of a phase I11 clinical trial carried out on
ticuloendothelial system with no evidence of 693 elective surgery patients in Europe, the
renal filtration. The volume of distribution is United States, Canada, and South Africa (see
dose independent and roughly corresponds to Section 5). During the clinical trial, Hemo-
that of plasma, ranging from 3 to 4 L. In con- pure, administered at doses up to 240 g,
trast, both the half-life and clearance are dose proved to be effective in reducing the number
dependent. The half-life for a dose of 45 g is of patients who received allogeneic red blood
celk and confirmed the good safety profile
about 20 hand the clearance is about 0.11 L/h.
shown during phase I and I1 clinical trials.
Upon 8 h from administration of HBOC-201, a
During the study more than 35% of patients
peak in serum iron was observed, followed, receiving Hemopure did not need any alloge-
upon 16 h, by an increase up to sixfold over neic blood donation during the whole fol-
baseline of erythropoietin and, upon 40 h, by a low-up period of 45 days.
peak in ferritin levels. 3.3.4.2 PolyHeme (Poly SFH-P). PolyHeme
3.3.4.7.2 Physiology and Pharmacology. is a glutaraldehyde-polymerized pyridox-
Preclinical studies showed that HBOC-201 is alated human hemoglobin formulated at a
capable of restoring normal levels of oxygen 10 g/dL concentration in physiological solu-
delivery to tissues after complete isovolemic tion. The solution is isooncotic with plasma
hemodilution (185). The improved oxygen- and exhibits a p50 of about 30 Torr (189). The
ation potential of HBOC-201 with respect to solution is stable for more than 1 year. The
RBC hemoglobin was assessed through direct steps involved in the production of ~ o l ~ ~ e m
measurements of skeletal muscle tissue oxy- are the purification of human hemoglobin,
genation (184). In dogs, it was found that the pyridoxalation, and subsequent polymeriza-
dose of blood substitute required to restore tion with glutaraldehyde. After purification of
oxygenation at baseline levels was about one- the polymerized protein, the content of tet-
third that of RBC hemoglobin. The increase in ramers is drastically reduced to less than 1%
muscle oxygenation was a consequence of a (189). Differently from Hemopure, PolyHeme
high extraction ratio that resulted in a steeper is produced from human hemoglobin that has
gradient between arterial and venous oxygen a lower p50 value than that of bovine hemo-
globin, but, according to the manufacturer, it
pressure. Enhanced oxygen delivery to tissues
is preferable to avoid possible immune re-
was also observed in clinical trials on healthy
sponses and transmission of viruses. The pyri-
volunteers. HBOC-201 administration in a
doxalation is intended to improve the oxygen-
normovolemic hemodilution setting results in delivery capacity of the product to a
reduced lactate levels and reduced heart rate physiologically suitable value.
following exercise stress with respect to con- 3.3.4.2.1 Pyridoxal Phosphate as a 2,3-DPG
trols (186). Furthermore, the higher efficacy Analog. In 1969 pyridoxal phosphate (PLP)
of HBOC-201 in restoring the exercise capac- (8) was proved to bind in the 2,3-DPG binding
ity in comparison with autologous transfusion pocket of hemoglobin and to lower oxygen
was confirmed: 1 g of HBOC-201 has the same affmity of stripped hemoglobin to the same
physiological effects as 3 g of RBC hemoglo- extent as that of its natural effector (76).
bin. HBOC-201 was effective in increasing the PLP binds preferentially to deoxyhemoglobin
oxygen transport in sickle-cell anemia pa- through a Schiff base linkage with the N-ter-
tients, thereby showing its potential applica- mind amino groups of the P-chains in a ratio
tion in sickle-cell anemia crisis (187). of one molecule of PLP for one p-chain (190).
HBOC-201 is also effective in stimulating The Schiff base linkage can be reduced by
erythropoiesis, both in men and women (179). NaBH, to obtain a stable pyridoxalated hemo-
In animal studies, HBOC-201 was proved to globin. The preferential binding to the deoxy
enhance tumor oxygenation and increase the form of hemoglobin and the covalent nature of
1Z 3 Blood Substitutes: Modified Hemoglobins and Perfh

Ib
[ the arninic bond between PLP and the protein 3.3.4.2.3 Pharmacology. A higher hemo-
I
result in a permanent low oxygen affinity, con- globin concentration can increase the oxygen-
sequent to the stabilization of the T state carrying capacity of plasma, thereby resulting
(190). in a better tissue oxygenation. Unfortunately,
this result is achieved only at the expense
- of an
increase in oncotic pressure and thus in
plasma volume and MAP. PolyHeme shows
the favorable effects of high hemoglobin con-
centrations up to 10 g/dL, and a physiological
oncotic pressure resulting from a controlled
polymerization procedure. Clinical trials in
acute blood-loss settings demonstrated that
PolyHeme is at least as effective as red blood
cell hemoglobin in oxygen delivery, and that
3.3.4.2.2 Clinical Use of Poly SFH-P. Poly- oxygen extraction ratio from PolyHeme is as
Heme has been tested during phase I1 clinical high as that from red blood cells (1921, because
trials in acute blood loss and during phase I11 of the increased p50, following the polymeriza-
clinical trials in elective surgery to avoid or tion and pyridoxalation. Furthermore, Poly-
decrease the amount of transfused allogeneic Heme seems to be effective in reducing- the
blood. Recently, a study of PolyHeme in hem- number of allogeneic - blood units transfused
orragic shock patients indicated that the prod- during emergency following traumatic or
uct could be effective in lowering the risk of acute blood losses (189, 192).
multiple organ failure (191). This is m e of the 3.3.4.3 Hemolink. Hemolink is the manu-
major causes of late postinjury death and facturer's name for o-rafinose-crosslinked
seems to be related to the presence of biologic human hemoglobin. Presently, phase I11 clin-
mediators, such as phospholipids and cyto- ical trials in coronary artery bypass grafting
kines in stored blood. Administration of Poly- are ongoing for 299 patients (193). Available
Heme appeared to be associated with a de- data refer to phase I and I1 clinical trials. The
crease of deaths caused by multiple organ Marketing Authorization Application for the
failure. However, because of the small number product was submitted and was awaiting the
of patients studied, no general and definitive review by the UK Health Authority, whereas
conclusion can be drawn on the efficacy of Health Canada's Therapeutic Products Pro-
PolyHeme in lowering the risk of multiple or- gram was reviewing the product at the end of
gan failure. the year 2000.
3.3.4.2.2.1 Adverse Effects. No adverse ef- ~ e m o l i n kis produced by treatment of hu-
fects were reported during phase I and I1 clin- man hemoglobin from outdated blood with
ical trials (189, 192). Heart rate and MAP did o-raffinose (194) (Fig. 8.7), followed by elimi-
not increase significantly after infusion and nation of nonpolymerized, dimeric hemoglo-
they remained in physiological ranges up to 3 bin. Hemoglobin is carefully purified by self-
days after administration. There was no evi- displacement chromatography to eliminate
dence of organ toxicity, as demonstrated by contaminants that can have toxic effects
physiological values of creatinine and liver en- (membrane debris, endotoxins). After poly-
zymes. PolyHeme administration did not af- merization, the unpolymerized, dimeric mole-
fect temperature. A peak in bilirubin concen- cules are separated from the final product
tration was reported upon 3 days after through ultrafiltration. The distribution of
administration. This finding was interpreted molecular weights (193) is as follows: 4%
as a consequence of hemoglobin metabolism dimeric hemoglobin; 34-43% tetrameric he-
and not as a sign of liver damage (192). moglobin; 54-62% polymerized hemoglobin
3.3.4.2.2.2 Pharmacokinetics. The half-life, up to 500 kD; <3% 500 kD polymerized hemo-
reported for PolyHeme clearance, is about globin. The product is formulated in Ringer's
24 h (189). lactate solution at a concentration of 10 g/dL.
41 6 Oxygen Delivery by Allosteric Effectors of Hemoglobin, Blood Substitutes, and Plasma Expanders

The formulation exhibits an oncotic pressure The conclusion of clinical trials I and I1 is
of 24 mmHg, a viscosity of 1 cP, and a p50 of that Hemolink is a safe and efficacious product
34 Torr (195). useful during autologous blood donation pro-
3.3.4.3.1 Clinical Use of Hemolink. Hemolink grams in decreasing the amount of donors'
has potential clinical applications in surgery blood administered in cardiac surgery (193).
as an alternative to allogeneic blood donation 3.3.4.3.1.2 Pharmacokinetics. Pharmaco-
and to replace patient's blood after harvesting kinetic data were reported for phase I clinical
in an autologous blood donation program. trials (194, 195, 198). Hemolink is primarily
3.3.4.3.1.1 Adverse Effects. Phase I clini- metabolized by the spleen and liver (194), as
cal trials were intended to assess the safety of assessed by preclinical studies on rats. The de-
cay of Hemolink in the plasma of human sub-
the product and involved 42 subjects to whom
jects is monoexponential, dose dependent, and
up to 600 mgikg of Hemolink was adminis-
associated with the molecular weight distribu-
tered in a top-loading experimental model
tion of hemoglobin polymers in the drug. In
without causing serious adverse effects (194- humans t,/, is about 1.6 h for a 25 mgikg dose
196). MAP was not significantly affected by and increases to a mean value of about 14.2 h
the administration of Hemolink, although a for a 500 m a g dose (194, 195). This mean
10% increase was observed in phase I clinical value is the result of two contributions: a t,/, of
trials. In phase I1 clinical trials, 13 out of 30 about 7.6 h for tetrameric hemoglobin and of
patients (193) exhibited elevated pressure, about 18.7 h for polymeric hemoglobin. In the
but, according to investigators' opinion, this kidney, both low molecular weight products
was not significantly different from that of the and small amounts of intact hemoglobin are
control group. In phase I studies this effect present (194). The low molecular weight mol-
was accompanied by heart rate decrease, a ecules are degradation products, whereas
symptom reported for many other hemoglo- traces of intact hemoglobin are attributed to
bin-based blood substitutes (194). filtration of a small fraction of dissociable,
Both phase I and phase I1 studies showed noncrosslinked hemoglobin that is unlikely to
neither renal nor respiratory adverse effects, cause severe renal damages.
nor negative interactions with coagulation or 3.3.4.3.2 Physiology and Pharmacology. In
other hematological parameters. Increased contrast with other blood substitutes tested so
levels of some liver enzymes, particularly as- far, the administration of up to 4 g of
partate aminotransferase and amylase, did Hemolink in rats did not affect the cardiac
not appear to be linked to liver or pancreas output and had only mild effects on systemic
damage and were not associated with symp- vascular resistance (199). It is not known to
toms of acute pancreatitis (193). what extent the absence of effects on cardiac
The most severe effects were associated output is also true in humans, but it could be
with the action of Hemolink on the smooth important in determining the final oxygen de-
muscles cells in the GI tract (194) (see Section livery to the tissues. Phase I1 studies demon-
3.3.1.3 for details). Some cases of jaundice strated that Hemolink is capable of maintain-
were reported for patients in phase I1 clinical ing tissues oxygenation during coronary
trials. The first effect is of mild intensity and artery bypass grafting (193).
probably the result of NO scavenging by he- 3.3.4.3.3 Structure-Function Relationships.
moglobin in the GI tract, in particular from o-Raffhose is a hexaldehyde obtained by oxi-
nonadrenergic, noncholinergic inhibitory dation of raffmose with sodium periodate. It
neurons (194, 197). Jaundice and the peak in forms a Schiff base with the exposed amino
the bilirubin concentration observed in phase groups of the protein (200) (Fig. 8.7). Subse-
I1 clinical trials are a consequence of hemoglo- quent reduction of the Schiff base linkage fur-
bin metabolism. A peak in the lactate concen- ther stabilizes the bond. The polymerization
tration that did not coincide with metabolic reaction is carried out on the deoxy form of
acidosis is considered a normal consequence of hemoglobin to stabilize the T state and de-
drug administration and not a symptom of tis- crease the oxygen affinity of the final product
sue hypoxia (193). (194). The crosslinking reaction takes place
3 Blood Substitutes: Modified Hemoglobins and Perflua

both in the 2,3-DPG pocket, forming a stable with bovine hemoglobin (202). The product
tetrameric hemoglobin with reduced oxygen has a molecular weight of 120 kD and it is a
affinity, and on the surface of the protein, stabilized tetramer without evidence of intra-
forming a wide range of polymerized mole- or intermolecular crosslinking (128). PEG-Hb
cules from about 100 to about 500 kD with is formulated in 150 rnM NaC1,5 mM sodium
traces of higher molecular weight polymers. phosphate, and 5 mM sodium bicarbonate at a
Recent studies (199) demonstrated that the concentration of 6 g/dL (202). The solution
peculiar characteristics of Hemolink on sys- has an oncotic pressure of 118 mmHg and a
temic and renal hemodynamics are only mar- viscosity of about 3 cP. The p50 of PEG-Hb
ginally influenced by the elimination of low ranges between 9 and 16 Torr (203). Phase Ib
molecular weight molecules, but are likely a
clinical trials have been halted and data re-
direct consequence of the o-raffinose decora-
ported in the following sections mainly refer to
tion of the molecule that reduces NO access to
preclinical studies and phase Ia clinical trials.
3.3.4.4 Recent Developments. Hemosol is 3.3.5.7.7Clinical Use of PEG-Hb. After
presently involved in the development of some phase Ia clinical trials on healthy volunteers,
new hemoglobin-based blood substitutes with the product was administered to cancer pa-
improved pharmacological properties with re- tients during phase Ib clinical trials. In fact,
spect to fmt-generation modified hemoglobins. solid hypoxic tumors respond better to radio-
o-Raffmose-polymerized hemoglobin conju- and chemotherapy if the malignant cells are
gated with Troxolon is among these new prod- well oxygenated (38).
ucts. Troxolon (9) is a derivative of vitamin E 3.3.5.1.1.1Adverse Effects. PEG-Hb did
and has strong antioxidant properties. The not show any significant adverse effects both
proposed clinical use is in hemorrhage to pre- during preclinical trials on animals (202) and
vent reperfusion injury (201). during phase I clinical trials (203). In animals,
a vacuolization in liver, kidney, and bone mar-
row cells was reported. It is likely correlated to
phagocytosis and subsequent degradation of
PEG-Hb in the reticuloendothelial system
(204) and seems to be transient without affect-
ing the safety profile of the product (205). The
only reported side effect of PEG-Hb adminis-
tration in phase I clinical trials is gastrointes-
tinal discomfort associated with the highest
doses administered. Some investigators pro-
posed that gastrointestinal pain might be a
3.3.5 Conjugated Hemoglobins. Conjugated consequence of PEG-Hb extravasation, fol-
or surface-modified hemoglobins are tet- lowed by a transient inflammatory response of
americ hemoglobins decorated with different the intestine (206).
acromolecules, such as dextran (78), PEG 3.3.5.1.1.2 Pharmacokinetics. Little is
791, and polyoxyethylene (POE) (80). Conju- known about the pharmacokinetics of PEG-
tion results in an increase in the molecular Hb. Its degradation might take place in the
reticuloendothelial system (204). As a conse-
quence of polyethylene glycol decoration,
retention times and lower immunogenic PEG-Hb has one of the highest vascular reten-
tion times among hemoglobin-based blood
3.3.5.1 PEG-Hb. PEG is a polymer of gen- substitutes. In humans, the highest adminis-
formula H(OCH,CH,),OH, where n is tered dose of 38 g exhibits a half-life of about
ater than or equal to 4. The greater the 48 h (203). The slow elimination of PEG-Hb
ue of n, the higher the molecular weight of was pointed out to be compatible with a
olymer. PEG-Hb produced by Enzon is weekly administration in cancer patients to
ned by the reaction of succinimidyl PEG improve hypoxic tumor oxygenation (133).
418 Oxygen Delivery by Allosteric Effectors of Hemoglobin, Blood Substitutes, and Plasma Expanders

Raffinose OH
I NaQ
CH20H
I
t
CHO CHO 0 CHO

CHO CHO
o-Raffinose

IStroma free
hemoglobin Val1 -pl
I
CH
I
20H

~~s82-pj
Val 1-pl

\
Figure 8.7. Preparation of o-
raffinose hemoglobin. Aldehy-
dic groups on o-raffmose react
with amino groups of hemoglo-
bin both in the 2,3-DPG pocket
and on the protein surface (not
shown). The three-dimensional
structure of human hemoglo-
bin in the presence of 2,3-DPG
(PDB code 1B86) shows the rel-
ative positions of p,Vall, p,Lys82,
and &Lys82, shown in ball-
and-stick mode, with respect to
2,3-DPG, shown in space-filling
mode.
3 Blood Substitutes: Modified Hemoglobins and Perfluor

3.3.5.1.2 Physiology and Pharmacology. protein. Even though the presence of reduced
PEG decoration, which leads to a n increased glutathione in the plasma might accelerate the
molecular weight, an increased hydrodynamic release of NO, the efficacy of SNO-PEG-Hb
volume, and an efficient masking of the pro- proved to be sustained with time (208).
tein, contributes to the good pharmacological 3.3.5.2 PUP (Pyridoxalated Hemoglobin-
profile of this product. In fact, differently from Polyoxyethylene Conjugate). Iwashita's group
other modified hemoglobins, PEG-Hb has only was the first to achieve the conjugation of hu-
mild vasoconstrictive effects and, thus, only man pyridoxalated hemoglobin with polyoxy-
mild consequences on MAP (207, 208), likely ethylene to obtain an oxygen carrier in the
resulting from the lower tendency to extrava- 1980s (213). Currently, PHP is produced by
sate or the physical hindrance to NO binding
Apex and is being developed as an NO scaven-
(128). PEG-Hb has proved to sustain physio-
ger
- in the treatment of shock associated with
logical functions during exchange-transfusion
systemic inflammatory response syndrome
experiments in rats (209). Furthermore, the
high oncotic pressure and viscosity of PEG-HZ, (95) and it is going to enter phase I11 clinical
solutions make this blood substitute a good trials (132). The product is only briefly pre-
volume expander for the treatment of severe sented in this section, its main *pharmaceutical
blood losses (210). Phase Ib clinical trials eval- application being shock treatment rather than
uated the efficacy of the product as an en- oxygen delivery.
hancer in radiotherapy of solid hypoxic tu- Differently from PEG-Hb, the polymer
mors in humans (133). used to modify the surface of the protein is a
3.3.5.1.3 Things to Come bifunctional reagent (alpha-carboxymethyl,
3.3.5.1.3.1 Hemospan. Sangart Inc. is de- omega-carboxymethoxy1 polyoxyethylene) and
veloping Hemospan, a blood substitute pre- thus both conjugation and crosslinking reac-
pared from outdated human blood hemoglobin tions take place. The modified hemoglobin is
and maleimide polyethylene glycol. Hemospan mainly in the conjugated form, but a small
has a higher oxygen affinity than that of other amount of crosslinked species is present (2141,
blood substitutes presently on clinical trials. It accounting for about 10% of the total protein,
has a higher oncotic pressure, a higher viscos- as reported in early works (213). The conjuga-
ity, and better flow properties. These features tion reaction takes place on marginally puri-
are likely to improve the hemodynamic re- fied hemoglobin solutions, which still contain
sponse to the blood substitute administration, superoxide dismutase and catalase, thus lead-
decreasing the usually reported vasoconstric- ing to a final product in which more than 90%
tive effect and enhancing tissue perfusion and of the catalase and superoxide dismutase ac-
oxygen extraction (211,212). The product has tivities found in the red blood cells is
been tested on animals, giving encouraging re- crosslinked to hemoglobin (132, 214). The
above-mentioned features are likely responsi-
3.3.5.1.3.2 SNO-PEG-Hb. The reduction ble for the low toxicity and for the potential
of the vascular response to the administration applications of PHP. Pyridoxal phosphate acts
of hemoglobin-based blood substitutes has re- as a 2,3-DPG analog, lowering oxygen affinity
cently been addressed with the preparation of (see Section 3.3.4.2.1).
a S-nitrosylated PEG-Hb (SNO-PEG-Hb). 3.3.5.2.1 Clinical Use of PHP. PHP is be-
The NO group is introduced through reaction ing administered in clinical trials to patients
of PEG-Hb with nitrosoglutathione, leading to suffering from volume refractory shock asso-
the formation of hemoglobin nitrosylated ciated with NO overproduction, but its appli-
nly at Cys93 in the P-chains, with the met- cation in the treatment of reperfusion injury
moglobin content kept at less than 10%. and hemorrhagic shock is envisaged because
xperiments on rats demonstrated that SNO- of its reduced prooxidant properties (132).
EG-Hb is effective in reducing the hyperten- Phase I clinical trials showed a good safety
on normally found upon administration of profile with no evidence of organ toxicity or
odified hemoglobins, both attributed to the changes in hemodynamics (95). The safety of
release of NO and to PEG decoration of the the product was also confirmed during phase
420 Oxygen Delivery by Allosteric Effectors of Hemoglobin, Blood Substitutes, and Plasma Expanders

I1 clinical trials up to a dose of 180 g. The complex functions of red blood cells and, for
plasma concentration of PHP is dose depen- this reason, their therapeutic applications
dent. will be limited.
3.3.5.2.2 Physiology and Pharmacology. 3.3.6.1 Encapsulated Hemoglobins. Encap-
Phase I and I1 clinical trials on patients with sulated hemoglobins are generally referred to
shock secondary to sepsis demonstrated the as third-generation blood substitutes and are
efficacy of PHP in increasing or maintaining expected to have physical and biochemical
blood pressure with a concomitant decrease in properties very similar to those of hemoglobin
heart rate. Furthermore, PHP is less suscep- inside red blood cells. Encapsulation of hemo-
tible to oxidation by hydrogen peroxide and to globin in artificial vesicles, liposome, or nano-
capsule is a strategy to mimic red blood cells.
formation of ferrylhemoglobin (see Section
These new delivery vehicles were developed to
3.3.1.4), thereby having potential applications
circumvent adverse effects related to adminis-
in ischemia and subsequent reperfusion injury tration of solutions of free hemoglobin. In ad-
(132). Notably, PHP has no hypertensive ef- dition, inclusion in artificial vesicles protects
fects on healthy subjects. Differently from hemoglobin from oxidation and degradation,
other commonly used drugs in septic shock, as, resulting in longer circulation persistence and
for example, NO synthase (NOS) inhibitors, milder untoward effects. Membranes are
PHP exerts its hypertensive effects only on semipermeable, allowing free diffusion of
subjects suffering from hypotension, having gases and small molecules and preventing dif-
no effects on healthy subjects. This selectivity fusion of large or charged molecules inside or
makes PHP a better choice than NOS inhibi- outside. Hemoglobin is thus maintained at a
tors in the treatment of shock (95). In addition high concentration inside the vesicles and the
to its NO scavenging activity, PHP is an NO equilibrium between tetramers and dimers is
donor because of the formation of S-nitrosohe- shifted toward the former. Moreover, compo-
moglobin in a way very similar to that of he- nents essential for hemoglobin function, such
moglobin A (132). as allosteric effectors and enzymes, can be in-
cluded in the formulation (215).
3.3.6 Recent Developments of Modified He- The first study on encapsulated hemoglo-
moglobins. Modified hemoglobin solutions can bin was carried out in 1957 by Chang (216).
be considered oxygen carriers rather than Artificial red blood cells were prepared using
real blood substitutes. In fact, their short synthetic membranes and encapsulated hemo-
half-life limits their use as a short-term re- globin was reported to have an oxygen dissoci-
suscitative fluid in emergency and surgery. ation curve similar to that of hemoglobin in
They cannot replace blood in many patho- red blood cells. Enzymes, such as catalase and
logical situations, such as chronic anemia. carbonic anhydrase, were included in the arti-
Furthermore, hemoglobin solutions still ficial red blood cells and were shown to retain
show some adverse effects, hypertension, their activity. Different polymers, polysaccha-
gastrointestinal discomfort, and potential rides, lipids, and proteins have been used to
oxidative damage, which would prevent modify the surface characteristics of the mem-
their application in the clinical routine. Sec- brane and to reduce the removal from the
ond-generation blood substitutes are being bloodstream. Removal of sialic acid, one of the
developed with the aim of reducing the pres- components of red blood cell membranes, was
sor effect (Sections 3.3.3.1.4 and 3.3.5.1.3.2) shown to reduce persistence in the circulation.
and the oxidative potential of free hemoglo- However, because of their mean diameter that
bin (Section 3.3.1.4), and optimizing oxygen ranges between 1 and 5 pm, microcapsules are
delivery to tissues through the modulation rapidly cleared from the bloodstream by the
of the flow properties of the formulation reticuloendothelial system and have too short
(Section 3.3.5.1.3.1). Even though second- a circulating time for clinical application
generation blood substitutes have better (217).
physiological and pharmacological proper- Small lipid-membrane artificial red blood
ties, they still do not possess many of the cells have been prepared since 1980 using
3 Blood Substitutes: Modified Hemoglobins and Perfluo~

lipid-membrane liposomes. In the first lipo- Hemoglobin content is generally similar to


some formulation, vesicles had a mean diame- that present in red blood cells. The reported
ter of about 0.2 pm and a slower clearance values of p5O and Bohr and Hill coefficients for
rate, but the uptake by RES was still rapid encapsulated bovine hemoglobin were similar
(218). Since then, different formulations of li- to those of free bovine hemoglobin in solution.
posome-encapsulated hemoglobin have been Enzymes involved in oxygen radical scaveng-
developed (217). Phospholipids and sterols are ing can be included in nanocapsules. Because
the main components of phospholipids-based li- nanocapsules are permeable to small hydro-
posomes. Cholesterol is added to increase lipo- philic molecules, they allow free diffusion of
some stability and flexibility and to reduce the glucose, required by the metHb-reductase sys-
tem, or reducing agents. In vitro studies and
tendency to fusion of vesicles. Different types
preliminary tests in animal models showed
of phospholipids were tested to modify lipo-
promising results (223).
some surface characteristics and to reduce rec- 3.3.6.2 Albumin-Heme. Recentlv," , it has
ognition and removal by the RES. As compo- been reported that albumin complexed with
nents of natural membrane, phospholipids are heme (224) or with a heme derivative (225,
well tolerated but they are rapidly metabo- 226) is capable of reversibly binding oxygen.
lized by lipase and phospholipase. In addition, The protein has potential applications as a
they are easily subjected to oxidation and are blood substitute, given its oxygen-carrying
unstable during storage (215). Thus, manu- properties, and as a volume expander, given
facture of phospholipid-based liposomes is ex- the high oncotic pressure exerted by its solu-
pensive and requires a high degree of mastery. tions.
Liposome-encapsulated hemoglobin was tested
in animals with minimal adverse effects after 3.4 Perfluorochemicals
administration of small or moderate doses
(219). 3.4.1 Current Drugs. Presently, none of
A different approach uses nonphospholipid the perfluorocarbon-based oxygen carriers is
amphiphiles to form liposomes. These mole- available for clinical use in the United States.
cules are produced from a large variety of in- Perfluorochemical-based blood substitutes
expensive raw materials and a broader spec- that have entered clinical trials are listed in
trum of chemical structures is available. Table 8.5.
These amphiphiles may be composed of ionic 3.4.1.1 First-Generation Fluorocarbon-Based
and nonionic polar headgroups, whereas the Blood Substitutes. The first injectable perfluo-
hydrophobic tails can be formed by many dif- rochemical (PFC) emulsion to be produced
ferent structural features, such as long-chain commercially was Fluosol (Green Cross Corp.,
fatty acids, acyl derivatives of fatty acids, long- Osaka, Japan), which consisted of 14% (wlv)
chain alcohols, and amides. Many of these perfluorodecalin (10) and 6% (w/v) perfluoro-
compounds proved to be toxic, but amphi- tri-n-propylamine (111, emulsified with Plu-
philes belonging to the alkyl polyoxyethylene ronic F-68 (poloxamer 188, a copolymer con-
ether nonionic surfactant series were found to sisting of two polyoxyethylene chains flanking
be suitable for pharmaceutical application a polyoxypropylene segment), together with
(215). Polyoxyethylene-2 cetyl ether was used small amounts of egg yolk phospholipids and
in a new formulation of nonphospholipid lipo- potassium oleate.
some-encapsulated hemoglobin (220), but li-
posomes were found to aggregate or react with
erythrocytes (215).
Hemoglobin-containing nanocapsules with
a mean diameter between 80 and 200 nm have
been recently produced using different biode-
gradable polymers (221, 222). Because poly-
mers are stronger and more porous, smaller
amounts of membrane material are necessary.
422 Oxygen Delivery by Allosteric Effectors of Hemoglobin, Blood Substitutes, and Plasma Expanders

Table 8.5 List of Perfluorocarbon-Based Blood Substitutes


Trade Name Generic Name (Structure) Originator Dose
Fluosol Perfluorodecalin (10) Green Cross Co. 5 dki?
Perftoran Perfluorodecalin (10) Perftoran Co. 5-30 mLikgb
Oxyfluor Perfluoro-1,8- HemaGenPFC 1.7 gikg
dichloroodane (16)
Oxygent Perfluorooctyl bromide Alliance 0.9-2.7 g/l@
(13) Pharmaceuticals Co.
aMaximum dose administered during clinical trials (233).
bAccordingto data provided by the company (see Section 5).
OThe reported dose refers to that used in clinical trials [Oxyfluor (88);Oxygent (239; 246)l.

substitute currently available on the market


but no other country, except Russia, has yet
approved its use.
3.4.1.2 Second-Generation Perfluorochemi-
cals. Oxygent (Alliance Pharmaceutical Corp.,
San Diego, CA, USA) is a 60% (wlv) emulsion
[58% perfluorooctyl bromide (13)and 2% per-
fluorodecyl bromide (1411 stabilized with 3.6%
(w/v) egg yolk phospholipids.

Other first-generation PFC emulsions are


Emulsion No. I1 developed in China and Perf-
toran (previously named Ftorosan) from Rus-
sia. Emulsion No. I1 (Institute for Organic
Chemistry, Shanghai, China) is a 20% (w/v)
emulsion based on the same PFCs used in
Fluosol, in which the surfactant is a polox-
amer-type compound. It was clinically tested
in surgical patients and war casualties. Little
is known about the current clinical and com-
mercial status. Perftoran (Perftoran Co.,
Pushchino, Russia) contains 14% (w/v) perflu-
orodecalin (10) and 6% (w/v) perfluoro-N-(4- The average particle size of the emulsion is
methylcyclohexy1)piperidine (12) emulsified about 0.16-0.18 pm. It was evaluated in 18
with the synthetic poloxamer Proxanol268. clinical human studies involving more than
1200 patients. Phase I1 studies showed that
Oxygent was more effective than a unit of
blood in reversing transfusion triggers and in
delaying the need for donor blood usage (89).
An international, multicenter European
phase I11 transfusion avoidance study in gen-
eral surgery patients (492 enrolled subjects)
The Russian health regulatory authorities was completed in May 2000 and demonstrated
approved Perftoran in 1995-1996 as a tempo- the efficacy of Oxygent in reducing or avoiding
rary intravascular oxygen carrier in the the need for allogeneic transfusion when used
treatment of traumatic hemorrhagic shock, in conjunction with acute normovolemic he-
microcirculatory disturbances, cardioplegia, modilution (227). A second multicenter phase
cardiopulmonary bypass, and perfusion of iso- I11 study in cardiac surgery patients is being
lated human organs (89). It is the only blood conducted primarily in the United States.
3 Blood Substitutes: Modified Hemoglobins and Perfluorochemicals

Nonclinical studies are ongoing to evaluate tions, toxicity was ascribed to high perfluoro-
P the potential use of Oxygent in the treatment chemical vapor pressure, large emulsion
of severe decompressionsickness (2281, sickle- particle size, and inadequate osmolarity. The
cell anemia (229, 230), and to reduce inflam- presence of by-products or impurities from the
matory response during extra corporeal circu- synthesis reaction was also suspected (234).
lation (231). Progress in selective synthesis, purification,
A European research team has recently de- and methods of emulsification has led to
veloped a series of perfluorodecalin (10)-based higher quality and purity product with a more
emulsions, under the provisional commercial favorable tolerability profile.
name of Fluxon (232). These emulsions use Because they are immiscible in water, fluo-
lecithin as surfactant and in some of them 1% rocarbons need to be in an emulsified form to
(wlv) of perfluoro-l,3-dimorpholinopropane be injected into the bloodstream. Surfactants
(15) was added. or emulsifiers must be added to facilitate the
formation of the emulsion. Surfactants and
emulsifiers play a key role in determining the
characteristics and bioacceptability of the
emulsion (235). Many side effects displayed by
Fluosol were attributed to the surfactant. Plu-
ronic F-68 was reported to elicit anaphylactic
reactions resulting from complement activa-
tion and to alter human leucocyte function,
At present, no clinical trials have been car- causing a depression of host resistance to in-
ried out on these formulations and their po- fection (234). Poor purification, a low cloud
tential use as oxygen carriers during perfusion point that limited heat sterilization, and the
of animal organs is under evaluation. polydisperse nature of Pluronic F-68 might ac-
Oxyfluor (HemaGen, St. Louis, MO, USA) count for these adverse effects. In fact, atten-
consists of 76% (w/v) of perfluoro-1,8-dichlo- uation or absence of side effects shown by
rooctane (16). Egg yolk phospholipids are Fluosol were reported when purer poloxamers
added as stabilizer and safflower oil is used as or other surfactants or emulsifiers were intro-
surfactant. duced. Egg yolk phospholipids, the emulsifier
used in second-generation PFC-based prod-
ucts, do not cause complement activation and
are well accepted in medical practice (235).
Extensive toxicology studies demonstrated
that PFCs emulsions are well tolerated with
no serious adverse events at clinically relevant
doses (233). Transient side effects were re-
Animal studies assessed its efficacy as a ported in clinical studies following infusion of
transient oxygen carrier. Phase 1/11 safety Oxygent and Oxyfluor (88). They consisted of
study in low-blood-loss surgical patients were an immediate response, characterized by fa-
completed. Oxfluor was under study as an cial flushing, headache, and lower back pain
agent to remove microemboli during cardio- during or immediately after the infusion, and
pulmonary bypass, but the current status of of a delayed increase in temperature, nausea,
clinical trials has not been reported (88). and chills. At higher doses, a dose-related
thrombocytopenia often occurred 2-3 days af-
3.4.2 General Side Effects. No toxicity or ter administration without any effect on plate-
either carcinogenic or teratogenic effects have let function and bleeding time. Increased
been established so far for any fluorocarbon platelet clearance was related to alteration of
(233). No changes in liver and pulmonary platelet surface caused by PFC emulsion
function, no immunologic or allergic reaction, (236). All patients showed spontaneous re-
and no vasoconstriction or hemodynamic vari- gression of the symptoms within hours or
ations were detected (89). In early formula- days.
424 Oxygen Delivery by Allosteric Effectors of Hemoglobin, Blood Substitutes, and Plasma Expanders

Temporary splenomegaly and hepatomeg- nary toxicity related to IPRV has ever been
aly without any evidence of inflammation and reported during PFC infusion in humans.
transient alterations in serum levels of liver
enzymes were described in patients treated 3.4.3 Pharmacokinetics. Once injected into
with PFCs emulsion (233). The duration and the bloodstream, PFC emulsion particles un-
magnitude of these effects appear to be species dergo opsonization (89, 233). They are recog-
specific and dose dependent. No permanent nized by phagocytic cells from the RES and
tissue alteration was observed after autopsy in removed from circulation. No fecal or urinary
animal studies. All changes were found to be elimination was reported, whereas immediate
fully reversible and thought to be a conse- vaporization and exhalation through the
quence of the physiological response to the lungs contribute in minor extent to initial
clearance of PFCs from the bloodstream. In
presence of emulsion particles in the
the RES the surfactant is degraded, whereas
bloodstream.
the PFCs are temporarily stored. No evidence
Phagocytosis of particulate materials re- of metabolism, enzymatic cleavage, oxidation,
sults in stimulation of macrophages in the or free-radicals reactions has ever been re-
spleen and Kupffer cells in the liver and acti- ported for PFCs. Depending on their lipophilic
vation of the arachidonic acid cascade with re- character, these molecules slowly diffuse from
lease of prostaglandins and pyrogenically ac- RES cells back in the bloodstream, carried by
tive cytokines. These effects can be prevented plasma lipids, and finally reach the lungs
by prophylaxis with long-acting cyclooxygen- where they are excreted as vapor in the ex-
ase inhibitors or corticosteroids (89,233). pired air.
The external appearance, mainly deter- A four-compartment model representing
mined by the surfactant, together with the emulsion of PFCs in blood, in RES tissues, in
particle size and the particle size distribution non-RES tissues, and PFCs dissolved in lipids
of the emulsion droplets influence the recogni- was proposed to describe the pharmacokinet-
tion of PFC droplets by the reticuloendothelial ics of perfluorooctyl bromide emulsions (238).
system (RES) cells and eventually the inci- Being slightly soluble in lipids, redistribution
dence and magnitude of adverse effects (89). from primary sites of storage to adipose tis-
New formulations with smaller particle size sues occurs during the first week postdosing
have proved to produce milder or no side ef- (233). The removal from circulation operated
fects, being less detectable by the cells of the by cells in the RES is responsible for the lim-
RES (237). ited intravascular residence of these com-
In some studies in animals, pulmonary tox- pounds. As previously described, emulsion
icity was associated with the administration of characteristics were recognized to greatly in-
PFCs emulsions (89,233). In such cases, lungs fluence the clearance process. Experiments
were reported not to deflate normally. This carried out on rats (237) showed that a de-
phenomenon, referred to as increased pulmo- crease in particle size and a narrow particle
nary relative volume (IPRV), was ascribed to size distribution reduce the scavenging of
retention of air in the alveoli. Interactions be- PFCs droplets through phagocytosis and pro-
tween lung surfactant and perfluorochemicals longs their presence in the bloodstream. Half-
are thought to favor air trapping and be re- life in the bloodstream is further dependent on
sponsible for IPRV. Lung toxicity was found to infused dose. Blood half-life of Fluosol 20%
be species dependent and related to physio- was reported to change from 7.5 to 22 h when
logic and morphologic characteristics. Insensi- the dose increased from 4 to 6 g/kg ( I l l ) ,
tive species generally exhibit large airways di- whereas the circulating half-time of Oxygent
mensions, high transpulmonary pressure, and was shown to vary from 6 h, when the admin-
effective initial RES clearance mechanisms istered dose is 1.2 gtkg, to 9 h for a 1.8 glkg
that reduce perfluorochemicals delivery to the dose (239).
lungs. Because human lungs possess all these A progressive saturation and a consequent
determinants, IPRV does not seem to be oper- loss of efficiency of RES-mediated clearance
ative in human. No evidence of serious pulmo- capacity was suggested to be responsible for
3 Blood Substitutes: Modified Hemoglobins and Perfluorochemicals

Table 8.6 Perfluorochemical Compounds Used as Blood Substitutes


Organ
O%JWn Molecular Retention
Solubility (mL Weight Chemical Time
Chemical Name (Structure) 0J100 mL) (Da) Formula (days) Reference
Perfluorodecalin (10) 40 462 CmFm 7 234
Perfluorodecyl bromide (14) NA 599 C1d721Br 23 232,111
Perfluoro-1,s-dichlorooctane 43 470 Cd7~&12 8 89
(16)
Perfluoro-1,3- 43 610 C11F22N202 55 232
dimorpholinopropane (15)
Perfluoro-N-(4- NA 595 C12F23N 60 232
, methylcyclohexy1)piperidine
I (12)
;
i Perfluorooctyl bromide (13) 50 499 C8F14r 4 235
Perfluorotri-n-propylamine 45 521 C9Fd 65 234
(11)

the increase in intravascular half-life and ac- 3.4.4 Physiology and Pharmacology. Fluo-
cumulation in non-RES tissues when increas- rocarbons are synthetic aromatic or aliphatic
ing the loading (233). compounds in which hydrogen atoms are
The organ persistence increases with in- substituted by fluorine atoms (89). The fluo-
creasing molecular weight within a homolo- rine-carbon bond is the strongest in organic
gous series (240). On the basis of the depen- chemistry. Moreover, fluorine, which is more
dency between excretion rate and molecular electronegative than hydrogen, forms a pro-
weight, a range of molecular weights suitable tective electron coating that reduces reactivity
for PFC administration was established. The of potential functional sites and prevents flu-
lower limit was set at 460 to ensure a vapor orocarbon molecules from chemical attack. As
pressure high enough to prevent risk of lung a result, PFCs are chemically and biologically
emphysema, whereas the value of 520 was
inert under physiological conditions. Liquid
chosen as the upper limit to ensure acceptable
perfluorochemicals are uniquely character-
organ retention times.
No influence of structural properties of ized by weak intermolecular van der Wads
PFCs on excretion rate was established, other forces that facilitate the insertion of gas mol-
than that associated with the change they ecules and account for high dissolving capac-
cause in the molecular weight (240) (Table ity. Thus, the gas is physically dissolved in the
8.6). Linear and cyclic compounds with similar PFCs. This property is not specific for oxygen
molecular weight have comparable organ re- but applies to any low polarity gas, with the
tention time. Addition of heteroatoms to the solubility decreasing with the decrease in mo-
existing carbon chain increases body persis- lecular volume of the solute (241).
tence because they increase the mass units. Among clinically useful PFC compounds,
However, heteroatoms decrease organ reten- carbon dioxide solubility is reported to vary
tion when they replace CF, or CF groups and between 140 and 230 mL per 100 mL, whereas
cause a loss of mass units. Excretion rates oxygen solubility ranges from 30 to 50 mL per
faster than expected on the sole basis of mo- 100 mL, when exposed to a partial pressure of
lecular weight effect are observed for com- 760 mmHg at 25°C (241). Oxygen solubility
pounds that have short hydrogenated frag- values for some of the formulations previously
ments at the end of the chain or atoms such as described are reported: Perftoran dissolves
bromine or chlorine on the terminal carbon. In 6-7 vol % of oxygen (data provided from the
fact, a lipophilic terminus is thought to favor company; see section 5); Fluosol20% (wlv)dis-
the uptake by plasma lipids and to accelerate solves 5.7 vol % (235);Oxygent with 60% PFCs
excretion (89). (w/v) dissolves 17 vol % (235). Oxyfluor con-
Oxygen Delivery by Allosteric Effectors of Hemoglobin, Blood Substitutes, and Plasma Expanders

Figure 8.8. Oxygen binding


curves of whole blood and various
PFC emulsions (with permission T
from Ref. 89). PiO2

taining 76% (wlv)of PFCs dissolves 17.2 vol % The oxygen solubility of PFCs is not influ-
of oxygen (242). enced by changes in temperature (88). Hence,
The oxygen-carrying capacity of PFCs is at the low temperatures experienced during
linearly related to oxygen partial pressure ac- preservation of isolated organs and hypother-
cording to Henry's law (88,89) (Fig. 8.8). For a mic cardiopulmonary bypass the oxygen-deliv-
given oxygen pressure, solubility increases ery capacity of PFCs does not change, whereas
with increasing concentration of perfluoro- Hb displays an increase in oxygen affinity and
chemicals. Therefore, the oxygen content of lower oxygen release. Oxygen delivery by flu-
PFC emulsion can be increased by augment- orocarbons is unaffected by changes of pH
ing either the PFC content in the emulsion or (234). Administration of PFCs can thus be
the oxygen concentration in the inspired air. A useful in case of hemorrhagic shock to ensure
higher partial pressure of oxygen in the in-
tissue oxygenation without loss of efficiency
spired air results in higher arterial oxygen
resulting from acidosis, and to remove carbon
pressure and in a larger oxygen tension gradi-
ent between plasma and tissues, providing an dioxide, thus restoring physiological pH values.
effective driving force to oxygen diffusion. A With a mean diameter of about 0.1-0.3 pm,
higher PFC content increases emulsion viscos- PFC emulsion particles are much smaller than
ity and affects fluidity. Highly concentrated red blood cells (7-8 pm). In larger arteries and
formulations with adequate viscosity for injec- arterioles red blood cells circulate in the cen-
tion were obtained by improved mastery of tral portion of the vessel and PFC droplets are
emulsion technology. Under atmospheric oxy- assumed to flow in the plasma layer close to
gen pressure, even highly concentrated PFC vessel wall (near-wall-excess phenomenon)
emulsions display lower oxygen capacity than (88,89).As the vessels get narrower, the aver-
that of hemoglobin on a gram per gram basis, age hematocrit decreases and the space among
although the absence of chemical bonds be- erythrocytes increases, with droplets filling
tween fluorocarbon molecules and oxygen and the plasma gaps. Hence, in the microcircula-
a large surface area for the gas exchange accel- tion PFC particles may perfuse even those
erate the loading and unloading processes. narrow capillaries that exclude red blood cells,
Thus, extraction ratios are higher for PFCs thus enhancing tissue oxygenation and pre-
(90%)than for Hb (2540%)and oxygen carried venting local ischemia resulting from inade-
by PFCs is more readily available. As a conse- quate perfusion during major blood loss or se-
quence, when both PFCs and hemoglobin are vere anemia. Moreover, treatment with PFCs
present, oxygen is preferentially released from during cardiopulmonary bypass may have a
PFCs and oxygen bound to Hb is preserved. protective effect against neurological deficits
3 Blood Substitutes: Modified Hemoglobins and Perfluorochemicals 427

caused by brain hypoxia, because PFCs act by withdrawn to reach relatively low hemoglobin
both favoring gaseous microemboli resorption levels before surgery and it is successively re-
and improving oxygenation of ischemic areas. infused during surgery or in the postoperative
In addition to oxygen transport, PFCs are period. During the operation, solutions of vol-
thought to contribute to oxygen transfer be- ume expanders are infused to replace collected
tween red blood cells and tissues. In fact, gas blood. When surgical bleeding further de-
exchange between blood and tissues requires creases hemoglobin concentration, patients
oxygen to diffuse through the so-called car- are treated with a perfluorocarbon emulsion
rier-free-region (plasma, endothelium, and in- that allows preservation of tissue oxygen-
terstitium) (243). Under normal circum- ation. Preclinical experiments showed that
stances no specific oxygen carrier is present in PFC-treated animals can be subjected to more
this region and the diffusion rate is limited by profound hemodilution. Acute normovolemic
oxygen solubility in plasma. Because fluoro- hemodilution supplemented by administra-
carbon molecules distribute in the acellular tion of a perfluorocarbon-based oxygen carrier
fraction, they increase oxygen solubility in and 100% oxygen ventilation was tested in
plasma and favor oxygen diffusion from red Phase 3 clinical trials for Oxygent and was
blood cells to tissues, acting as "stepping reported to be effective in reducing the need
stones" (89). for blood transfusion (246).
3.4.4.1 Hemodynamics. Hemorrhage, sur-
gical bleeding, anemia, or hemodilution re- 3.4.5 Structure-Activity Relationship. The
duce red blood cell mass. Consequently, blood gas-dissolving capacity of PFCs was attributed
viscosity and arterial oxygen content decrease. to the existence of cavities within the solvent
Thus, the stroke volume increases, leading to where the solute molecules can be hosted (240,
higher cardiac output and the total oxygen 241). Both molecular weight and chemical
consumption is maintained. With more severe structure of fluorocarbons influence oxygen-
levels of anemia there is a critical point at dissolving capacity (Table 8.6). Oxygen solu-
which the compensatory mechanisms cease to bility decreases with increasing molecular
be effective and oxygen consumption becomes weight within a homologous series. Linear
supply dependent (88). compounds have higher oxygen-dissolving ca-
Infusion of PFCs was proposed to be able to pacity than that of cyclic and aromatic com-
provide adequate tissue oxygenation, nor- pounds with similar molecular weight. This
mally achieved through blood transfusion. Ad- reduction in the amount of dissolved oxygen
ministration of PFC-based oxygen carriers appears to correlate with the decrease in cav-
was seen to increase plasma dissolved oxygen ity size. In fact, linear structures are thought
content, tissue oxygenation, and mixed ve- to create larger channels, whereas inclusion of
nous oxygen partial pressure. No negative ef- solute molecules seems to be less favorable in
fect on hemodynamics was observed. No planar structures that form closely inter-
change in microcirculation perfusion and vas- locked layers. Similarly, the presence of a dou-
cular resistance was reported. PFCs do not in- ble bond in the central position confers higher
terfere with compensatory cardiac output in- dissolving capacity over that of saturated an-
crease in response to hemodilution or anemia. alogs or unsaturated compounds with double
Oxygen delivery benefits by the increase in bond at the end of the chain because it seems
cardiac output, given that blood circulates to create a "notch" in the PFC skeleton and to
faster and reoxygenation of PFCs in the lungs facilitate cavity formation.
occurs more frequently (88).
A new strategy to preserve patients' blood 3.4.6 Emulsion Stability. Emulsions do not
and to minimize the need for donor blood form spontaneously and may break down into
transfusion consists of infusion of PFCs in separate phases, when subjected to increasing
t conjunction with acute normovolemic hemodi- temperature, alteration in salt composition,
lution. In Alliance's patented procedure, interfacial film structure, or decrease in vis-
termed "Augmented-Acute Normovolemic cosity of the continuous phase that may occur
i Hemodilution A-ANH" (244, 2451, blood is during heat sterilization, handling, and stor-
428 Oxygen Delivery by Allosteric Effectors of Hemoglobin, Blood Substitutes, and Plasma Expanders

age. Coalescence is one of the phenomena re- diblock compounds of the CnF2,+lCmH2m+l
sponsible for emulsion separation in two type can be added to phospholipids. The pres-
phases (234, 240). The other major cause of ence of a fluorinated portion and of a hydroge-
coarsening of emulsion particles is molecular nated segment reinforce the adhesion be-
diffusion or Ostwald ripening. Because of the tween phospholipids and PFCs droplets (89,
Kelvin effect, individual molecules of PFCs 111,247) because they act as a "dowel" at the
tend to leave the smaller droplets, diffuse interface.
through the external phase, and join the
larger ones, with an increase in the average 3.4.7 Recent Developments. Great effort
particle sizes. The droplet growth essentially has been made to solve the stability issue of
depends on the particle size and on the type of classical fluorocarbon-based emulsions. As
PFC. Solubility and diffusibility of PFCs in the previously described, different strategies have
continuous phase increase with decreasing been adopted to reduce emulsion degradation.
molecular weight. In addition, other factors Microemulsions were considered as alterna-
such as a highly dispersed particle size distri- tives to classical formulations because they
bution and an increasing temperature are are thermodynamically stable systems (111,
thought to favor Ostwald ripening and con- 234, 235). Unlike macroemulsions, they are
tribute to destabilize the emulsion. transparent and form spontaneously when a
Given that emulsion particle size was rec- suitable surfactant is added to a mixture of
ognized to play a major role in determining the water and fluorocarbons. Different strategies
persistence of PFCs in the bloodstream, caus- have been pursued in the attempt to develop
ing adverse effects and affecting oxygen deliv- microemulsions to be used as blood substi-
ery efficiency, improvement of emulsion sta- tutes. In one case, fluorocarbons were the ba-
bility by hindering molecular diffusion has sis for microemulsions (248). Fluorinated sur-
become a central issue in the development of factants were required because fluorinated
the formulation of PFC-based oxygen carriers. and hydrogenated chains tend to segregate.
Different strategies were adopted (111). Small Many new surfactants were synthesized and
amounts of high molecular weight PFCs, such examined in pursuit of biocompatible com-
as perfluorotri-n-propylamine and perfluoro- pounds. Different combinations of fluorinated
N-(4-methylcyclohexyl)piperidine,were added surfactants and fluorocarbons have been
in Fluosol and Perftoran, respectively, to act tested to elucidate physicochemical and bio-
as stabilizer and to reduce solubility in the logical properties of these ternary mixtures.
aqueous phase. Unfortunately, while enhanc- Microemulsions of water, fluorocarbon, and
ing emulsion stability, high molecular weight perfloroalkylpoly(oxyethylene) surfactants
prolongs PFCs body persistence. In Oxygent, have been reported to dissolve oxygen but
this problem was circumvented by the use of their toxicity profile has not been yet clearly
perfluorodecyl bromide as additive PFC. De- established (234).
spite its higher molecular weight, it is rapidly Recently, a different approach has been fol-
excreted, given the lipophilic character con- lowed to circumvent the difficulty of finding a
ferred by the bromine atom. Improvement of nontoxic fluorinated surfactant. Microemul-
stability may also be achieved using fluori- sions were produced by mixing partially fluor-
nated surfactants. These surfactants have one inated compounds with hydrogenated non-
or more F-alkyl hydrophobic chains and are ionic surfactants that were known to be
expected to be better emulsifiers for PFC biocompatible (248). The fluorinated portion
emulsions because they are more surface ac- conferred to them an oxygen-dissolvingcapac-
tive than their hydrogenated analogs. Thus, ity, whereas the hydrogenated part enabled
they reduce the interfacial tension between them to be microemulsified. Micwemulsions
water and PFCs to lower values. Several mol- containing water, Montanox 80, a widely used,
ecules have been synthesized and proved to be low-toxicity surfactant of the polysorbate fam-
effective, although their pharmacology and ily, and C,F,,CH,CH=CHCHC,H, were
toxicity still have to be clearly evaluated (111, found to dissolve oxygen. The measured
247). Alternatively, fluorocarbon-hydrocarbon amount of dissolved oxygen was higher than
4 Plasma Volume Expanders

the theoretical one, suggesting that micellar tion being other globulins. All protein solu-
organization might enhance oxygen-carrying tions are extracted from human donor plasma
capacity. The biocompatibility of these dis- and heat-treated to eliminate the risk associ-
perse systems has still. to be completely as- ated with viral or bacterial contamination.
sessed but preliminary toxicity tests are en- Hydroxyethyl starch (HES) is a synthetic
couraging and show promise for the polymer produced by hydroxyethyl substitu-
development of microemulsions as blood sub- tion at the C2, C3, and C6 positions on the
stitutes. glucose residues of amylopectin polymers. Dif-
ferent degrees of substitution can be obtained
4 PLASMA VOLUME EXPANDERS by varying the reaction time with ethylene ox-
ide, whereas the duration of acid hydrolysis
4.1 Clinical Use affects the molecular weight distribution. The
solution results in a heterogeneous mixture of
Plasma volume expanders are infused to re-
place blood losses and maintain intravascular particles with different molecular weights and
degrees of hydroxylation. Its weight-average
; volume and tissue perfusion. Plasma volume
molecular weight is about 450,000 Da. It is
is regulated very finely through several com-
plex mechanisms. Reduction in normal circu- clinically available as a 6% solution in normal
lating blood volume may occur as a consequence saline. Many different formulations of HES
of hemorrhagic trauma, surgical procedures, with lower molecular weights and degrees of
or blood-salvage strategies, such as acute nor- substitution are under development. Among
movolemic hemodilution. In these situations, these, pentastarch (weight-average molecular
changes in blood volume cannot be immedi- weight, 264,000 Da) has been used as plasma
ately compensated for by the regulatory mech- expander in Canada and some European coun-
anisms. Therefore, volume therapy is aimed at tries. In the United States, this product is ap-
temporarily increasing plasma volume until proved only for use in leukapheresis. It is
those mechanisms can correct the hypovole- available as a 10% solution.
mia. Different products are available for the Dextrans are glucose polymers produced by
therapy based either on crystalloids or col- the bacterium Leuconostoc mesenteroides
loids. The choice of the appropriate agent in when grown on a sucrose media. The most
the treatment of hypovolemia has not yet been common preparations for clinical use are dex-
univocally settled. Plasma expanders have tran 40 (weight-average molecular weight,
been evaluated in conjunction with perfluoro- 40,000 Da) and dextran 70 (weight-average
chemicals and hemoglobin-based oxygen car- molecular weight, 70,000 Da). They are pro-
riers to reduce the need for blood transfusion duced by acid hydrolysis of the parent mole-
1
and ensure tissue oxygenation. cules and are polydisperse. Dextran 40 is avail-
i
able as a 10% solution and dextran 70 is
4.1.1 Current Drugs on the Market. Two formulated as a 6% solution.
families of products have been used for this Gelatins used as plasma expanders are
purpose: crystalloids and colloids. Crystalloids chemically modified derivatives of bovine col-
consist of electrolytes (e.g., sodium, chloride, lagen. The thermally degraded and chemically
and potassium) in water solutions. The most hydrolyzed collagen can be crosslinked with
frequently used crystalloids are normal saline hexamethyl di-isocyanate to form urea-
and Ringer's solutions. Different commercial bridged gelatin or can be treated with succinic
formulations are available, varying in electro- anhydride to form succinylated gelatins. The
lyte concentration, tonicity, and osmolarity. former is reported to have a weight-average
The class of colloids includes albumin, molecular weight of 35,000 Da and it is avail-
starches, dextrans, and gelatins (Table 8.7). able as a 3.5% solution. The weight-average
Albumin is available as 5% and 25% solutions molecular weight of the latter is 30,000 Da and
in isotonic saline. A purified protein fraction is it is formulated as a 4% solution. They are
also commercially available and consists of at commonly available in Europe and Australia,
; least 83% albumin, with the remaining frac- but not at present in the United States.
430 Oxygen Delivery by Allosteric Effectors of Hemoglobin, Blood Substitutes, and Plasma Expanders

Table 8.7 List of Plasma Volume Expanders


Trade
Generic Name Chemical Class Name Originator Dosea
Albumin Protein Buminate Baxter
Healthcare Co.
Albuminar Aventis Behring
Albutein Alpha
Therapeutics
Co.
Dextran 40 Modified polysaccharides Gentran Baxter
40 Healthcare Co.
LMD Abbott
Dextran 70 Modified polysaccharides Gentran Baxter
70 Healthcare Co.
Hydroxyethylstarch Modified polysaccharides Hespan B. BraunMcGaw
Hetastarch Gensia Sicorl
Baxter
Hextend BioTime Inc.1
Abbott
Modified bovine collagen Haemaccel Aventis
gelatin
Succinylated Modiiied bovine collagen Gelafundin B. Braun
gelatin
"The reported dose is the madmum recommended dose (251).

4.1.2 Side Effects. Overload is a general ad- of lactate to bicarbonate, both following mas-
verse effect of administration of plasma ex- sive infusion of Ringer's lactate solution.
panders and is independent of the infused Generally, hypertonic solutions do not
fluid, given that it is generally the result of cause edema but their higher serum osmolar-
inadequate monitoring of the circulatory sys- ity and sodium and chloride levels may cause
tem. Hypervolemia caused by colloids is more metabolic acidosis and hypokalemia. Colloids
difficult to treat than is crystalloid overload are contraindicated for volume replacement in
(249). Crystalloids are considered to be safe clinical situations where endothelial perme-
and devoid of toxic or allergic reactions (250). ability is altered because colloids may leak into
The principal complications are progression of the interstitial space. They exert an osmotic
shock and renal failure when inadequate vol- gradient and withdraw water from the vascu-
umes are infused, and tissue and pulmonary lar space to the interstitium, causing tissue
edema consequent to excess volume infusion. and pulmonary edema (251).
When large volumes of crystalloids are in- Allergic reactions were reported for the
fused, electrolyte abnormalities have to be whole class of colloids, with different com-
considered. A mild alkalosis may occur with pounds varying in frequency of occurrence
Ringer's lactate solution because of lactate be- and severity (252). Incidence of allergic reac-
ing metabolized to bicarbonate. Administra- tions for dextran 40 and dextran 70 is reported
tion of sodium chloride solution may cause hy- to be 0.007 and 0.069%, respectively (252),al-
perchloremia or hypernatremia. Adverse though according to other authors it ranges
effects may occur in specific patients in rela- from 0.03 to 5% (250). Allergy to dextrans is
tion to specific pathological status, such as hy- thought to involve immune complex reactions
perkalemia in patients with renal failure or attributed to preexisting antibodies against
lactate accumulation in patients with liver dextrans stimulated by exposure to these poly-
failure because of impairment in metabolism saccharides produced by bacteria or present in
4 Plasma Volume Expanders

food products. Monovalent hapten-dextran


-
motic pressure to a level sufficient to offset the
was used to bind the antibody and reduce risk hydraulic filtration pressure and suppress fil-
of allergy. Other factors are probably required tration (256). The precise mechanism of renal
for severe reactions to occur, the nature of toxicity is not known and it has not been
which remains unclear. clearly established whether all plasma ex-
Urticaria, fever, chills, or hypotension have panders cause renal dysfunction.
been observed after infusion of albumin. Over- All colloids have been reported to alter co-
all incidence of allergic reaction is 0.011% agulation profiles primarily by hemodilution
(252). Because the purified protein fraction (250, 251). Blood coagulation is mostly im-
contains greater quantities of kinins or pre- paired by dextran and HES. The effects of the
kallikrein activator, it carries a greater risk of latter was shown to depend on its molecular
hypotension than does human serum albumin weight and rate of elimination. High molecu-
(253).Allergic reactions to HES were reported lar weight and highly substituted HES seem to
to occur rarely, with an incidence of 0.085% have detrimental effects on coagulation, given
(252). No evidence of any histamine-mediated that larger molecules are slowly degraded and
process was found and the presence of anti- persist longer in the plasma. It seem less likely
HES antibody was not correlated with allergy that rapidly degradable HES induces coagula-
(249). tion disorders (257). Thrombocytopenia, pro-
Gelatin was associated with allergic reac- longation in partial thromboplastin time,
tions with incidence estimated at 0.146% for bleeding time, and prothrombin time were ob-
urea-bridged gelatins and at 0.066% for modi- served in patients treated with hetastarch.
fied fluid gelatine (252). Histamine release HES is reported to impair the coagulation sys-
was associated with rapid infusion, probably tem through a significant drop in factor VIIII
because of a direct effect of gelatins on mast von Willebrand factor complex, to reduce
cells and basophils (249, 250). True anaphy- platelet adhesiveness, and to produce less sta-
lactic reactions may " also result from mediator ble thrombus because it accelerates the con-
activation induced by immune complex be- version of fibrin to fibrinogen. However, when
tween gelatin and antigelatin antibody (254). the infused dose is lower than 20 mL kg-'
After infusion of albumin solution in day-*, coagulation parameters seem not to be
trauma and burn-injured patients, different significantly affected (251).
studies reported a decrease in glomerular fil- Both dextrans 40 and 70 affect coagulation
tration rates (GFR) and renal function and a (250). The high molecular weight dextrans ex-
lower urine output, despite a 40% increase of ert more detrimental effects. They are re-
plasma volume (251). The mechanism of albu- ported to prolong bleeding time and to reduce
min-induced reduction in GFR is unknown, platelet adhesiveness and aggregation result-
although it may be attributable to an increase ing from a decrease of factor VIII. Thus, hemo-
in the oncotic pressure within the peritubular stasis abnormalities are similar to those seen
vessels that may cause a decrease in sodium in type I von Willebrand syndrome. In addi-
and water excretion. tion, they are reported to coat blood vessel wall
The effect of plasma expanders on renal and cellular elements and to impair polymer-
function is still a debated issue. Development ization of fibrin, elasticity, and tensile
of acute renal failure was described after infu- strength of fibrin clots. Clinically significant
sion of dextrans 40 (255), which might be be- disturbance of hemostasis is unlikely to occur
cause of an accumulation of low molecular when the infused dose is less than 1.5 g kg-'
weight fractions in the tubule. Consequently, day-' or 20 mL/day. No effect on hemostasis
highly viscous urine is produced and dextrans other than hemodilution was observed for gel-
may precipitate, causing renal damage. Renal atins. Hemostatic effects of albumin other
dysfunctions or clinical conditions that aug- than those related to hemodilution are still
ment urine viscosity might worsen the situa- controversial.
tion (250). It was suggested that an accumula- Trace amounts of aluminum may contami-
tion in plasma of any unfilterable, osmotically nate albumin solutions and aluminum toxicity
active substance might raise the colloid os- may represent a risk for premature infants
432 Oxygen Delivery by Allosteric Effectors of Hemoglobin, Blood Substitutes, and Plasma Expanders

and patients subjected to long-term infusion Because polydisperse formulations of dex-


of this colloid (258). Furthermore, because al- trans are composed of different molecular
bumin binds calcium and reduces the fraction weight fractions, each of them exhibits its own
of ionized calcium, hypocalcemia and myocar- pharmacokinetic profile (260). Smaller mole-
dial depression may arise when albumin ther- cules undergo renal excretion or they may en-
apy is used for fluid resuscitation (259). ter the interstitial space and return to the
bloodstream through lymphatic drainage.
4.1.3 Pharmacokinetics. The pharmacoki- Larger polymers persist longer in the blood-
netics profile of exogenous albumin follows stream and are successively stored in hepatic,
that of the endogenous protein (250, 251). Its renal tubular, or reticuloendothelial system
serum half-life has been reported to be 16 h. cells (250).Dextrans are slowly metabolized to
Albumin infused in the intravascular space carbon dioxide and water by dextranase local-
gradually leaves the bloodstream and redis- ized in spleen, liver, lung, and kidney (260).
tributes to the interstitial sites. About a third The variety of routes of elimination and the
of albumin is in the intravascular space, influence of molecular weight on excretion
whereas extravascular interstitial sites con- profiles account for the complexity of pharma-
tain 60% of total albumin. The normal rate of cokinetics of dextrans. Following intravenous
transcapillary escape is 5%/h, but can be administration of dextran 40, approximately
higher in many pathological states. Some al- 60% of the dose given is eliminated within 6 h,
bumin is tissue bound, whereas the free pro- whereas administration of dextran 70 results
tein may reenter the intravascular space in 35% of the dose cleared within 12 h (251).
through lymphatic circulation to maintain se- Similarly to other heterogeneous mixtures,
rum levels and compensate for transcapillary low molecular weight molecules of gelatins are
leak and catabolism. The reticuloendothelial rapidly cleared by glomerular filtration and
system plays a major role in the removal of exhibit short half-lives (251). Small amounts
albumin from the body and metabolism. Ap- move from the intravascular compartment
proximately 10% of albumin is degraded daily into the interstitial space, returning to the
to amino acids and returns to the liver. bloodstream through the lymphatic circula-
Pharmacokinetics of HES is complex be- tion. Gelatins may also be cleaved by proteases
cause of its heterogeneity in size and substitu- into small peptides and amino acids. No accu-
tion (250,251,260). Clearance rate and serum mulation in the body has been reported (260).
half-life are both affected by hydroxyethyla-
4.2 Physiology and Pharmacology
tion and molecular weight. The main route of
elimination of hydroxyethyl starches is uri- Body water is divided into compartments: in-
nary. Smaller molecules are rapidly excreted tracellular water accounts for 70% of total
in urine. Larger particles remain in the circu- body water, whereas the extracellular com-
lation longer, where they can be either par- partment contains 30% of total body water
tially hydrolyzed to small- or medium-sized and includes the interstitial space (75% of the
molecules by serum a-amylase or slowly re- extracellular fluid) and the intravascular
moved by the reticuloendothelial system, space (25%). Intracellular and extracellular
where they accumulate. The extent of enzy- fluids have the same osmolarity. Thus, when
matic degradation depends on the degree of extracellular osmolarity changes, movement
hydroxylation because the added alcohol of water between these two sectors occurs. Iso-
groups protect from enzymatic cleavage. Me- tonic solutions have the same osmolarity as
tabolism of hydroxyethyl starches was re- that of body fluids and distribute between the
ported to occur without formation of glucose intravascular and the interstitial spaces. Gen-
or hydroxyethyl glucose (261). Because the erally, the volume of isotonic solution required
dispersion of the molecular weight of the mol- for fluid replacement has to be four to five
ecules changes over time, several phases of times greater than the volume of blood loss
elimination with different corresponding half- (250). The effect is relatively short lasting, so
lifes have been reported (260). Accumulation that significant amounts of additional solution
of HES within the tissues was reported (262). are required over time. Hence, the use of iso-
5 Web Site Addresses

tonic solutions results in the increase of the volume expansion. Thus, a 25% albumin solu-
volume of interstitial fluid and causes a cer- tion has an oncotic pressure of 100 mmHg and
tain degree of edema formation, attributed to determines a n increase of 300-500 mL in in-
accumulation of fluids in the interstitial space travascular volume for 100 mL of infused so-
that is not completely drained by the lym- lution. A 5% solution has a colloid osmotic
phatic circulation. pressure of 19 mmHg and increases the intra-
Crystalloids have been tested in different vascular volume by the same amount of vol-
clinical studies and were reported to be effec- ume infused. Reported oncotic pressures for a
tive in resuscitation, even if larger volumes 6% hetastarch, 10% pentastarch, and 10%
were reauired and resuscitation times were dextrans 40 are about 28, 40, and 30 mmHg,
longer compared to those of colloids. Hyper- respectively (251).
tonic crystalloids increase the osmolarity of In a polydisperse mixture, the low molecu-
extracellular fluids, resulting in the move- lar weight fraction exerts a greater initial on-
ment of water from the intracellular space to cotic effect that tends to be short lasting be-
the interstitial and vascular compartments. cause of the rapid renal elimination. Larger
Thus, they expand the extracellular fluid vol- molecules generate a smaller colloid osmotic
ume by a greater amount than the volume in- pressure that is sustained longer, given that
fused and less fluid has to be administered they persist longer in the circulation (250,
than that using isotonic crystalloid solutions 251).
(250). In addition, dextrans have been reported to
The concentration of proteins is much alter the viscosity of whole blood. The effect
higher in plasma than that in the interstitial depends on molecular weight of the polymers.
space because membranes between the two Aggregation of red blood cells occurs when
compartments allow for only limited passage. dextrans with molecular weight higher than
According to Starling's law, the factor that de- 60,000 Da are administered. Below this value,
termines flows of fluid between the intravas- dextrans tend to disaggregate them, thus im-
cular and interstitial spaces and regulates proving peripheral flow properties especially
compartment volume is the difference be- under abnormal circulatory conditions (251).
tween the oncotic pressure gradient generated
by the asymmetric distribution of colloids on
5 WEB SITE ADDRESSES
each side of the endothelium and the opposing
hydrostatic pressure gradient. The primary
Alliance Pharmaceutical Corp. (San Diego,
protein in human plasma, albumin, comprises
7540%of the normal colloid oncotic pressure, CA, USA): http://www.allp.com
0 Allos Therapeutics (Denver, CO, USA):
and about 50-60% of the protein content.
Albumin is produced in the liver and its syn- http:/h.allos.com
thesis is regulated to keep colloid osmotic Apex Bioscience Inc. (Research Triangle
pressure constant. At physiological concentra- Park, NC, USA):
tions, albumin ensures an oncotic pressure of http://www.apexbioscience.com
about 18 to 22 mmHg. Administration of col- Baxter Healthcare Corporation (Deerfield,
loids in the vascular compartment increases IL, USA): http://www.baxter.com
the oncotic pressure gradient and draws water 0 Biopurem Corporation (Cambridge, MA,
from the interstitial space. Because osmotic USA): http://www.biopure.com
pressure depends on the number of particles
Enzon Inc. (Piscataway, NJ, USA): http://
in solution, solutions containing larger
www.enzon.com
amounts of colloids or particles with lower mo-
0 Hemosol Inc. (Toronto, Canada): http://
lecular weight exert a higher colloid osmotic
pressure and result in a more effective volume www.hemosol.com/home_page.html
restoring (251). Solutions isooncotic with 0 Northfield Laboratories Inc. (Evanston, IL,

plasma exert an initial volume expansion ap- USA): http://www.northfieldlabs.com


proximately equal to the volume infused, 0 Perftoran (Pushchino, Russia): http://
whereas hyperoncotic ones produce higher perftoran.ru/
434 Oxygen Delivery by Allosteric Effectors of Hemoglobin, Blood Substitutes, and Plasma Expanders

Sangart (San Diego, CA, USA): http://www. M . F. Perutz and C. Poyart, Lancet, 2,881-882
sangart.com/ (1983).
SynZyme Technologies LLC (Irvine, CA, C. Ropars, M. Chassaigne, and G. Avenard,
USA): http://www.synzyme.com Med. Biol. Eng. Comput., 36,508-512 (1998).
D. J . Abraham, M . F. Perutz, and S. E. Phillips,
Proc. Natl. Acad. Sci. USA, 80, 324-328
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Chichester, UK, 1988, pp. 94-129. Kim, G. Onesti, 0. Ramirez, and A. N. Brest,
J. G. Riess, Vox Sang., 61,225-239 (1991). N. Engl. J. Med., 277,1113-1118 (1967).
M. G. Scott, D. F. Kucik, L. T. Goodnough, and M. Moran and C. Kapsner, N. Engl. J. Med.,
T. G. Monk, Clin. Chem., 43, 1724-1731 317,150-153 (1987).
(1997). J. Treib and J. F. Baron, Intens. Care Med., 25,
P. E. Keipert, S. Otto, S. F. Flaim, J. G. Weer, 258-268 (1999).
E. A. Schutt, T. J. Pelura, D. H. Klein, and T. L. G. L. Klein, Nutr. Rev., 49,74-79 (1991).
References 441

259. S.G.Kovalik, A. M. Ledgerwood, C. E. Lucas, RECOMMENDED READINGS


and R' F' Higgins' J'Trauma' 219 275-279 Chang, T. M. S.,Blood Substitutes: Principles, Meth-
(1981).
ods, Products and Clinical Trials, Vols. 1-11,
260. U.Klotz and H. Kroemer, Clin. Pharmacoki- Karger Landes System, Basel, Switzerland,
t
net., 12,123-135(1987). 1997, 1998.
261. J. D. Hulse and A. Yacobi, Drug Intell. Clin.
6 Pharm., 17,334341(1983). Riess, J. G.,Chem. Rev.101,2797-2920 (2001).
262. C.Sirtl, H. Laubenthal, V. Zumtobel, D. Kraft, Winslow, R. M., Vandegriff, K. D., and Intaglietta,
and W. Jurecka, Br. J. Anaesth., 82,510-515 M . , Eds., Blood Substitutes: New Challenges,
(1999). Birkhaeuser, Boston, 1996.
Inhibition of Sickle Hemoglobin
Polymerization as a Basis for
~ h e r a ~ e u t Approaches
ic to
sickle--cell Anemia
CONSTANCE TOM NOGUCHI
ALAN N. SCHECHTER
National Institute of Diabetes and Digestive and Kidney Diseases,
National Institutes of Health
Laboratory of Chemical Biology
Bethesda, Maryland

JOHN D. HALEY
OSI Pharmaceuticals Inc.
Farmingdale, New York

DONALD J. ABRAHAM
Virginia Commonwealth University
Department of Medicinal Chemistry
Richmond, Virginia

Contents
1 Introduction, 444
2 Pathophysiology of Sickle-Cell Anemia, 444
2.1 Abnormal Hemoglobin, 444
2.2 Abnormal /3-Globin Gene, 445
2.3 Hemoglobin and Oxygen Binding, 447
2.4 Hemoglobin Molecular Size and Nonideality,
447
2.5 Sickle Hemoglobin Polymerization, 447
2.6 Hemoglobin S Polymerization and Disease
Manifestations, 448
2.7 Hemoglobin S Polymer Structure, 449
2.8 Hemoglobin S Polymerization Kinetics, 450
2.9 Deoxygenated Hemoglobin S Solubility and
Hemoglobin Polymerization, 450
2.10 Dense SS Erythrocytes and Irreversibly
Burger's Medicinal Chemistry and Drug Discovery Sickled Cells, 451
Sixth Edition, Volume 3: Cardiovascular Agents and 3 Modifiers of Sickle-Cell Anemia and the Sickle-
Endocrines Cell Disease Syndromes, 452
Edited by Donald J. Abraham 3.1 Origins of Sickle-Cell Anemia, 452
ISBN 0-471-37029-0 O 2003 John Wiley & Sons, Inc. 3.2 Fetal Hemoglobin, 452
443
444 Inhibition of HbS Polymerization as a Basis for Therapeutic Approaches to Sickle-Cell Anemia

3.3 Genetic Modifiers of Sickle-Cell Anemia, 453 5.2 P- and E-Selectin and Sickle-Cell Animal
3.4 Sickle-Cell Anemia and a-Thalassemia, 454 Models, 462
3.5 Sickle-Cell Anemia and P-Thalassemia, 456 5.3 Improved Intravascular Blood Flow and
3.6 The "Sparing" Effect of Fetal Hemoglobin, Oxygen Delivery, 462
456 5.4 Flocor and Sickle-Cell Anemia, 462
3.7 Sickle-Cell Anemia and Hereditary 5.5 Nitric Oxide and Sickle-Cell Anemia, 462
Persistence of Fetal Hemoglobin, 457 6 Therapeutic Induction of Hemoglobin F, 463
3.8 Sickle Trait, 457 6.1 5-Azacytidine, 464
3.9 Physiologic Modifying Factors, 458 6.2 Hydroxyurea, 464
3.10 Interactions with Endothelium, 458 6.3 Butyrate, 465
3.11 Animal Model Systems, 459 7 Bone Marrow Transplantation, Globin Gene
4 Rational Approaches to Sickle-Cell Therapy, 459 Expression, and Gene Transfer, 465
4.1 Inhibition of Hemoglobin S Polymerization, 7.1 Allogeneic Bone Marrow Transplantation,
459 465
4.2 Cyanate and Sickle-Cell Anemia, 460 7.2 Regulation of Globin Gene Expression, 466
4.3 Chemical Modifiers of Hemoglobin S, 461 7.3 Modification of Endogenous Globin Gene
4.4 Vanillin and Hemoglobin S Polymerization, Expression, 468
461 7.4 Gene Transfer of Recombinant Globin Gene
4.5 Increasing Sickle Erythrocyte Hydration and Vectors, 468
Membrane Active Agents, 461 7.5 Modification of Hematopoietic Stem Cell
5 Therapeutic Decrease of Microvascular Response, 469
Entrapment, 462 7.6 Improvement in Gene Transfer Technology,
5.1 Antiendothelial Receptor Antibodies, 462 470

1 INTRODUCTION acute painful crisis, organ damage, and ge-


netic inheritance of the disease (2-5). Red
Understanding the molecular defect in sickle- blood cells from individuals with sickle-cell
cell anemia has led to the most important anemia undergo morphologic deformation
current strategies for disease therapy, based from the normal biconcave-shaped disk when
on inhibiting intracellular polymerization of deprived of oxygen, many adopting the char-
sickle hemoglobin leading to pathophysiology. acteristic sickle shape (Fig. 9.lb) (6, 7). Al-
There have been more than 50 years of inves- though much of the early focus was on the
tigation of the biochemical and biophysical distortion of the red cell, it is now known that
processes associated with sickle-cell anemia. the important molecular lesion occurs intra-
Under current investigation are new thera- cellularly, as shown by electron microscopy
peutic strategies based on molecular interac- (Fig. 9 . 1 ~ (6).
)
tions of the abnormal hemoglobin, the patho-
physiologic behavior of the sickle cells in the 2.1 Abnormal Hemoglobin
circulation, and the genetic program of the ab- Hemoglobin makes up 98% of the intracellular
normal erythroid cells. protein of the red blood cell. In 1949 Linus
Pauling and colleagues determined that the
2 PATHOPHYSIOLOGY OF SICKLE-CELL primary defect in sickle-cell anemia, giving
ANEMIA rise to the characteristic change in cell shape,
was not the result of an inherent membrane
Almost a century ago, James B. Herrick noted defect, but was a consequence of a molecular
"peculiar, elongated and sickle shaped" red change on the hemoglobin molecule itself (8).
blood cells in a West Indian student with se- This molecular defect resulted in a change in
vere anemia (Fig. 9.la) (1). Before the under- the overall charge of the hemoglobin molecule
standing of the molecular basis of the disease, that was discernable by a change in electro-
this characteristic morphology observed by phoretic mobility (Fig. 9.2a). This change dif-
light microscopy was a useful diagnostic tool ference between sickle hemoglobin (HbS) and
in efforts to understand the chronic anemia, normal hemoglobin A (HbA) has become the
2 Pathophysiology of Sickle-Cell Anemia

acids and is encoded in a gene cluster located


on chromosome 16pter-p13.3 (12-14). The
P-globin chain is 144 amino acids long and is
encoded in a gene cluster on chromosome
llp15.5 (15-17). The substitution of a valine
residue for glutamic acid in the sixth amino
acid of each of the P-globin chains is the mu-
tation in HbS and accounts for the change in
electric charge between HbS and HbA (Fig.
9.2b). However, the mutation also causes a
change-a marked decrease in solubility upon
deoxygenation (18, 19)-that accounts specif-
ically for the pathophysiology of the disease.
2.2 Abnormal P-Globin Gene
In the adult, red cells are produced in the bone
marrow. Hematopoietic stem cells are acti-
vated by a variety of cytokines. Stimulation of
differentiation along the erythroid lineage re-
quires erythropoietin, a hematopoietic cyto-
kine produced principally by the kidney (20-
22). Erythropoietin binding to the surface of
erythroid progenitor cells promotes their sur-
vival, and proliferation and differentiation
leading to the production of red blood cells
(Fig. 9.3a). During terminal differentiation,
Figure 9.1. Sickle erythrocytes. (a) James Herrick the erythroid precursor cells cease to divide
was the first to report elongated and other mal- and lose their nuclei, leading to reticulocyte
formed sickle erythrocytes that were observed in formation, whereas protein synthesis contin-
blood taken from a dental student from Grenada.
ues with translation of the globin genes and
[From J. B. Herrick, Arch. Intern. Med., 6, 517
(1910).1 (b) Scanning electron micrographs of hemoglobin production before formation of
oxygenated (left) and deoxygenated (right) sickle the mature red blood cell. With the identifica-
erythrocytes from an individual with homozygous tion of the messenger RNA (mRNA)for p-glo-
sickle-cell anemia (SS)show the characteristic mor- bin, a single nucleotide substitution in the
phologic difference between the normal discoid sixth codon of the p-globin gene of GTG for
shape of the oxygenated erythrocyte and the classic GAG was determined as the genetic lesion in
sickle shape of the deoxygenated SS erythrocyte. (c) sickle-cell anemia (23). Sickle-cell disease cor-
Transmission electron micrograph of a deoxygen- responds to the homozygous (SS)condition for
xted SS erythrocyte shows long fibers of intracellu- this p-globin A to T nucleotide mutation;
lar hemoglobin polymer.
sickle-cell trait (AS) corresponds to the het-
erozygous or carrier state for this mutation
basis of the current primary diagnostic tool for (5).
sickle-cell anemia (9, 10). Vernon Ingram The HbS mutation is located on the surface
Identified the genetic alteration giving rise to of the hemoglobin molecule and HbS exhibits
HbS as an amino acid substitution on one of similar binding to oxygen and 2,3-DPG in di-
;wo polypeptides making up hemoglobin (11). lute solution. During hemoglobin synthesis,
'Iemoglobin is a protein consisting of four the mutant pS chain has a slightly lower affin-
~olypeptidechains (a pair of a-globinlp-globin ity for the a-globin chain in formation of a-glo-
limers). Each globin chain surrounds an iron bidp-globin dimers. This leads to selective de-
~orphyrinmolecule, or heme group, capable of struction of the pS chain and a 40:60 ratio for
*eversiblebinding to molecular oxygen. The HbS:HbA in individuals with sickle trait (AS)
u-globin chain is a polypeptide of 141 amino (24).
446 Inhibition of HbS Polymerization as a Basis for Therapeutic Approaches to Sickle-Cell Anel

- +
Normal (AA) t
Figure 9.2. Sickle hemo-
globin. (a) Electrophoresis
Sickle trait (AS)
8
Sickle cell (SS)
pattern of hemolysates from
normal (AA), sickle trait
(AS), and sickle-cell anemia
(SS)
. . red blood cells shows
the separation of normal he-
moglobin (HbA) from sickle
hemoglobin (HbS) based on
charge arising from the sub-
stitute of a charged glutamic
acid residue in HbA by a
neutral valine residue in
HbS in the p6 position. AS
cells have both types of he-
moglobin with more HbA
than HbS. (b) Representa-
tion of a tetrameric hemo-
globin molecule based on the
crystal structure shows the
position of p6 and other res-
idues implicated in intermo-
lecular contacts of the de-
oxygenated hemoglobin S
polymer. (From the estate of
Irving Geis, with permis-
sion.)

Etythropoietin

Multipote
stem
-- \
/
To circulation
Life span
- 120
/ Red
cells days
blood
cells

Bone 200 trillion cellslday


marrow

Figure 9.3. Red cell and hemoglobin production. Erythropoietin stimulates the hematopoietic stem
cells in the bone marrow to proliferate and differentiate along the erythroid lineage. The multipo-
tential stem cells can self-"renew" as well as differentiate into erythroid progenitor cells. Erythro-
poietin is required for the viability, proliferation, and differentiation of erythroid progenitor cells into
erythroid precursor cells. During differentiation to become red cells, transcription ceases and the
erythroid precursor cells lose their nuclei and become reticulocytes. The cells continue to translate
protein from globin mRNA. With terminal differentiation, translation ceases and the mature eryth-
rocytes are formed, with hemoglobin making up 98% of the intracellular protein.
' Pathophysiology of Sickle-Cell Anemia 447

Figure 9.4. Diagram for oxygenated (Oxy) and deoxygenated (Deoxy) hemoglobins based on the
crystal structure viewed down the dyad axis of symmetry. The a-carbon backbone of the p-chains is
shown. The .f16 .position is indicated by open circles. The major conformation change for the deoxy-
~ -

genated T-state from the oxygenated R-state is the shift and rotation of the a,& dimer relative to the
a#, dimer. The P-chains move further apart and create a binding site for 2,3-DPG. (From the estate
of Irving Geis, with permission.)

.3 Hemoglobin and Oxygen Binding tons) accounts for the allosteric effects of
these modulators of oxygenation.
'he primary function of the red blood cell is to
my oxygen from the lungs to the tissues
25). Molecular oxygen taken up by the red 2.4 Hemoglobin Molecular Size and
lood cell is associated with the iron on the Nonideality
erne group. Each hemoglobin molecule made The relatively large size of hemoglobin (64 A
p of four globin chains is capable of coopera- in diameter) and high hemoglobin concentra-
ve binding of up to four molecules of oxygen. tion in the red blood cell of 32-34 g/dL results
:-ray crystallography provides evidence sug- in extensive crowding. The molecular separa-
esting that the oxygenated forms of HbS and tion between hemoglobin molecules in an in-
ormal hemoglobin or hemoglobin A (HbA) tact red cell is less than one molecular diame-
re isomorphous and similar in overall struc- ter (Fig. 9.5a). At this protein concentration
Ire (18, 26, 27). The crystallographic struc- there is a major departure from ideal condi-
ire of hemoglobin suggests that the molecule tions, and a high degree of nonideal behavior.
self "breathes" and undergoes a conforma- Based on the fact that the activity coefficient
on change between its deoxygenated form increases exponentially with protein concen-
l'-state) and its fully liganded (or oxygenated) tration (Fig. 9.5b), hemoglobin acts 50 times
r m (R-state) (Fig. 9.4) (28). The allosteric more concentrated inside the red blood cell
mctural changes between the T-state and because of its physical size than if it were a
ie R-state are a shift in relative orientation of point particle. Nevertheless, HbA remains sol-
le a-lp-globin chain dimers. On deoxygen- uble at these high protein concentrations un-
tion, the p-globin chains move further apart, der all physiological conditions.
roviding a binding site for 2,3-DPG in the
mtral cavity of the hemoglobin molecule, re- 2.5 Sickle Hemoglobin Polymerization
dting in stabilization of the deoxygenated
emoglobin conformation. This structural The allosteric shift in conformation between
lange accounts for the cooperativity of oxy- oxygenated and deoxygenated hemoglobin
?nbinding, leading to the characteristic sig- when there is a substitution of a d i n e residue
loidal shape of the oxygen binding isotherm. for glutamic acid on the p-chain of HbS mark-
he differential binding of 2,3-DPG (and pro- edly affects the ability of deoxygenated HbS to
448 Inhibition of HbS Polymerization as a Basis for Therapeutic Approaches to Sickle-Cell Anelmia

Figure 9.5. Hemoglobin and nonideality.


(a) Artistic representation shows the
crowding between hemoglobin molecules
separated by less than one molecular diam-
eter apart within the red blood cell. (b)The (b) 600 3
I /
/
activity coefficient (7) (solid line: linear
scale; dashed line: logarithmic scale) de-
scribes the departure from ideal solution
behavior and is the ratio of the hemoglobin
activity to its measured concentration. At
I 400 - 0/
-2
0=
low concentrations, the activity coefficient 2,
0
/ '
is close to 1. At physiologic hemoglobin con- / /
centrations found inside the red blood cell 200 - 0/* - 1
(32 to 34 g/dL), the activity coefficient indi- */ )
/ @

cates that hemoglobin behaves as though it


is 50 times more concentrated because of its
physical size than if it were a point particle. O
016 32 48O
[From C. T. Noguchi, Biophys. J . , 45,1153
(1984).] Hb concentration (gldl)

remain in solution (29,301. Under physiologic 2.6 Hemoglobin S Polymerization and


conditions, the solubility of deoxygenated HbS Disease Manifestations
is about half the hemoglobin concentration in
the red blood cell, or around 16 g/dL (31-33). At physiologic conditions the dramatic (le-
As a result, when HbS is deoxygenated, the crease in intracellular HbS solubility when (ie-
shift in conformation to the deoxygenated con- oxygenated and its subsequent intracellu:lar
formation favors the aggregated or polymer aggregation results in significant changes in
state and HbS gels. (This process forms semi- the cell's viscoelastic properties (31, 40, 41).
crystalline structures that are called liquid Intracellular HbS aggregation decreases r,ed
crystals or tactoids.) Gelation of HbS leads to cell flexibility and increases abnormal red cell
alteration of the physical properties of HbS, rheology during transit in the circulatic)n,
resulting in marked increases in the intracel- causing injury to tissues by microvascular ()C-
lular red cell viscosity compared to that of cells clusion, hemolysis, and decreased oxygen cle-
with oxygenated HbS or deoxygenated or oxy- livery (Fig. 9.6a). The premature red cell cie-
genated HbA (34-36). Growth and alignment struction results in moderate to sevc!re
of HbS polymer domains also give rise to the hemolytic anemia and related clinical comp11i-
marked changes in cell morphology character- cations, with hemoglobin values from 6 to 10
istic of sickle-cell anemia (Fig. 9.lb) (6, 7). gldL compared with a normal level of 12 g/ciL
These intracellular changes also lead to rela- (9,42). Clinical symptoms can include chrorlic
tive rigidity of the cell, making it difficult to as well as acute painful episodes characterized
traverse small blood vessels (37-39). This ul- by extreme pain in the extremities, bac:k,
timately leads to irreversible biochemical and chest, or abdomen (Fig. 9.6b) (43). Spleen in-
physiological changes to the sickle red blood farction and resorption decreases immu ne
cell, even though the polymerization process is function and increases infection. Growth a]nd
completely reversible (39). development are delayed. Cardiovascular, p~11-
Pathophysiology of Sickle-Cell Anemia

(a) Production Circulation Obstnrction

- -
-02

+02
7

lntracellular HbS
polymerization
Bone marrow

(b) Neurological
complications Ophthalmological
complications

= Cardiovascular
manifestations

Renal and Splenic sequestration


urogenital
manifestations

Musculoskeletal

Skin
ulcers

Figure 9.6. Cellular pathophysiology of sickle-cell anemia. (a) Sickle-cell anemia is attributed to the
hemoglobin composition in mature red blood cells of virtually total mutant hemoglobin S (HbS) that
has low solubility in its deoxygenated form. As HbS gives up oxygen, HbS polymerizes. The increase
in intracellular viscosity increases red cell fragility and decreases cell deformability, particularly at
low oxygen tensions in the microcirculation, causing occlusion in the arterioles. Occlusion of the red
cells in the circulation, which is triggered by mechanisms still poorly understood, but which is
primarily dependent on oxygen concentration, results in insufficient oxygen delivery and tissue
damage. (b) In sickle-cell anemia almost any organ in the body can be affected by vaso-occlusion and
lack of oxygen delivery to the tissues, a s shown here.

onary, and renal insufficiency and other or- work of aligned hemoglobin molecules (Fig.
m involvement can lead to end-organ failure 9.7a) (44). The aggregated HbS molecules ap-
~ddeath. The acute and chronic pathophys- pear as bundles of fibers. The HbS fibers con-
logy of sickle-cell disease can be explained sist of assembled strands of hemoglobin mole-
sincipally by the polymerization tendency of cules bundled as a 14-strand polymer, with a
bS and impaired transit of SS red cells. How- slight helical twist (350-nm repeat distance)
.er, many other processes related to the red (Fig. 9.7b). X-ray crystallography of deoxygen-
11 and its interaction with other cells may ated HbS in a physiologic buffer provides in-
so contribute to these pathophysiological sight into the intermolecular contact regions
ocesses, especially to the induction of patho- between HbS molecules in the fiber structure
gcal events. (Fig. 9.7d) (45-47). The 14-strand polymer is
grouped as seven pairs of half-staggered
7 Hemoglobin S Polymer Structure strands of deoxygenated HbS molecules (Fig.
ectron microscopy analysis of aggregated 9.7~).In the crystal structure, deo-xygenated
oxygenated HbS reveals a long fibrous net- HbS molecules are oriented as half-staggered
450 Inhibition of HbS Polymerization as a Basis for Therapeutic Approaches to Sickle-Cell Anemia

oxygenated HbA with glutamic acid in the


sixth position of P-globin cannot participate in
the hydrophobic interaction with p85Phebe-
cause of its size and charge (Fig. 9.7e). How-
ever, the availability of pSSPheon HbA is able
to provide the hydrophobic pocket for docking
of the mutant pSGVal that stabilizes the deoxy-
genated HbS polymer. Although the HbA tet-
ramer cannot fit into the polymer structure,
the hybrid HbA/HbS molecule or hemoglobin
tetramer, consisting of mixed dimers of alpA
and alpS, can be accommodated with the nor-
mal pA-globinchain providing the p85Phe.De-
oxygenated HbA molecules therefore have 112
the probability of HbS for participating in the
deoxygenated polymer phase (48-50).

2.8 Hemoglobin S Polymerization Kinetics


The polymerization kinetics of deoxygenated
HbS solutions exceeding the solubility is pre-
ceded by a delay period in which no change
in physical parameters such as viscosity, tur-
bidity, heat absorption, or nuclear magnetic
resonance (water-proton relaxation) are ob-
served (31,51,52). In the absence of preexist-
Figure 9.7. The deoxygenated hemoglobin S poly- ingpolymer, the delay time for polymerization
mer. (a)A 14-strand model for the HbS polymer was is inversely dependent on the solubility to a
determined from electron micrographs (44, 276). A high power (210) (Fig. 9.8a). Detailed analysis
single gel fiber is about 210 A in diameter, with a of the exponential progress curves for poly-
slight helical twist with a repeat of about 350 nm merization kinetics suggests a double-nucle-
along the fiber axis. (b) A cross section showing pos- ation model (Fig. 9.8b) (53,541.The formation
sible pairing of hemoglobin molecules in the 14- of a critical nucleus proceeds by homogeneous
strand model based on the double strands seen in nucleation. With existing polymer fibers, the
the crystal of deoxygenated HbS (45, 277). (c) Ar- polymerization process can proceed by heter-
rangement of hemoglobin tetramers illustrating the
double-strand arrangement observed in the crystal ogeneous nucleation. Fibers grow by addition
of deoxygenated HbS. Only one pSGVdof each hemo- of hemoglobin at the ends. As hemoglobin con-
globin tetramer is involved in an intermolecular centrations increase, homogeneous nucle-
contact. (d) Artistic rendition of the pSGV"contact ation dominates (55).
region showing possible arrangement of the hydro-
phobic pocket involving pPhe and on an ad- 2.9 Deoxygenated Hemoglobin S Solubility
jacent molecule. (e)The normal pGGIU is charged and and Hemoglobin Polymerization
too bulky to allow the same geometry. (d and e from
the estate of Irving Geis, with permission.) The primary determinants of HbS aggrega-
tion or polymerization in the intact red cell are
hemoglobin composition, hemoglobin concen-
pairs, with only one pSGVdmutation on each tration, and oxygen saturation (56). Maximal
HbS molecule fitting in a hydrophobic pocket HbS polymerization is achieved a t complete
of an adjacent 0-globin chain in the vicinity of deoxygenation. At 0% oxygen, HbS at physio-
pssphe.A number of other close contact sites logic concentration partitions into a polymer-
are consistent with other studies of naturally phase fraction of 0.7 and a solution-phase frac-
occurring hemoglobin mutants that affect tion of 0.3 (Fig. 9.9) (33). HbS polymerization
HbS aggregation (Fig. 9.7d). Interestingly, de- can be treated as a phase transition (57, 58).
Pathophysiology of Sickle-Cell Anemia 451

o0*---
0 Oo
Homogeneous
nucleation
_ t - -
Growth
___C

+
-
Heterogeneous
nucleation
+

Figure 9.8. Kinetics of hemoglobin S polymerization. The double nucleation model for HbS poly-
merization suggests two pathways for nucleation (53).The initiation of HbS (circle) polymerization
proceeds through the thermodynamically unfavorable aggregates until the formation of a critical
nucleus. Subsequent additions of hemoglobin molecules are thermodynamically favorable. The first
polymer is formed by homogeneous nucleation. Nucleation of additional polymers can proceed either
by homogeneous nucleation or by addition of polymer to preexisting polymer, termed heterogeneous

gen binding to HbS in the solution phase ized HbS led to the appreciation of the rela-
oceeds cooperatively as it does for binding to tionship between the polymer structure and
HbA, but the HbS polymer phase does not the morphological abnormality characteristic
bind oxygen cooperatively (59,60). Increasing of sickle-cell anemia (6,65).Growth and align-
oxygen saturation decreases the concentra- ment of the HbS polymer fiber can distort the
tion of deoxygenated HbS and the extent of cell, although polymerization can proceed
HbS polymerization decreases (29, 33). Al- without changes in cell morphology. High he-
though the solubility of deoxygenated HbS is moglobin concentrations and low deoxygen-
around 16 g/dL, or half of the corpuscular he- ated HbS solubility favor homogeneous nucle-
moglobin concentration, significant amounts ation and small polymer domains (55).Studies
of HbS polymer can be detected at oxygen lev- of ion transport in SS red blood cells indicated
els well above 50% oxygen saturation. Because that cycles of polymerization and depolymer-
of the broad distribution of corpuscular hemo- ization altered membrane transport, leading
globin concentration of red blood cells from an to changes in ionic and water composition (66,
individual homozygous for sickle-cell anemia 67).
(SS), HbS polymerization can be detected in
2.10 Dense SS Erythrocytes and Irreversibly
some SS red cells, even at oxygen saturation
Sickled Cells
above 90% (61). Although increasing oxygen
saturation increases HbS solubility, the in- In the heterogeneous distribution of corpuscu-
crease in solubility is more gradual than would lar hemoglobin concentrations of SS red blood
be predicted for an ideal solution. This is a cells, the most dense cell fraction, with the
direct consequence of HbS crowding or the greatest hemoglobin concentration (MCHC >
high activity (nonideality) of HbS at physio- 37 g/dL), is the subpopulation with the great-
logic concentrations. The low oxygen affinity est HbS polymerization tendency (Fig. 9.10)
of the HbS polymer decreases the overall oxy- (41, 61). This dense cell fraction contributes
gen affinity of SS red blood cells and shifts the disproportionately to the abnormal rheology,
oxygen-binding curve to the left (62,63). This deformability, or filterability (Fig. 9.11a) (41,
shift can be used as an index of the extent of 68). Even when exposed to air, these dense
polymer formation (30, 64). cells contain significant amounts of polymer to
The low deoxygenated HbS solubility is one decrease filterability in uitro that can be fur-
of the limiting factors in determining HbS po- ther "melted" upon the addition of carbon
lymerization. Increasing HbS concentration monoxide (Fig. 9.11b) (39, 68). Irreversible
further increases the polymerization tendency membrane changes lead to membrane rigidity
(41, 61). Studies of the structure of polymer- and the formation of irreversibly sickled cells
Inhibition of HbS Polymerization as a Basis for Therapeutic Approaches to Sickle-Cell Anem

directly with the extent of HbS polymerizatic


for the bulk population (Fig. 9.12).

0% 40% 70% 100% 3 MODIFIERS O F SICKLE-CELL ANEMIA


Oxygen saturation A N D THE SICKLE-CELL DISEASE
SYNDROMES

3.1 Origins of Sickle-Cell Anemia


Sickle-cell anemia is one of the most prevalei
genetic diseases and has the highest frequent
in individuals from equatorial Africa. Baa
on genetic polymorphisms in the P-globin ger
cluster associated with the pS globin gene, tl
genetic mutation leading to sickle-cell anem
appears to have originated in three indepei
dent African regions: Senegal, Benin, ar
"0 50 100 Bantu (Fig. 9.13)(74-76). A group of restri
% Oxygen saturation tion enzyme sites characteristic of one or mo?
of these genetic polymorphisms is used to d
Figure 9.9. Oxygen content is a primary determi- fine the Senegal, Benin, and Bantu genet
nant of hemoglobin S polymerization. (a) Artistic haplotypes, as well as that associated wil
representation of the HbS gel illustrates the rela- sickle-cell anemia in Saudi ArabiaIIndia (Fi
tionship between oxygenated hemoglobin tetramers
(open circles) and deoxygenated tetramers (filled
9.13) (77). In the p-globin cluster, the prev,
circles) which may be in the polymer or aligned lence of the sickle-cell anemia genetic mut,
phase, or in the solution phase in equilibrium with tion (with some AS frequencies of 25% (
it. (b) 13C-NMRmeasurements of the fraction of po- higher) overlaps with the geographic distribi
lymerized HbS at equilibrium versus oxygen satura- tion of malaria. In spite of the severe clinic,
tion for SS erythrocytes. Polymer is detected even at manifestations of sickle-cell anemia, the hip
very high oxygen saturation in SS erythrocytes, and frequency of the HbS gene correlates with ii
increases with decreasing oxygen to a fraction of 0.7 creased resistance to Plasmodium falciparui
at complete deoxygenation. For AS erythrocytes, malaria in the AS subpopulation (78-80).11
polymer is detected as oxygen saturation falls below deed, sickle-cell anemia has low prevalence i
65% and is maximal at about 0.4 at 0% saturation.
nearby arid regions.
For SS and AS erythrocytes, polymer fraction in-
creases with decreasing oxygen and is maximal at
complete deoxygenation. AS samples are repre- 3.2 Fetal Hemoglobin
sented by "A" and "+"; all other symbols represent
Clinical manifestations of sickle-cell anem-
SS. [From C. T. Noguchi et al., Proc. Natl. Acad. Sci.
USA, 77,487 (1980) and C. T. Noguchi, Biophys. J., vary markedly and range from mild, with mil
45, 1153 (1984).] imal symptoms, to severe, characterized 1:
multiple painful vaso-occlusive crises per yer
and multiple organ damage (43). Patients wit
symptomatic disease had the highest fr~
that retain a fixed morphology and are no quency of early mortality, and low fetal hem1
longer deformable, regardless of the oxygen globin (HbF) levels correlated with a more se-
saturation, even in the absence of HbS poly- vere course in early childhood with increased
merization (69-72). In contrast, dense cells risk of stroke (81,82). Fetal hemoglobin is ex-
are not observed in red blood cell populations pressed at high levels before birth, and is
of normal individuals, individuals with sickle downregulated after birth as adult hemoglo-
trait (AS), or individuals with the mild sickle bin (HbA) expression increases, becoming 2%
syndrome of S-P'-thalassemia (73). In these or less in normal adult individuals. Many
cell populations, loss of filterability correlates other modifying factors also contribute to the
Modifiers of Sickle-Cell Anemia and the Sickle-Cell Disease Syndromes

1 .o
(a)

C
.-
0
C
0
!?
.,-
-0g> 0.5
a

0
0 0.5 1 .O
Oxygen saturation
40 I I I I I I I I I
(b)
= - 29.5 - - 32.7 -
u
5 - - - -
C
a -
-
0
0)
- -
<
-
0
20- -
c
v -
a3
.-
N
6 - -
E
-02.
a - -

0 0.5 10 0.5 1
Oxygen saturation
Figure 9.10. Dense cells have the highest polymerization potential. (a) Theoretical curves for illus-
trating polymer fraction as a function of oxygen saturation for various intracellular HbS concentra-
tions in g/dL as indicated based on a model two-phase model of HbS polymerization. [From C. T.
Noguchi, Biophys. J., 45,1153 (1984).1(b) 13C-NMR measurements of HbS polymerization of intact
SS erythrocytes separated by density gradient to give subpopulations with a narrow density range at
the average intracellular hemoglobin concentration (indicated in g/dL) validate the theoretical pre-
dictions based on hemoglobin composition, intracellular hemoglobin concentration, and oxygen sat-
uration (solid lines). [From C. T. Noguchi, et al., J. Clin. Invest., 72,846 (1983).]

clinical variability of the disease, including greatly change its manifestation. The most
physiologic, psychosocial, and environmental readily identified genetic effectors are those
conditions (83). In addition to levels of HbF, that modify globin gene expression, that is,
genetic and other factors, many yet to be iden- the thalassemic syndromes including heredi-
tified, contribute to the heterogeneous Presen- tary persistence of fetal hemoglobin (42). In-
tation in clinical severity of sickle-cell disease. formation on many of these are in a
These include epistatic genetic modifiers un- hemog~obinopathy database (http://globin.
linked to the beta- or alpha-globin clusters. cse.psu.edu) (84). Variation in hemoglobin
composition or hemoglobin concentration
3.3 Genetic Modifiers of Sickle-Cell Anemia
within the intad red cell caused bymincident
Sickle-cell anemia is not really a monogenetic thalassemia or other mutations gives rise to a
disease, given that other genes can have very full range of sickle syndromes (Table 9.1). The
large effects on the severity of the disease and sickle syndromes exhibit varying severity,
Inhibition of HbS Polymerization as a Basis for Therapeutic Approaches to Sickle-Cell Anemia

Figure 9.11. Dense SS red cells contribute dis-


proportionately to abnormal rheology. (a) Fil-
tration measurements of subpopulations of
density-separated SS erythrocytes shows im- I I I I I I
pairment of filtration detected at higher oxygen 0 5 10 15 20 25
saturation for cells with greater corpuscular he- Oxygen tension (kPa)
moglobin concentration [from least to most
dense, fractions 1 (square), 2 (triangle), 3 (cir-
cle), and 4 (diamond)].[From M. A. Green et al.,
J. Clin. Invest., 81, 1669 (1988).1 (b) Hemoglo-
bin S polymerization in the dense cell fraction
contributes disproportionately to impaired rhe-
ology. Dense SS cells have a high polymeriza-
tion tendency and polymer can still be detected
at high oxygen saturation. Dilute SS cell sus-
pensions exposed to room air still exhibit im-
pairment to filtration through
. - 5-micron filters
proportional to the percentage of dense cells. In
.
marked contrast. im~airment to filtration is
significantly reduced to that of near-normal AA
cells when cells are exposed to CO that com-
pletely melts out the polymerized HbS. [From
H. Hiruma et al., Am. J. Physiol. Heart Circ. 1 .o 2.0
Physiol., 268, H2003 (1995).1 % Dense cells

ranging from the benign sickle trait (AS) to occur and lead to more severe a-thalassemia
the most severe African form of homozygous syndromes. A decrease in a-globin chain pro-
SS sickle-cell anemia. HbS polymerization duction decreases the overall intracellular he-
tendency as calculated from these parameters moglobin concentration. This gene deletion is
correlates strongly with the general degree of found at a frequency of up to 30%associated
anemia and relative severity representative of with sickle-cell anemia in African populations.
these different sickle syndromes (42). The genetic combination of a-thalassemia and
homozygous SS results in a reduction of cor-
3.4 Sickle-Cell Anemia and a-Thalassemia
puscular hemoglobin concentration, leading
In the a-globin gene cluster, the adult a-globin to a reduction in the HbS polymerization ten-
gene is duplicated (aa). Deletion of one of dency (86,871. Increased deformability of the
these a-globin genes (-a) decreases a-globin SS erythrocyte with the two a-gene deletion
mRNA expression, a form of a-thalassemia (-a/- a) genotype increases red cell survival
(85). Deletions of two or more a-genes can also and total blood hemoglobin level. The in-
3 Modifiers of Sickle-Cell Anemia and the Sickle-Cell Disease Syndromes

-
.-
0
SS-a-thal

.-m

0.0
0.0 0.1 0.2 0
Polymer fraction

0.1 0.2
Polymer fraction

Fi;gure 9.12. HbS polymerization correlates directly with abnormal rheology in the absence of dense
cel1s. Populations of SS cells [with (open squares) or without (star) coexisting a-thalassemial with
nificant dense cell fraction show a marked impairment to filtration that does not correlate with
lymer fraction determined for the bulk population based on the mean corpuscular hemoglobin
centr ration (MCHC). When the dense cell fraction is removed, as in sickle trait (AS) erythrocytes
)en and filled triangles) or erythrocytes from individuals with S-p+-thalassemia (open circles),
!asured impairment of filtration correlates directly with polymerization tendency [predicted poly-
me!r fraction based on mean values for hemoglobin composition, mean corpuscular hemoglobin
COIicentration (MCHC), and oxygen saturation]. [From H. Hiruma et al., Am. J. Hematol., 48, 19
(1s

1 Figure 9.13. Geographic origins


of the sickle-cell mutation. Popu-
lation genetic analyses indicate
four major haplotypes
. " . associated
with sickle-cell anemia that largely
- - localize to four distinct geographic
areas (74,77).These are the Sene-
gal, Benin, Bantu, and Saudi Ara-
bia-Indian haplotypes.
456 Inhibition of HbS Polymerization as a Basis for Therapeutic Approaches to Sickle-Cell Anemia

Table 9.1 Some Sickling Disorders


Hb MCHC HbF HbS
Severity Condition (g/dL) (g/dL) (%) (%)
Asymptomatic 1. HbAS (African)
2. HbS-GFy/30-HPFH(African)
Mild 3. H ~ s - ~ ~ ~ " P O - H P(African)
FH
4. ~bs-~y-pO-HpFH (African)
5. HbSS (Saudi Arabia)
6. HbSS (Indian)
7. HbS-P+-thalassemia(African)
8. H~s-Po-thalassemia(Saudi Arabia)
Severe 9. HbSS-a-thalassemia(African)
(a-/a- genotype)
10. HbS-Po-thalassemia(African)
11. HbSS-a-thalassemia(African)
(a-/aa genotype)
12. HbSS (African)

creased hematocrit and blood viscosity appear to be a direct consequence of the high level of
to offset the potential benefit of improved red HbF associated with SS disease in Saudi Ara-
cell parameters (881, and a clear clinical bene- bia and India. The "sparing" effect of HbF has
fit of a-thalassemia in homozygous SS disease been known since the early 1950s (19).Replac-
has been difficult to demonstrate. ing HbS with HbF reduces deoxygenated he-
moglobin polymerization to a greater extent
3.5 Sickle-Cell Anemia and P-Thalassemia than replacing HbS with HbA (48,501. Quan-
Mutations or deletions in the P-globin gene titatively, this is related to the formation of
cluster can decrease P-globin gene expression mixed hybrids or the combination of HbSIHbF
and give rise to P-thalassemia (89, 90). De- heterodimers to form a hemoglobin tetramer
creased P-globin gene expression results in de- (a,pSy). The inability of the HbSJHbF hybrid
creased intracellular hemoglobin production to participate in the HbS polymer structure
and intracellular hemoglobin. Two y-globin further increases the hemoglobin solubility
genes are also encoded in the p-like globin compared with comparable mixtures of HbS
gene cluster, G-y- and A-y-globin. The y-glo- and HbA, in which the hybrid can enter the
bin chains in combination with a-globin make polymer phase, as described in section . This
up fetal hemoglobin (HbF). In sickle-cell dis- effect can be seen in direct comparison of he-
ease, different forms of p-thalassemia can al- moglobin solubility in mixtures of HbS and
ter the percentage of HbS (%HbS), with re- non-HbS as the proportion of non-HbS in-
sultant increased HbF or HbA,. Because of the creases (Fig. 9.14). An increase in %HbF to
decreased polymerization tendency with in-
30% or 40% would increase deoxygenated he-
creased HbF or HbA, (48, 501, populations
moglobin solubility comparable to a 40:60
with these sickle syndromes present overall a mixture of HbS:HbA found in sickle trait (95)
more mild form of sickle-cell disease compared
and give almost total amelioration of disease
with the African homozygous SS disease (Ta-
manifestation. Note, however, that unlike
ble 9.1)(42).
HbS and HbA, HbF is not necessarily uni-
formly distributed among the population of
3.6 The "Sparing" Effect of Fetal
erythrocytes (96, 97). In sickle-cell anemia,
Hemoglobin
whereas HbF in a single individual may be up
Individuals with sickle-cell anemia in Saudi to 8%or greater, HbF can be readily detected
Arabia and India exhibit significantly more in some but not all of the red blood cells. This
mild disease manifestation compared with SS heterogeneous distribution of HbF com-
individuals in Africa (76,91-94). This appears pounded with the heterogeneous distribution
Modifiers of Sickle-Cell Anemia and the Sickle-Cell Disease Syndr

bin gene cluster, giving high levels of y-globin


c
0
- 35
mRNA, results in high %HbF (98-101). In
sickle-cell disease, the resultant reduced HbS
5 30 polymerization tendency is also correlated
--
50
.-0 25
with a relatively mild clinical course (42).
Some specific genetic polyrnorphisms in the
p-globin gene cluster have been associated
a3
- with elevated HbF levels. A significant one is
C
20 the C to T polymorphism at position -158 5'
5 upstream of the G-y-globin gene that in-
-0rn creases G- y-globin gene expression (102,103).
2 15 This polymorphism is known as the Xmnl
I" G-y-polymorphism and can be identified by
10 the ability of the Xmnl restriction enzyme to
cut genomic DNA at this site. Sickle-cell ane-
Fraction HbX mias with the Saudi ArabianIIndian and the
Senegal genetic background are associated
Figure 9.14. Hemoglobin composition modifies with high levels of HbF and have this Xmnl
the extent of hemoglobin S polymerization. Deoxy-
genated hemoglobin solubility increases with in-
G-y-polymorphism. Other genetic loci not
creasing non-S hemoglobins. The sparing effect of linked to the globin clusters have also been
HbF (a2y2)is greater than HbA (a2P2)because of associated with determination of HbF produc-
the differences between the y-chain and the p-chain. tion (104). These include a region on chromo-
The minor adult hemoglobin, hemoglobin HbA, some Xp22.2 and another on chromosome
(a$,), has a similar sparing effect on increasing 6q23 (105-107).
HbS solubility as HbF. Hemoglobin C (HbC; a2pC2),
another mutant hemoglobin with a p6G'u'Ly"substi-
tution behaves similarly to HbA in mixtures with 3.8 Sickle Trait
HbS. Individuals with sickle trait (AS) and SC dis- As with SS red blood cells, the fraction of po-
ease have one ps-globin gene and one normal p-glo- lymerized hemoglobin in AS erythrocytes is
bin gene or the mutant pc-globin gene. In sickle
trait, the HbS:HbA ratio is about 40:60 because of
maximal at complete deoxygenation (49). The
the higher affmity of the a-chain to the normal hemoglobin solubility in AS erythrocytes de-
fi-chaincompared with the mutant ps-chain. In SC creases to 24 g/dL at complete deoxygenation.
disease, the HbS:HbC ratio is about 50:50, and the The polymerization potential decreases with
charge difference of HbC causes red cell dehydra- increasing oxygen saturation with little or no
tion, with an increase in intracellular hemoglobin polymer detected at physiologic oxygen levels
ncentration. These changes result in increased above 50% oxygen saturation (Fig. 9.9). Al-
polymerization tendency and disease manifestation though sickle trait or carriers for sickle-cell
in SC disease not observed in sickle trait (AS). anemia do not show clinical symptoms, there
[From W. N. Poillon et al., Proc. Natl. Acad. Sci. is a urine-concentrating defect (108).The high
USA, 90,5039 (1993).1
osmolality and low oxygen saturation of the
renal medulla are conditions that favor HbS
of intracellular hemoglobin concentration polymerization. The urine-concentrating abil-
adds another level of complexity in assessing ity correlates inversely with the polymeriza-
determinants of disease severity. It should tion potential of AS erythrocytes. The genetic
also be noted that HbA, ( ( ~ ~ 6does
, ) not enter combination of AS with a-thalassemia reduces
the polymer phase and has the same sparing the %HbS and polymerization potential as
effect as that of HbF (48, 50). well as the urine-concentrating defect (Fig.
9.15). Nevertheless, the absence of pathophys-
3.7 Sickle-Cell Anemia and Hereditary
iology associated with AS suggests that reduc-
Persistence of Fetal Hemoglobin
tion of HbS polymerization tendency to that of
ereditary persistence of HbF (HPFH) aris- AS erythrocytes would be an appropriate ther-
ing from mutations/deletions within the p-glo- apeutic goal.
458 Inhibition of HbS Polymerization as a Basis for Therapeutic Approaches to Sickle-Cell Anemia

I I I I I J
35 50
Percent hemoglobin S
Figure 9.15. Sickle trait (AS) individuals exhibit a urine-concentrating defect correlating with
percentage of HbS. The high osmolality and low oxygen saturation of the renal medulla are conditions
that favor polymerization. In AS individuals, %HbS (and HbS polymerization tendency) correlates
inversely with urine-concentratingability. Coinheritanceof AS with a-thalassemia reduces %HbSas
well as the .polymerization potential and the urine-concentrating defect. [From A. K. Gupta et al.,
~

J. Clin. Invest., 88, 1963 (1991).1

3.9 Physiologic Modifying Factors contribute to the variable tissue response of


the disease. In vitro measurements of SS
Impaired flow of SS red blood cells arises from
the acute and chronic effects of HbS polymer- erythrocyte adherence to endothelium corre-
ization as cells transit the circulation from the late with clinical disease severity (109) and
lung, through the tissue microvascular beds, vascular occlusion has been proposed to be ini-
and return. Variation in intracellular hemo- tiated by adhesion of the least-dense SS reticu-
globin composition and intracellular hemoglo- locyte with relatively high endothelium adhe-
bin concentration are only two of the modify- sion (110). Several receptors and other
ing factors that give rise to the heterogeneous molecules on the surface of red blood cells and
nature of SS red blood cells and the broad dis- endothelium have the potential to contribute
tribution of clinical severity. Microvascular directly to erythrocyte-endothelial adhesion.
occlusion is attributed primarily to HbS poly- These include on the red cell the integrin a4pl
merization, which alters SS erythrocyte de- (Ill),thrombospondin receptor CD36 (1121,
formability and causes blockage in the micro- the very late antigen activator 4 (VLA-4)
vasculature. However, the extent of HbS (113),aggregated band 3 (114),sulfated glyco-
polymerization alone cannot predict the be- lipids (1151, and increased phosphotidylserine
havior of cells in the macro- and microvascular (PS) on the surface of SS red blood cells (116).
beds and underscores the importance of direct On endothelial cells, association with red cell-
rheological measurements of SS erythrocytes. endothelium adhesion has been made with the
Other factors such as plasma proteins and in- vitronectin receptor integrin aVp3 (1171, vas-
teractions of blood cells (SS red cells, platelets, cular adhesion molecule 1 (VCAM-1) (113),
and leukocytes) with endothelium affect blood
and possibly CD36 and glycoprotein (GP) IB
flow. The influence of vasomotion and inter- (118). VCAM-1 is not constitutively expressed
mittent flow, neural and humoral controls, on endothelium but its expression can be acti-
and adherence of blood cells to the vasculature vated by exposure to several agonists such as
act to modify passage of SS erythrocytes cytokines &d hypoxia (113). Other plasma
through the circulation. factors (and their corresponding receptors on
erythrocytes and endothelial cells), such as
3.1 0 Interactions with Endothelium
thrombospondin (119, 120), von Willebrand
Variation in local oxygen saturation as well as factor (1211, and laminin (122), also influence
tissue demand for oxygen and vascular tone blood cell adherence to endothelium. The role
4 Rational Approaches to Sickle-Cell Therapy

of these interactions in sickle-cell anemia gene for p-globin gives rise to the valine sub-
pathophysiology in vivo remain uncertain, stitution for glutamic acid, resulting in a
that is, whether they contribute to chronic marked decrease in HbS solubility at physio-
events in the microcirculation and organ pa- logic conditions as oxygen is removed from the
thology or whether they contribute more to red blood cell (Fig. 9.6). The resultant transi-
initiating episodic painful crises. Recently, it tion from the soluble to polymer phase of HbS
has been proposed that it is sickle celllwhite (Fig. 9.7) alters the viscoelastic properties of
cell interactions and not sickle celltendothe- HbS inside the SS erythrocyte (Fig. 9.11).
lium interactions that are the triggering Changes in red cell deformability lead to ab-
events in the pathophysiological mechanism normal rheology or blood flow, increased red
(123). cell destruction, compromised oxygen deliv-
ery, microvascular obstruction, and subse-
3.11 Animal Model Systems
quent downstream clinical manifestations. In
addition to symptomatic treatment, therapeu-
In addition to in vitro systems, animal models tic strategies have targeted hemoglobin poly-
and the recent development of mice express- merization, red cell circulation and the micro-
ing exclusively human hemoglobins, particu- vasculature, and red ceU/hemoglobin production
larly HbS, have been used to assess SS red cell (Table 9.2).
rheology, pathophysiology, and treatment
(124, 125). Intravital microscopy with video 4.1 Inhibition of Hemoglobin S
image analysis has been used to document the Polymerization
rheological behavior of SS red blood cells and Early efforts at therapeutic strategies focused
their interaction with vascular endothelium on increasing deoxygenated HbS solubility
(123,126,127).Ex vivo preparations of the rat (130-132). Rather than the ambitious goal of
mesocecum vasculature infused with a bolus increasing solubility to match the corpuscular
of red blood cells have been used to determine hemoglobin concentration, a reasonable end-
flow of SS erythrocytes in the microvascula- point would be increasing the solubility to
ture (110, 115). These studies indicated that mimic that associated with the generally
vaso-occlusion of oxygenated SS erythrocytes symptom-free AS phenotype (49). Recognizing
resulted from dense SS cells causing precapil- the hydrophobic substitution of valine for the
lary obstruction and the less-dense reticulo- charged glutamic acid, early efforts focused on
cytes and young discocytes preferentially ad- disrupting hydrophobic interactions. Agents
hering to the immediate postcapillary venules, such as urea, known to perturb solvent inter-
causing blockage of dense, irreversibly sickled actions, were proposed but their effects were
cells (128). Magnetic resonance imaging of the too small to be useful at levels necessary for
rat leg infused with technetium-99m-labeled therapeutic intervention (133-138). Ste-
erythrocytes provided further evidence of the reospecific competitors such as peptides and
importance of the densest SS red blood cell in modified amino acids were also able to in-
producing vaso-occlusion (129). crease solubility (139-142). The hydrophobic
aromatic amino acids proved to be the most
effective. Chemical modification to increase
4 RATIONAL APPROACHES TO solubility of the amino acid itself or other oli-
SICKLE-CELL THERAPY gopeptides increased their potency. X-ray dif-
fraction and solution techniques have been
Description of the biophysics of HbS polymer- useful in identifying their binding sites (143).
ization provides the basis for understanding However, specific uptake of these compounds
the pathophysiology of sickle-cell disease. The by red cells at sufficient concentrations neces-
production of red cells containing a mutant sary to therapeutically increase HbS solubility
gene product leads to the potential for in- remain problematic. The nonideal behavior of
traerythrocytic hemoglobin polymerization even relatively small molecules such as pep-
that can cause obstruction in the microvascu- tides reduced their effectiveness, and changes
lature. The single-nucleotide mutation in the in solubility were low (142). Furthermore,
460 Inhibition of HbS Polymerization as a Basis for Therapeutic Approaches to Sickle-Cell Anemia

Table 9.2 Rational Therapeutic Design for Sickle-Cell Anemia


Mechanism Target Examples
Inhibit HbS polymer Hemoglobin Noncovalent modifiers (urea, peptides, vanillin)
interactions Covalent modifiers to increase solubility and/or
oxygen affinity (acetylatingagents, cyanate,
ethacrynic acid)
Hydration Erythrocyte Hyponatremia (DDAVP)
Membranelion transport modifiers
Inhibit Gardos Channel (cetiedil, clotrimazole)
Inhibit (KC11 cotransport (Mg2+)
Inhibit C1 conductance (NS3623)
Decrease abnormal Red cell replacement Exchange transfusion
RBC
Decrease Vascular tone Vasodilators (nifedipine, arginine, nitric oxide)
microvascular Endothelial adhesion Decrease red cell stasis (Flocor)
entrapment Decrease endothelial cell adhesion (anti-aVp3, anti-
P-selectin)
Increase endogenous Endogenous 7-globin gene DNA hypomethylation (5-azacytidine)
HbF production expression Cell cycle inhibitors (hydroxyurea)
Differentiating agents (butyrate, short chain fatty
acids, phenylacetate)
Replace HbS Hematopoietic stem-cell Allogeneic bone marrow transplantation
production replacement
Reduce HbS Endogenous Increase non-ps-globin gene expression by
production hematopoietic stem retrovirus-mediated gene transfer (7-globin)
cells Increase non-ps-globin gene expression by
lentivirus-mediated gene transfer (p-mutant-
globin)
Reduce ps-globin by ribozymes (ps-ribozyme, PA-
transribozyme)

several oligopeptides mimicking the region oxygenated HbS solubility. Although in vitro
surrounding the pSGV*mutation actually de- assays showed promise in these assays, during
creased deoxygenated HbS solubility, presum- clinical treatment of sickle-cell patients with
ably because of the effects of nonideality and oral administration of potassium cyanate,
excluded volume (141). adverse side effects developed. Cataract
formation and peripheral neuropathy as a con-
4.2 Cyanate and Sickle-Cell Anemia sequence of oral potassium cyanate adminis-
In addition to corpuscular hemoglobin concen- tration resulted in discontinued use. Extra-
tration and the low deoxygenated HbS solubil- corporeal treatment was attempted to
ity, oxygen saturation is one of the major de- overcome these complications, but did not
terminants of polymerization tendency. To show clear benefit on painful crisis frequency
increase oxygen saturation, strategies aimed (147-150). More generally, however, it is not
at increasing oxygen affinity were developed. clear that increasing oxygen affinity would
Cyanate, a by-product of urea in solution, was have overall clinical benefit. Red cells must
found to increase oxygen affinity and reduce deliver a constant amount of oxygen to indi-
sickling of partially deoxygenated SS erythro- vidual tissues and either nonmodified hemo-
cytes (144, 145). Cyanate covalently modifies globin molecules will deliver oxygen (and then
hemoglobin by carbamoylation of the a-amino polymerize) or oxygen tension will fall suffi-
groups of the globin chains, increasing oxygen ciently that even the modified hemoglobin
affinity (146). Carbamoylation of the P-globin molecules will transfer their oxygen and poly-
chain specifically also has a small effect on de- merize.
Rational Approaches to Sickle-Cell Therapy

.3 Chemical Modifiers of Hemoglobin S for HbS polymerization. Vanillin would be


suitable for further testing in animal models
ther chemical modifiers such as nitrogen
of sickle-cell disease.
ustards (151), alkylating agents (152), alde-
des (1531, and bis[3,5-dibromo salicyllfum-
rate that binds in the 2,3-DPG pocket and 4.5 Increasing Sickle Erythrocyte Hydration
ross links the P-82 lysines (154) also in- and Membrane Active Agents
reased deoxyhemoglobin S solubility and/or Increasing red cell hydration and decreasing
creased oxygen affinity in vitro. However, intracellular hemoglobin concentratiodmodi-
espite their effects, specificity and potency on fication of intracellular HbS polymerization
tact red blood cells are markedly dimin- by decreasing intracellular hemoglobin con-
hed. Covalent modification of hemoglobin in centration has been attempted by regimens
tact erythrocytes is particularly challenging designed to cause cell swelling. The use of fluid
because of potential toxicity and undesirable restriction and desmopressin acetate (DDAVP)
side reactions. Even with extracorporeal ad- to induce hyponatremia in a small number of
ministration, additional problems such as the sickle-cell patients under close observation in
potential for immunogenic adducts on blood a metabolic ward resulted in a decrease in
cells arise. Investigation of phenoxy and ben- MCHC (159). In this limited study - there was
zyloxy agents that increased deoxygenated an apparent decrease in painful crisis fre-
HbS solubility led to studies of the antilipi- quency. Although impractical for general ap-
demic drug clofibrate (155) and the diuretic plication because of the severity of this treat-
agent ethacrynic acid (156). The difficulty in ment, the feasibility of the approach was
identifying potential therapeutic inhibitors of demonstrated. Pharmacological agents that
HbS polymerization was exemplified by stud- affect ion and proton pumps to cause red cell
ies of ethacrynic acid that could inhibit HbS swelling have also been proposed such as ce-
polymerization in cell-free systems. However, tiedil and, more recently, clotrimazole, an im-
as a renal diuretic, treatment of SS cells re- idazole blocker of the red cell Ca+-activated
sulted in ion and water loss. Given that cell Kf (Gardos) channel (160). Clotrimazole is
shrinkage promotes, rather than inhibits, used to treat mycotic infections through inhi-
HbS polymerization the loss of cell water bition of cytochrome P450 activity. Prelimi-
would adversely influence red cell rheology. A nary studies in a few sickle-cell anemia pa-
mass spectrometry screening method has tients showed a reduction in MCHC and
been proposed as a high throughput method- erythrocyte density, with mostly mild side ef-
ology to identifjr new covalent modifiers of he- fects at its lowest dosage (161). Its inhibition
moglobin (157). of cytochrome P450 may account for the tox-
icity observed at higher doses. Oral M$+ sup-
plementation in preliminary studies of SS pa-
4.4 Vanillin and Hemoglobin S
tients showed a decrease in K-C1 cotransport
Polymerization
activity, the principal mediator of red cell de-
In search for therapeutic agents that could be hydration in sickle-cell disease, and a decrease
tolerated at high levels, the food additive van- in dense SS red blood cells. However, success
illin was explored (158). Vanillin was found to and side effects varied with different M$+
inhibit deoxygenated-induced SS cell sickling supplements (162, 163). Recent studies of
and possibly increase deoxygenated HbS solu- NS3623, an inhibitor of erythrocyte C1- con-
bility. Vanillin binds specifically to hemoglo- ductance, showed in vivo hydration of eryth-
bin in the central water cavity and to a region rocytes in an animal model (SAD) for sickle-
implicated as a contact site in the HbS poly- cell disease, with an increase in intracellular
mer. Its mode of action is suggested to be a Na+ and K' (164). Hematocrit increased and
dual mechanism of allosteric modulation to a there was a selective loss of the densest eryth-
high oxygen aMinity HbS molecule and by ste- rocyte population. The highest dose used gave
reospecific inhibition of the T-state required rise to echinocytosis.
462 Inhibition of HbS Polymerization as a Basis for Therapeutic Approaches to Sickle-Cell Anemia

5 THERAPEUTIC DECREASE OF the possibility that drugs targeting leukocyte


MICROVASCULAR ENTRAPMENT interactions with endothelium or SS red blood
cells may be useful in preventing or treating
5.1 Antiendothelial Receptor Antibodies sickle-cell vaso-occlusion.
Plasma factors, von Willebrand factor and
5.3 Improved lntravascular Blood Flow and
thrombospondin, contribute to the interaction
Oxygen Delivery
of SS erythrocytes with vascular endothelium.
These factors can bind to receptors on SS Oxygenated perflubron-based fluorocarbon
erythrocytes and on the surface of endothelial emulsion (PFE) has been proposed as a strat-
cells. Antibodies to aVP3 integrin on the sur- egy to improve oxygen delivery of partially
face of endothelial cells were effective in inhib- occluded vessels (127).PFE reduced microvas-
iting platelet-activating factor-induced SS red cular obstruction of deoxygenated SS erythro-
blood cell adhesion to endothelium (117). In- cytes in an ex vivo preparation of the rat meso-
fusion studies in certain preparations of the cecum vasculature. In contrast, deoxygenated
rat ex vivo mesocecum vasculature demon- PFE was not effective in reducing widespread
strated a reduction in adhesion of SS erythro- adhesion and postcapillary blockage. PFE has
cytes to the venules and postcapillary occlu- a lo-fold greater capacity to dissolve oxygen
sion upon pretreatment with monoclonal compared with that of plasma and appeared to
antibodies (MoAb) 7E3 and LM609. MoAb be effective in unsickling SS erythrocytes in
7E3 also recognizes aIIbP3 (GPII/IIIa), but partially occluded vessels rather than alter
antibodies specific to aIIbP3 had no effect. The blood cell adhesion or vascular tone.
resultant decrease in SS red blood cell-endo-
thelium interactions by blockage of aVP3 in- 5.4 Flocor and Sickle-Cell Anemia
teractions suggests that integrin receptors
may be useful targets for therapeutic inter- Flocor was developed to improve intravascu-
vention of blood cell-endothelium interac- lar blood flow by lowering blood viscosity and
tions. Anti-integrin receptor therapeutics "friction" between blood cells and the vessel
have already been shown to be therapeutically wall. Developed by the company CytRx, Flocor
useful in acute coronary syndromes (165). is a polymer shown to reduce slightly the du-
ration and severity of vaso-occlusive crisis in
5.2 P- and E-Selectin and Sickle-Cell Animal sickle-cell disease patients. Previously exam-
Models ined for treatment of acute myocardial infarc-
tion, this rheologic/antithrombotic agent ap-
In transgenic mouse models expressing hu- parently exerts its effects primarily by
man sickle hemoglobins, in vivo microcircula- enhancing blood flow in oxygen-starved tis-
tory studies used the cremaster muscle prepa- sues. Clinical trials with Flocor suggested a
ration to visualize adhesion of red blood cells small increase in resolution of vaso-occlusive
to postcapillary venules (166). Transgenic crisis.
sickle-cell mice demonstrated an inflamma-
tory response to hypoxia,/reoxygenation with 5.5 Nitric Oxide and Sickle-Cell Anemia
increased leukocyte adherence, suggesting a
model for reperfusion injury associated with The common gas nitric oxide (NO) plays many
human sickle-cell disease (167). This inflam- roles in the body, including relaxation of blood
matory response was inhibited by infusion of vessels. Researchers studying the effects of ni-
antibody to P-selectin, but not to anti-E-selec- tric oxide inhalation have shown that it can
tin antibody. Mice deficient in P- and E-selec- effectively treat several life-threatening lung
tins exhibit defective leukocyte-endothelium conditions (168). The gas is successful in ex-
adhesion. When these mice were modeled to panding constricted blood vessels in the lung
express exclusively human HbS, they were without affecting the rest of the body's circu-
protected from vaso-occlusion. These data latory system. The effect is limited to the
provide a possible new approach for the patho- lungs because the gas binds with hemoglobin
genesis of microvascular occlusion and raise upon entering the bloodstream, neutralizing
6 Therapeutic Induction of Hemoglobin F 463

Embryonic I
I
Fetal I
I
Adult
Hb gower 1

FiguIre 9.16. Hemoglobin development. Hemoglobin is a tetrameric protein consisting of two a-like
and two 0-like globin chains. The genetic information for the d i k e and 0-like globin gene are
localized into gene clusters on chromosomes 16 and 11,respectively. The a-globin cluster contains the
emblyonic 5- and two adult a-globin genes. The P-globin cluster contains the embryonic E-, two fetal
Y-,a nd a minor adult 6- and adult 0-globin genes. During development, hemoglobin expression
exhit)its a switch from embryonic to fetal to adult globin genes. Production of the respective globin
chairis leads to various combinations of hemoglobin tetramers giving rise to the embryonic (Hb
Gowc3r and Hb Portland), fetal (HbF), and adult (HbA and H b k ) hemoglobins.

its vessel-expanding properties (169-171). Re- mia (174). The observation of low L-arginine
searchers in Boston reported that it had no and nitric oxide metabolite (NOx) levels dur-
effect on normal hemoglobin, but increased ing or after vaso-occlusive crisis and acute
the oxygen affinity of sickle hemoglobin, lead- chest syndrome increased interest in the ther-
ing to an apparent reduction of sickling (172). apeutic potential of L-arginineas well as NO
initial studies showed in eight of nine sickle- for sickle-cell anemia (175, 176).
2ell disease patients that breathing nitric ox-
ide caused their red cells to give up oxygen less
readily than before, whereas the cells from 6 THERAPEUTIC INDUCTION OF
normal patients showed no change. However, HEMOGLOBIN F
Subsequent studies did not confirm these ob-
;ervations and showed no effect of NO treat- The hemoglobin tetramer requires two a-like
nent on oxygen affinity, other than that at- and two p-like globin chains (Fig. 9.3). The a-
iributed to the deleterious formation of and P-globin gene clusters encode other like
nethemoglobin (173). Effects of NO inhala- subunits of hemoglobin that are differentially
ion on pulmonary vasculature include a re- expressed during development (Fig. 9.16). The
luction in pulmonary pressures and increased a-globin gene cluster contains an embryonic
~xygenation,suggesting that NO may be ther- form 6-globin and the two adult a-globin
lpeutic for acute chest syndrome and second- genes. The p-globin gene cluster contains the
r y pulmonary hypertension in sickle-cell ane- embryonic &-globin,the fetal G-y-and A-y-glo-
464 Inhibition of HbS Polymerization as a Basis for Therapeutic Approaches to Sickle-Cell Anemia

bin genes, and the minor adult 8-globin and droxyurea is known to inhibit progression of
predominant adult p-globin genes. Sequential the cell cycle into S-phase and inhibit ribonu-
expression of these globin chains during devel- cleotide reductase. Although the exact mecha-
opment results in production of the embryonic nism of increasing HbF is not known, it is
Gower (L,eZ, aZcZ)and Portland (L2yz)hemo- thought to alter the proliferation of early RBC
globins, fetal hemoglobins (a2yz), and the precursors capable of increased HbF synthe-
adult hemoglobin A (a,&) and minor adult sis. Hydroxyurea may also increase cellular
hemoglobin A, (a,6,). Hemoglobin A, is gen- hydration and MCV. In the late 1980s,several
erally 1 2 % and is uniformly distributed small-scale studies determined the optimal
among adult erythrocytes. Because these he- protocols for administering hydroxyurea to
moglobins bind oxygen cooperatively, embry- sickle-cell patients to elevate HbF (186-190).
onic, fetal, and adult hemoglobins function
These studies led to the design and implemen-
similarly with possible variation in oxygen af-
tation of the Multicenter Study of Hydroxyu-
finity and 2,3-DPG binding, and can substi-
rea (MSH), which was ended in early 1995
tute for adult hemoglobin A in adults. A high
throughput screen based on increasing y-glo- (191). As a result of the MSH study, hydroxyu-
bin promoter activity has been designed to rea has recently been approved by the U.S.
identify new potential inducers of fetal hemo- Food and Drug Administration (FDA) for use
globin (HbF) (177). in adults with sickle-cell disease who have had
at least three crisis episodes in the preceding
year. In the MSH clinical trials of 299 patients,
hydroxyurea treatment decreased the number
In sickle-cell anemia, only the P-globin gene is of painful sickle-cell episodes and the fre-
mutated. Substitution of HbF for HbS is not quency of acute chest syndrome by 50%, the
only functional in the adult, but also provides number of patients transfused by 30%, and
an additional "sparing" effect for HbS poly- the number of transfusions by 37%. Hy-
merization (as discussed above). Activation of droxyurea's major mechanism of action is to
fetal globin gene expression has become an im- increase HbF (Fig. 9.17). There have been sug-
portant therapeutic strategy in treatment of gestions that other effects, such as decreasing
sickle-cell anemia (178-180). BAzacytidine, a white blood cell levels, may also contribute to
DNA methylation inhibitor, was the first such clinical benefit, but these have not been
agent to show significant increases in HbF ex- proved. In addition, the elevation in VCAM
pression in patients with sickle-cell anemia associated with SS disease and suggested to
(181, 182). Interestingly, although there was contribute to red celllendothelial interactions
an increase in HbF production, there were no appears to decrease with hydroxyurea. Hy-
marked increases in MCHC. Rather, there was droxyurea also produces nitric oxide both in
a normalization of the red cell density distri- vitro and in vivo (192-194). Myelotoxicity and
bution and a significant decrease in the dense neutropenia were observed with hydroxyurea,
cell population. The high teratogenic and/or and its carcinogenic potential is unknown,
tumorigenic risk associated with 5-azacyti- particularly for long-term administration in
dine therapy led to the development of other pediatric patients (195, 196).
inducers of HbF (183). Treatment with 5-aza- Preliminary evidence in sickle-cell patients
cytidine and related compounds remain of in-
suggested that combination therapy of hy-
terest, particularly in patients who exhibit lit-
droxyurea and erythropoietin under select
tle or no response to alternative agents such as
conditions could further increase HbF (1971,
hydroxyurea (184).
but results appeared to be dependent on treat-
ment regimen (197, 198). Other ribonucle-
6.2 Hydroxyurea
otide reductase inhibitors inducing HbF in-
Available as the anticancer therapeutic for clude Didox, which increased HbF in baboons
over 30 years, hydroxyurea was found to in- and in a transgenic mouse model (199, 2001,
duce a significant increase in HbF synthesis and Resveratrol, which increased HbF in ery-
about 15 years ago (185). In culture, hy- throid progenitor cell cultures (201).
Bone Marrow Transplantation, Globin Gene Expression, and Gene Transfer

:+ 6% - HbSIHbF hybrid
a-Hbs polymer 1
Figure 9.17. Hydroxyureacan induce the production of hemoglobin F in sickle-cell anemia patients
that respond to drug treatment. Illustrated is the expected decrease in polymerization potential for
HbS at physiologic total intracellular hemoglobin concentration of 34 g/dL if 25%of HbS is replaced
with HbF. Because the sparing effect of HbF is greater than that of HbA, induction of 25%HbF with
75%HbS is comparable to the polymerizationpotential of 60%HbA with 40%HbS expected for sickle
trait.

7 BONE MARROW TRANSPLANTATION,


GLOBIN GENE EXPRESSION, AND GENE
Butyrate and related short-chain fatty acids
TRANSFER
stimulate fetal hemoglobin gene expression in
erythroid cells (202-205). The butyrates are
7.1 Allogeneic Bone Marrow
perhaps the first class of drugs designed to
Transplantation
transcriptionally activate fetal globin genes
that are developmentally silenced. Although Modern therapy with transfusions and iron
the molecular basis for butyrate action is still chelation, as well as hydroxyurea in most
being defined, studies have shown that bind- cases of sickle-cell disease, has greatly im-
ing of putative regulatory proteins to a specific proved both the quality and length of life for
region of the y-globin promoter is altered i n patients with the hemoglobin disorders (43).
vivo in patients receiving butyrate therapy Nevertheless, progressive iron overload in or-
(206). In initial clinical studies, intravenous gans, hepatitis, and other randomly acquired
infusion of large doses (gram amounts) of bu- infections increase the risk of mortality with
tyrate were not consistently efficacious (207). age, especially in P-thalassemia (211). Alloge-
Intermittent or pulse therapy improved the neic bone marrow transplantation of normal
increase in levels of HbF. Recently, greater or unaffected (including AS) hematopoietic
clinical efficacy has been observed with pulsed stem cells from an HLA-matched donor repre-
intravenous dosing schedules for butyrate sents the only form of possible cure for these
(208), although this methodology is likely to diseases (Fig. 9.18) (212,213). The potential to
be insufficient for general clinical acceptance. be cured decreases with advancing age and
An oral form would alleviate problems associ- risk of death attributed to the procedure in-
ated with hospital visits for infusion, and bu- creases. Bone marrow transplantation has
tyrate may offer significant advantage when been investigated most thoroughly for use in
used in combination with other therapies such P-thalassemia, and more recently for sickle-
as hydroxyurea (178, 179, 209). Although cell disease, in Europe (214). The degree of
these compounds are relatively safe and with- morbidity, mortality, and the considerable
out generalized cytotoxicity in patients, drug cost are significant negative factors for wide-
tolerance develops in some patients after pro- spread application of this approach. Bone mar-
longed therapy. An oral form of phenylbu- row transplantation provides a potential cure
tyrate was also found to increase HbF levels for only about 5% or fewer of sickle-cell disease
patients (215).Patients must have a sickle-cell
466 Inhibition of HbS Polymerization as a Basis for Therapeutic Approaches to Sickle-Cell Anernia

Marrow aspirate

Red
blood
conditioning cells

Bone marrow

Figure 9.18. Bone marrow transplantation introduces hematopoietic stem cells from a disease-free
donor with an AA or AS genotype, as shown in the schematic drawing. Hematopoietic stem cells are
harvested from a normal donor. The affected individual is treated with a marrow-conditioning
regimen to reduce the pool of affected hematopoietic stem cells. The donor hematopoietic stem cells
are then infused into the affected individual. Successful transplantation would result in replacement
of the affected hematopoietic stem cells with donor hematopoietic stem cells and the production of
normal or unaffected red blood cells.

disease-free sibling who is an exact immuno- mortality associated with bone marrow trai
logical match. Because of the risk of the pro- plantation from a human leukocyte antig
cedure (8-10% risk of death attributed to (HLA)-matched donor for sickle-cell anen
graft vs. host disease and complications), up by use of milder marrow-ablative conditic
until now it has been reserved for patients ing.
who have failed other treatments. The initial The therapeutic potential of stable mix:ed
transplant for sickle-cell anemia was carried chimerism has important implications jfor
out as a treatment for coexisting leukemia gene-transfer approaches in the treatment of
(216). To date, more than 100 patients with sickle-cell anemia and p-thalassemia. Com-
sickle-cell anemia have been treated with bone plete modification of the pool of hematopoietic
marrow transplantation. stem cells, a formidable task, no longer 2IP-
Transplantation of allogeneic bone marrow pears to be the absolute requirement for thler-
through the use of a less-intense marrow-ab- apeutic efficacy. The difficulty in availability
lative conditioning regimen offers the advan- of HLA-matched donors and associated tox ic-
tage of reduced toxicity, but increases the po- ity limit treatment by allogeneic bone marrcDW
tential for hematopoietic mixed chimerism transplantation. An alternative approach is
(217). The longer red cell survival time of genetic manipulation of the diseased hema to-
normal erythrocytes is expected to provide a poietic stem cell by gene replacement/corrcec-
preferential survival to normal versus SS tion or gene addition to restore the nornla1
erythrocytes, and reduce the contribution of phenotype. For success, such changes w olld ~
remaining SS hematopoietic stem cells to the have to correct a significant proportion oft he
red cell population (218). The report of a small hematopoietic stem cells with a high level of
number (3) of SS patients with stable mixed expression of normal P-like globin genes, Ire-
chimerism (donor myeloid chimerism of 20- sulting in a marked decrease of HbS exprles-
75%) after bone marrow transplantation of sion.
HbA donor marrow provided evidence for the
7.2 Regulation of Clobin Gene Expression
selective survival of normal enrthrocvtes
(%HbSfrom 0% to 7%) and a significant k e - Hemoglobin production proceeds by acti~
liorative effect (213). These results offer tion of globin gene transcription (219-22
promise for improvement in the morbidity and The coding region for the a-like (encoding1
7 Bone Marrow Transplantation, Globin Gene Expression, and Gene Transfer 467

acids) and p-like (encoding 146 amino additional information on DNA regulatory el-
globin genes are interrupted by two in- ements that could confer a high level of eryth-
rvening sequences (introns). Globin tran- roid-specific gene expression. In transgenic
pts are processed or spliced to remove the mice, the human p-globin gene that includes
wo introns, and to add a poly-A tail. Mature the proximal promoter is expressed in a tissue-
obin mRNA transcripts are then trans- specific manner, but at low levels (232, 233).
ported out of the nucleus and act as templates DNase hypersensitive sites, particularly hy-
for globin polypeptide chain synthesis. Appro- persensitive site 2 (HS2), located 50 kb 5' of
priate protein folding and incorporation of the the P-globin gene within the p-globin cluster
iron-containing heme group allows for a-lp- (2341, significantly increase expression of the
globin dimer formation and association into p-globin or p-like globin transgene (235-238).
the hemoglobin tetramer. Globin gene tran- The five hypersensitive sites spanning 20 kb
scription is regulated principally by a proximal or more, and referred to as the locus control
promoter located 5' upstream of the coding region (LCR), when combined with the pglo-
region. Globin promoters are characterized by bin gene provide a high level of transgene ex-
an upstream "TATA" box that is located about pression in an erythroid-specific manner com-
30 base pairs (bp) upstream of the start site for parable to the endogenous mouse p-globin
transcription, where the transcription initia- gene. The LCR is able to upregulate other cis-
tion complex can assemble. Interactions with like erythroid genes in a copy-dependent man-
other protein complexes assembling upstream ner independent of the chromosomal location
on other promoter elements such as CCAAT of the transgene, suggesting that the LCR is
and CACCC motifs, binding sites for the critical for a high level of erythroid-specific
largely erythroid GATA-1 transcription factor transcription activity and contributes to de-
[(A/T)GATA(G/A)](223), or other enhancers, termining the chromatin structure of the
repressors, or regulatory motifs in distal P-like globin cluster. Construction of vectors
DNase hypersensitive sites (224) and beyond, for a high level of erythroid-specific expression
contribute to the frequency of transcription is likely to incorporate components of the
initiation of specific globin genes. Nuclear fac- LCR.
tor-erythroid 2 (NF-E2) (225, 226) and stem The LCR spans 20 kb or more, and its large
cell leukemia (SCL)/Tal-1 transcription fac- size limits its utilitv
" in vector constructs. To
tors (227, 228) further contribute to globin readily use the LCR in expression vectors, core
gene regulation. Gene-specific transcription elements have been determined for the DNase
factors include erythroid krupple-like factor hypersensitive sites 2, 3, and 4 (HS2, HS3,
(EKLF)that binds to the 5' region of the P-glo- HS4) that are able to enhance 6-globin gene
bin gene and is required for high level p-globin expression in erythroid cells many fold (239-
gene expression in adult erythroid progenitor 241). However, in stable cell transfection stud-
cells (2291, and possibly FKLF and FKLF-2 ies, expression varied from clone to clone, and
that are reported to exhibit preferential acti- in some cases, silenced after some time in cul-
vation of y- and c-globin genes (230, 231). Al- ture. Although the small truncated LCR was
teration of transcription factor levels provides able to provide appropriate gene regulation in
the potential for direct manipulation of globin transgenic mice, position-effect variation re-
gene transcription. mained problematic in long-term cell culture.
Naturally occurring mutations and large "Promoter suppression" can be blocked by use
deletions of the p-globin cluster (Table 9.1) of insulator elements such as the HS4 insula-
provided initial information on important reg- tor from the chicken P-globin cluster (242).
ulatory elements located in cis to the globin Inclusion of this insulator in a retrovirus con-
genes within the p- or a-globin gene clusters struct significantly improved transduction ef-
(219). These include point mutations in the 5' ficiency in hematopoietic stem cell cultures
flanking region of the y-globin genes that give and in mouse transplantation studies (243,
rise to the HPFH phenotype by modifying 244). Other strategies for erythroid gene ex-
transcription factor binding. Studies of globin pression have used other enhancer regulatory
gene regulation in transgenic mice provided elements as well as other erythroid-specific
468 inhibition of HbS Polymerization as a Basis for Therapeutic Approaches to Sickle-Cell Anemia

promoters (245,246). The DNase hypersensi- site-directed correction of the pS mutation


tive site in the a-globin cluster, HS-40 (247),is (256). However, success has been elusive,
another erythroid enhancer that has been in- given the initial reports of a 5-11% conversion
corporated into expression vectors (246,248). rate of GAG in the normal p-globin gene to the
GTG ps-globin mutation in an enriched
7.3 Modification of Endogenous Clobin
CD34+ hematopoietic cell population.
Gene Expression
7.4 Gene Transfer of Recombinant Clobin
Strategies to reduce globin-specific transcripts
Gene Vectors
include antisense oligonucleotides to block
specific globin chain synthesis (249), ri- Although large constructs including artificial
bozymes (2501, and multiribozymes (251) to chromosomes can be used in the construction
reduce the pool of mature transcripts for a spe- of transgenic mice, the amount of DNA that
cific globin, and transribozyme technology to can be incorporated into viral vectors is lim-
replace the mutant ps-globin transcript with a ited (248). Early efforts for genetic-based
y-globin transcript (252). In culture studies of treatment for sickle-cell anemia and other he-
globin-producing cells, expression of human moglobinopathies used replication-defective
p-globin antisense transcripts was able to re- retroviruses (257-259). Vectors designed to
duce p-chain biosynthesis and increase increase expression of normal p-globin were
y-chain synthesis fivefold (249). Through the used in mouse studies to target hematopoietic
use of hammerhead ribozyme, RNA that is ca- stem cells in vitro that were transplanted back
pable of sequence-specific cleavage of other into conditioned recipients (Fig. 9.19). The
RNA molecules, transgenic mice expressing low gene-transfer efficiency and low gene ex-
human ps-globin and a lobin in directed ri- pression in these studies were discouraging.
bozyme reduced the ps-globin chains by 10% Although sickle-cell anemia was the f i s t ge-
(250). In culture studies, a multiribozyme in- netic disease described more than five decades
corporating five specific globin cleavage sites ago, the difficulty in obtaining a high degree of
was able to reduce a-globin mRNA by 50% or gene transfer diminished the prospects of
more, suggesting a method for further in- treatment of sickle-ceI1anemia by this modal-
creasing ribozyme activity (251).Other uses of ity, and sickle-cell anemia was no longer con-
ribozyme technology are based on RNA repair sidered a primary candidate for initial gene
and make use of a trans-splicing group I ri- therapy trials in humans.
bozyme to convert ps-globin transcripts to Adenoviral vectors (Ad) were found to be
y-globin transcripts (252).Initial in vitro stud- useful in targeting CD34+ CD38- hematopoi-
ies claimed an 8% conversion of ps-globin etic cells (260). but their transient nature lim-
transcripts, demonstrating the potential of its their utility for globin gene expression in
this approach. Peptide nucleic acids or oligo- late erythroid precursor cells. Recombinant
nucleotidescapable of forming complexes with adeno-associated virus (AAV) could provide
the 5' region of the y-globin gene have been expression of human y-globin gene in ery-
proposed as inducers of HbF (253,254). throid cells, particularly in the presence of
Homologous recombination has been use- positive selection, and showed the potential of
ful in generating genomic mutations or modi- AAV to transfer globin gene expression (261,
fications at specific genetic loci. Such modifi- 262). Recently, a hybrid adenovirus (Ad)/ad-
cations in murine embryonic stem cells eno-associated virus (AAV) vector with a chi-
provide important models of gene-specific dis- meric Ad capsid for efficient gene transfer into
eases. Targeted deletion of the mouse a- and human hematopoietic cells and the AAV in-
p-globin genes are the basis of mouse models verted-terminal repeats for integration of vec-
expressing exclusively human hemoglobins, tor genome into the host genome was created
including HbS. However, the current low fre- to include an 11.6-kb y-globin expression cas-
quency of homologous recombination dimin- sette containing HS2 and HS3 of the LCR
ishes its value as a therapeutic strategy for (263). This vector was devoid of all viral genes
sickle-cell anemia (255). RNA-DNA oligonu- and provided stable transgene expression in
cleotides have been proposed as a strategy for hematopoietic cells. Although not yet tested in
7 Bone Marrow Transplantation, Globin Gene Expression, and Gene Transfer

Normal gene
w\

Red
blood
cells

Bone marrow

Figure 9.19. A gene-transfer approach to therapy introduces a normal or corrected hemoglobin


gene into the hematopoietic stem cell of the affected SS individual, as shown in the schematic
drawing. Hematopoietic stem cells are harvested and treated ex viuo. The individual is treated with a
marrow-conditioning regimen to reduce the pool of affected hematopoietic stem cells. The treated
stem cells containing the new genetic material are then infused back into the affected individual.
Successful transplantation would result in the replacement of affected hematopoietic stem cells with
treated hematopoietic stem cells and the production of normal or unaffected red blood cells.

human hematopoietic stem cells, this vector vectors to provide long-term expression of glo-
shows the potential of combining elements bin genes in affected erythroid progenitor cells
from multiple viruses to optimize vector de- and reducing the physiologic symptoms in
sign. these mouse models of hemoglobinopathies.
To increase the amount of DNA that can be
7.5 Modification of Hematopoietic Stem
incorporated into gene-transfer vectors, the
Cell Response
human immunodeficiency virus 1 (HIV-1)was
modified and used as a basis for vector con- The potential for erythropoietin administra-
struction because of its ability to include large tion to augment the increase in HbF in sickle-
DNA fragments and its RNA-splicing poten- cell anemia patients undergoing hydroxyurea
tial (246,264,265). A large genomic fragment therapy suggests an additional strategy for
containing the LCR core elements, p-globin gene-transfer techniques. In animal studies,
zene, and 3' p-globin enhancer were incorpo- direct muscle injection of an AAV vector ex-
rated into an attenuated HIV-1-derived vector pressing erythropoietin or a DNA plasmid ex-
and used successfully in gene-transfer experi- pression vector encoding erythropoietin ac-
~ e n t sto treat p-thalassemia in a mouse companied by electric pulses to stimulate cell
model, by providing therapeutic levels of hu- uptake provided long-term expression of
nan normal p-globin (264). However, expres- erythropoietin in uivo (266,267). Other gene-
lion of the transferred normal p-globin gene transfer approaches are based on modifying
Mas heterogeneous and low. As a strategy for the surface receptors on hematopoietic stem
ireatment of sickle-cell anemia, a modified cells and progenitor cells to enhance drug re-
'IN-1-based lentiviral vector was optimized sponse (268). Incorporation of drug-resistance
'or expression of a p-globin variant, P87Thr-Gln 9 genes into the gene-transfer vector provides a
;hat prevented HbS polymerization (265). In potential for competitive selection with drug
rene-targeting experiments in sickle-cell ane- treatment (269). This strategy biases against
nia mouse models, up to 52%of the hemoglo- untreated endogenous cells or transduced
)in was modified and distributed among 99% cells not expressing the transferred gene. In-
)f circulating erythrocytes, providing normal- clusion of other genes directed at increasing
zation of hematological parameters, urine- the pool of transduced hematopoietic stem
:oncentrating ability, and spleen size. These cells include HOXB4 (270) and possibly the
ltudies demonstrate the ability of lentiviral anti-apoptotic Bcl-2 (271). Expression of a
470 Inhibition of HbS Polymerization as a Basis for Therapeutic Approaches to Sickle-Cell Anemia

truncated erythropoietin receptor provided a their ultimate potential for treating human
selective advantage to transplanted hemato- beings with sickle-cell anemia and related he-
poietic stem cells in the presence of erythro- moglobinopathies.
poietin (272). Hybrid receptors have been
designed to incorporate some of the hemato-
poietic cytokine receptors such as receptors REFERENCES
for thrombopoietin, erythropoietin, or G-CSF. 1. J. B. Herrick, Arch. Intern. Med., 6, 517521
Through use of a drug-binding extracellular (1910).
domain with the cytoplasmic domains for 2. J. G. Huck, Bull. Johns Hopkins Hosp., 23,
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Iron Chelators and
Therapeutic Uses
1
e
i RAYMOND J. BERGERON
JAMES S. MCMANIS
WILL^ R. WEIMAR
JAN WIEGAND
EILEEN EILER-MCMANIS
College of Pharmacy
University of Florida
Gainesville, Florida

Contents
1 Introduction, 480
1.1Iron in the Biosphere, 480
1.2Iron Dynamics in Microorganisms, 481
1.2.1Metal Complex Formation and the
Chelate Effect, 481
1.2.2Structural Classes of Siderophores, 483
1.2.3Bacterial Iron Uptake and Processing,
488
1.3 Iron Dynamics in Humans, 495
1.3.1Iron Storage and Transport, 495
1.3.2Molecular Control of Iron Uptake,
Processing, and Storage, 499
1.3.3Iron Absorption, 501
1.3.4Iron-Mediated Damage, 501
1.4Iron-Mediated Diseases, 503
2 Clinical Use of Chelating Agents, 505
2.1Iron Chelators on the Market, 507
2.1.1Desferrioxamine (DFO, 9),507
2.1.1.1Side Effects of Desferrioxamine,
509
2.1.1.2Pharmacology of
Desferrioxamine, 510
2.1.2Diethylenetriamine Pentaacetic Acid
(DTPA, 361,511
2.1.2.1Side Effects of DTPA, 511
2.1.2.2Pharmacology of DTPA, 511
2.1.31,2-Dimethy1-3-hydroxypyridin-4-0ne
Burger's Medicinal Chemistry and Drug Discovery (Deferiprone, L1;33),512
Sixth Edition, Volume 3:Cardiovascular Agents and 2.1.3.1Side Effects of L1,512
Endocrines 2.1.3.2Pharmacology of L1,512
Edited by Donald J. Abraham 3 History of Chelation Therapy; Discovery of
ISBN 0-471-37029-0 O2003John Wiley & Sons, Inc. Agents with Iron-Chelating Activity, 514
480 Iron Chelators and Therapeutic Uses

4 Recent Developments, 515 5.3 Integration of Design, Synthesis, and


4.1 N,N1-Bis(2-hydroxybenzy1)ethylenediamine- Testing: Desferrithiocin Analogs, 540
N,N'-diacetic Acid (HBED, 39a),515 5.3.1Iron-Clearance Evaluations, 545
4.1.1 Parenteral Administration to Rodents, 5.3.1.1Changes In the Distances
515 Between the Ligating Centers
4.1.2 Parenteral Administration to Primates, (Series I, Compounds 105-107),
516 545
4.1.2.1Subcutaneous Administration, 5.3.1.2Thiazoline Ring Modification
517 (Series 11,Compounds
4.1.2.2 Intravenous Administration, 519 108-114),546
4.1.3 Preclinical Toxicity Trials, 520 5.3.1.3 Configurational [(R)-and (S)-I
5 Things to Come, 522
Changes at C-4 (Series 111,
5.1Synthetic Approaches, 522
Compound 115 versus 103,
5.1.1 Catecholamides, 522
5.1.2Hydroxamates, 524 Compound 116 versus 104,
5.1.3Desferrithiocin and its Analogs, 530 Compound 117 versus 118),547
5.1.4 Rhizoferrin, 532 5.3.1.4Benz-Fusion (Series IV,
5.2Animal Models Employed in Iron Compounds 96,119-1231,549
Metabolism and Iron Chelator Studies, 534 5.3.1.5Addition of Electron-Donating
5.2.1 Genetically Characterized Rodents, 534 and -Withdrawing Groups to
5.2.2 Wild-type Rodents, 535 the Aromatic Ring (of 104,
5.2.2.1 Non-Iron Overloaded Bile Duct- Series V A, Compounds 118,
Cannulated Rat, 535 124-126;of 93,Series V B,
5.2.2.2Iron-Overloaded Rodent Compound 941,549
Models, 536 5.3.2 Toxicity, 549
5.2.3Primate Models, 536 5.3.3 Metabolism of Desazadesferrithiocins,
5.2.3.1Cebus apella, 536 550
5.2.3.2 Marmosets (Callithrixjacchus), 6 Web Site Addresses and Recommended Reading
538 for Further Information, 551
5.2.3.3 Comparison of the C.apella 7 Acknowledgments, 551
and Marmoset (C.jacchus)
Models, 539

1 INTRODUCTION tial cofactor in a variety of biological redox


systems, for example, cytochromes, oxidases,
1.1 lron in the Biosphere peroxidases, and ribonucleotide reductase (2,
Although iron has many oxidation states 3). Paradoxically, even though iron is the sec-
available to it, ranging from -2 to +6, the +2 ond most abundant metal on earth, living sys-
and +3 valences are of the greatest impor- tems have had to develop sophisticated meth-
tance in biological systems. his statement is ods for its acquisition (4-6). In fact, with the
not meant to diminish the significance of possible exception of some lactobacilli, life
Fe(IV) (ferryl, found in cytochromes and without this metal is virtually unknown.
horseradish peroxidase), Fe(V) (perferryl), When the most primitive life forms devel-
and Fe(V1); however, the chemistry of these oped about 3.5 billion years ago, the atmo-
species is beyond the scope of this chapter. The sphere was basically anaerobic; in the absence
+2 and + 3 oxidation states, which are charac- of molecular oxygen, iron was mostly in the +2
terized, respectively, by their d6 and d5 oxidation state. This form of the metal is much
ground-state configurations, are exquisitely more soluble and accessible to biological sys-
sensitive to both pH and the nature of the li- tems than is Fe(II1) (7). It is for this reason
gating functionality (1).At a cellular level, this that oral iron supplements are generally in the
sensitivity has been exploited, inasmuch as Fe(I1) form (e.g., ferrous sulfate) and not as
this metal can function both as an electron the Fe(II1) salts. Upon evolution of the blue-
source and an electron sink. Iron is an essen- green algae, problems developed; these diffi-
f 1 Introduction
L

i culties were related directly to the availability


2 of this essential micronutrient. The oxygen
produced from these algae by photosynthesis
caused the conversion of the iron(I1) in the
biosphere to Fe(III), a species that is highly
insoluble in an aqueous environment. The sol-
ubility product of ferric hydroxide under phys-
iological conditions, 2 x lop3', corresponds to
a solution concentration of the free cation of
approximately1 X 10-"M (8,9). Under most
conditions that exist in the biosphere, iron(II1)
forms insoluble ferric hydroxide polymers.
Thus, in spite of the abundance of this metal,
primitive life forms needed to develop meth-
ods for rendering it usable.

1.2 Iron Dynamics in Microorganisms


Ultimately, bacteria adapted to the iron acces-
This same set of equilibria also can be ex-
sibility problem by producing relatively low
pressed in a non-stepwise fashion or as overall
molecular weight, virtually iron(II1)-specific,
equilibrium constants, as shown in Equations
ligands, siderophores (from the Greek sidero
10.2A-10.2F:
andphore, literally "iron carrier"), for the pur-
pose of acquiring this transition metal (5, 6 ,
10). Under conditions of low iron availability,
microorganisms biosynthesize and release up
to several times' their own weight of ligand
into the environment daily (11). These chela-
tors form soluble complexes with the metal;
the bacteria are then virtually immersed in
these iron chelates, a usable iron source.

1.2.1 Metal Complex Formation and the


Chelate Effect. This discussion is best opened
with an overview of the equilibria that de-
scribe metal complex formation. Consider a
transition metal [e.g., Fe(1II)I in the presence
of a monodentate ligand (L). The equilibria
depicting the formation of an octahedral,
hexacoordinate Fe(II1) complex are shown in
Equations 10.1A-10. IF; the constants K,-K,
are referred to as stepwise equilibrium con-
stants:

A simple algebraic manipulation de-


monstrates the relationship between these
Iron Chelators and Therapeutic Uses

two forms of expression (Equations 10.3A-


10.3D):

Thus, the generalized relationship between


the two expressions becomes (Equation 10.4):

When one compares equilibrium constants in


the literature, it is important to be certain
which equilibrium expression is being re-
ferred to. The stepwise constants are useful in
identifying which species islare present; these
constants generally diminish in value as the
1igand:metal ratio increases. In keeping with Figure 10.1. Structures of the catecholamide sid-
our focus on natural product iron chelators, erophore enterobactin (1) and an enterobactin
the application of complex formation equilib- model system, 2,3-dihydroxy-NJV-dimethylbenz-
ria is best seen in earlier work on the enter- amide (DHBA, 2).
obactin-Fe(II1)complex (12-14). Enterobactin
(1, Fig. 10.1),a siderophore produced by Esch-
erichia coli, forms a very tight octahedral
hexacoordinate complex with Fe(II1). A com-
parison of the iron binding properties of this
siderophore and a model bidentate ligand ex-
emplifies the importance of the chelate effect log Kz = 13.96
in complex formation.
When three moles of the bidentate ligand
2,3-dihydroxy-Nfl-dimethylbemamide(DHBA,
2), are reacted with Fe(III), (2) forms a stable
3:l 1igand:metal complex with the iron (13).
The stepwise reactions and their respective
-
log K3 = 8.51
equilibrium constants are shown in Equations
10.5A-10.5C:
Thus, P3 for this sequence of reactions is
calculated as P3 = KlK2K3,or log P3 as log P3
= log K , + log K2 + log K3 = 40.24 (13).
Examination of the same calculation for en-
terobactin (1)when compared to that for (2) is
interesting. The equilibrium expression for
log Kl = 17.77 this complex is (Equation 10.6):
1 Introduction

Fe(II1)+ % Fel-3 that determined for the fully deprotonated


form (Ip6) (14).Thus, to provide a more mean-
[Fel-3] ingfbl comparison among ligands, investiga-
(10.6)
KML = [Fe(III)][1-6] tors have suggested the use of pM values,
where pM = -log[Fe(III)] (15). This number
log K M L ( P =
) 52 describes the concentration of free Fe(II1) in
solution at a given pH, total iron, and total
The log K, for this complex was calculated to ligand concentration; lower free iron concen-
be52 (12,13),12 powers of 10 greater than the trations translate into higher pM values. Ta-
p, for the individual donor, DHBA. This enor- ble 10.1 provides several examples of key li-
mous difference in formation constants be- gands, both natural and synthetic, for the
tween a complex consisting of three uncon- reader's consideration (16-24). Note the ef-
nected bidentate ligands and Fe(II1) and one fect of denticity on the pM values (e.g., 1 ver-
consisting of Fe(II1) complexed with a single sus 2). The measurements were made at pH
hexacoordinate donor, in this example enter- 7.4, M Fe(III), and M ligand.
obactin, is attributed to the chelate effect. Re-
call that AG = -RT in K and that AG = AH - 1.2.2 Structural Classes of Siderophores.
TU. Although a large number of siderophores have
There are several ways of thinking about been isolated, they generally can be separated
this phenomenon, two of which are somewhat into two basic structural groups, the catechol-
simplistic, yet informative. When the three bi- amides and the hydroxamates. Interestingly,
dentate ligands replace the water that sur- many of these chelators have common struc-
rounds the Fe(III),six water molecules are dis- tural denominators. They can be considered as
placed, and a net of three molecules are either directly predicated on polyamine (e.g.,
liberated. When the hexacoordinate sid- putrescine, cadaverine, norspermidine, or
erophore (1)binds to Fe(III), again, six mole- spermidine) backbones or based on the bio-
cules of water are displaced; however, a net of chemical precursors to the polyamines (e.g.,
five molecules are liberated. Thus, there is a ornithine or lysine) (25). The catecholamide
greater increase in disorder (AS) in the case of chelators (Fig. 10.2),N1p-bis(2,3-dihydroxy-
(1)than in that of (2). An alternative thought benzoy1)spermidine (compound 11, 3) (261, L-
is that when (1)donates its first bidentate parabactin (4) (26, 271, L-agrobactin (5) (281,
L-fluviabactin (6) (29), L-vulnibactin (7) (30),
fragment, the second and third bidentate frag-
ments are held closely for donation, unlike the and L-vibriobactin (8) (31), all have bidentate
situation with the (2) donors. 2,3-dihydroxybenzoyl groups fixed directly to
One of the difficulties with comparing li- either a spermidine (3-6) or a norspermidine
gand formation constants is that, often, the (6-8) backbone. Obviously, (1)(Fig. 10.1), a
measurements are made under different con- siderophore based on a macrocyclic serine
ditions and are quite sensitive to the pK, of backbone, is an exception to this observation.
the particular donor groups. For example, in Chelators in the hydroxamate class contain
the case of enterobactin with Fe(III), the hy- bidentate N-hydroxy amide (i.e., hydroxamic
drogen ion dependency is seen in the expres- acid) groups. The designations "hydroxamic
sion (Equation 10.7): acid" and "hydroxamate" are interchangeable
in the arena of iron chelator nomenclature
Fe(II1) + H61 = Fel-3 + 6H' (32), although the latter term is sometimes
restricted to N-alkoxy (33) or N-silyloxy
[Fel-3][H+]6 amides (34). The cadaverine moieties of the
(10.7) hydroxamates (Fig. 10.3) desferrioxamine B
K M ~= [Fe(III)][H611
(DFO, 9) (35), desferrioxamine G (10) (361,
log KML(H61)
= -9.7 desferrioxamine E (nocardamine, 11) (371,
bisucaberin (12) (38), and arthrobactin (13)
The stability constant for the fully protonated (39) are apparent. Other polyamines or their
ligand (H,l) is substantially different from amino acid precursors are evident in various
Table 10.1 Iron pM Values for Selected Ligands
Compound
. Ligand
- Equilibrium Quotient log K log P 3 PW Reference(s)
(1) Enterobactin [Fe(III)-L3-I/([Fe(III)I[L6pl) 52 35.5 (12-14)
(2) 2,3-Dihydroxy-N,N-dimethylbenzamide [Fe(III)-L33-I/([Fe(III)I[L2-13) 40.2 15~ (14,161
(4) Parabadin [Fe(IIIbLI/([Fe(III)1[Ll) 48 - (17)
(9) Ferrioxamine B [Fe(IIIbLII([Fe(III)I [Ll) 30.99 25.9 (15)
(11) Ferrioxamine E [Fe(III)-LI/([Fe(III)l [Ll) 32.5 27.7 (13,21)
(14) Aerobactin [Fe(III)-LI/([Fe(III)I[LI) 23.1 23.3 (13,14)
Q
OJ (17) Ferrichrome [Fe(III)-LI/([Fe(III)l[Ll) 29.1 25.2 (14,22)
A
Nonec Transferrin 23 23.6 (13,23,24)
(32) EDTA [Fe(III)L-]/[Fe(III)I[L4pl 25.1 22.3 (15)
(33) 1,2-Dimethyl-3-hydroxypyridin-4-one [Fe(III)-L,I/([Fe(III)l[L-13) 35.92 18.3 (18,191
(L1)
(35) DTPA [Fe(III)-L2-I/[Fe(III)1[L5-I 28.0 23.8 (15)
(39) HBED [Fe(III)-L-]/[Fe(III)I[L4-] 39.68 26.74 (15,20)
"Calculated for 10 pH ligand, 1 pH Fe(III), at pH 7.4.
bCalculatedpM is below the lower limit determined by the K, of ferric hydroxide, indicating precipitation of iron under these conditions.
'See Fig. 10A for a diagram of this protein.
Figure 10.2. Other catechol-
amide siderophores: compound I1
(3), L-parabadin (4, R = HI,
L-agrobadin(5, R = OH), L-fluvi-
abadin (6),L -vulnibactin (7, R =
H), and L-vibriobactin(8, R = OH).
Polyamine backbones are high-
lighted by darkened bonds.
Iron Chelators and Therapeutic Uses

Figure 10.3. Hydroxamate siderophores: desferrioxamine B (DFO,9, R = CH,), desferrioxamineG


[(lo),R = (CH,),COOH], desferrioxamine E (nocardamine, 111, bisucaberin (12),arthrobactin (13,
R = H),aerobactin (14, R = CO,H), mycobactin S (15),nannochelin A (16),desferri-ferrichrome (17,
R = COCH,), rhodotorulic acid (18), schizokinen (191,and alcaligin (20).Polyamine backbones are
highlighted by darkened bonds.

hydroxamate ligands, such as aerobactin (14) the organism under high iron conditions,
(40), mycobactin S (15) (41), nannochelin whereas the catecholamide "backup" system
A (16) (42), desferri-ferrichrome (17) (431, is activated when iron concentrations are low.
rhodotorulic acid (18) (44), schizokinen (19) Logically, the catecholamide chelators typi-
(45), and alcaligin (20) (46). cally bind iron far more tightly than do hy-
The major functional contrast between the droxamates (Table 10.1). The formation con-
hydroxamate and catecholamide siderophores stants of the catecholamides can be as high as
is related to environmental iron concentration lo5' M-' for the (l):Fe(III) complex (12, 131,
(47). The hydroxamates are synthesized by whereas those for the hydroxamates are con-
Figure 10.3. (Continued.)
lron Chelators and Therapeutic Uses

OH
CH3
Figure 10.4. "Miscellaneous" sid-
erophores: rhizobactin (211, rhizo-
ferrin (22),pyochelin (23),and des-
ferrithiocin (24, DFT).Polyamine .H S .
backbones are highlighted by dark-
ened bonds.

siderably lower, on the order of lo3' M-I for amine-N,N'-bis(o-hydroxyphenylacetic acid),


desferrioxamine B (21,22, 48). has been well documented (57-59). Yet more
Although most siderophores do fall into one fascinating is the differential use of L- and D-
of the aforementioned classes, there are li- parabactin (25, Fig. 10.5) as measured by the
gands that do not belong to either family, such stimulation of bacterial growth under such
as rhizobactin (21) (49), rhizoferrin (22) (50), conditions. As expected, L-parabactin (4) fos-
pyochelin (23) (51-53), and (5')-4,5-dihydro-2- tered bacterial growth, whereas D-parabactin
(3-hydroxy-2-pyridinyl)-4-methyl-4-thiazo1e-(25) could not be used by the bacterial iron-
carboxylic acid (desferrithiocin, DFT, 24) (Fig. transport apparatus (Fig. 10.6) (57).
10.4). This chelator, isolated from Streptomy- The stereospecificity of the kinetics of iron
ces antibioticus (54), forms a stable (Kf = 4 x acquisition illustrates this phenomenon fur-
1OZ9M-l) 2:l complex with iron (55, 56). As ther (Fig. 10.7) (59). Iron accumulation data
discussed later in this chapter, DFT (24) rep- from [55Fe]ferric (25) fit a straight-line dou-
resents an excellent pharmacophore from ble-reciprocal plot and, thus, obey a simple
which to construct therapeutic orally active Michaelis-Menten model. However, the kinet-
iron chelators. ics of iron acquisition from ferric (4) are quite
different (Table 10.2). The presence of a high
1.2.3 Bacterial lron Uptake and Proces- affinity transport system for ferric (4) is re-
sing. One of the major questions concerning flected by the pronounced differences in up-
the iron-chelator complexes is the method for take rates at ligand concentrations below 1
processing them in vivo. Considering that the a. In fact, the ferric (4) data are both non-
siderophores bind iron quite tightly, how does linear and likely biphasic, as the enlargement
a microorganism extract iron from a com- of Fig. 10.7 illustrates (Fig. 10.8). The data for
pound that binds it so well? The purpose that 1 pit4 < [4] < 10 CJM fit one line, which sug-
the siderophores serve in providing microor- gests a low affinity system, yet the data for 0.1
ganisms with iron is illustrated here by the pA4 5 [4] 5 1 CJM fit another line, which is
parabactin-mediated iron transport appara- consistent with a high affinity system (Table
tus of Paracoccus denitrificans. The efficacy of 10.2, Fig. 10.8). The apparent K, of the high
L-parabactin (4) in supplying P. denitrificans affmity uptake (0.24 f l is comparable to the
with iron under artificially iron-lowered con- affinity constant reported for the purified fer-
ditions, such as in the presence of ethylenedi- ric (4) outer membrane receptor protein (58)
1 Introduction

Figure 10.5. Structure of D-parabactin(25), which is derived synthetically from D-threonine; L-


homoparabadin (26), a parabactin homolog; L-homofluviabactin(27), a fluviabactin homolog; D-
fluviabactin (28), the enantiomer of (6);and the open-chain threonyl ( " A ) forms of L- and D-para-
bactin (L-parabactinA, (29),and D-parabactin A, (30), respectively).
Iron Chelators and Therapeutic Uses

8.50
Figure 10.6. Growth rate of P. denitrifi-
cans [rendered as colony-forming units
(CFU)/mL,y-axis, versus time, x-axis] in
the presence of L-parabactin (0, 2 jd0,
D-parabactin (A, 2 jd0,or controls (0) 8.25
each in the presence of EDDHA (1.1 mM)
without added ferric nitrate. [Reprinted
with permission from R. J. Bergeron et al.,
J. Biol. Chem., 260, 7936-7944 (1985). 8.0
Copyright 1985 The American Society for
Biochemistry and Molecular Biology.] Time (hours)

and also is in keeping with the values reported lation of 66Fefrom ferric (8).Perhaps not sur-
for high affmity transport systems for fer- prisingly, ferric (25) at similar concentrations
richrome (17,0.15-0.25 and ferric entero- exerted no impact on 55Fe transport (59).
bactin (1,0.10-0.36 phf) in E. coli (60-62). In Thus, the high affinity ferric L-parabactin re-
other microorganisms, the Kmvalues reported ceptor seems to recognize and subsequently
for siderophore-mediated iron transport allow iron acquisition from these ferric L-ox-
range from relatively low aMinity apparati, azoline homologs, at least over the concentra-
such as that for ferric coprogen transport (K, tion range studied. The degree to which this
= 5 pM) in Neurospora crassa (63, 64) to an system also contributes to the low affmity
extremely high affinity assemblage (K, = 0.04 component of the biphasic kinetic profile is un-
pM) for ferric schizokinen (19) transport in clear. Biphasic kinetics have been observed for
species of the cyanobacterium Anabaena (65). many membrane transport systems. In some
Biphasic kinetics similar to those of ferric cases, the presence of two independent sys-
(41, including a high affinity component, have tems for transport of the same substrate ex-
been observed in P. denitrificans using plains the observed phenomenon (67). A plau-
L-agrobactin (5),L-fluviabactin (6),L-vibriobac- sible alternative is that negative allosteric
tin (8),and two synthetic homologs (L-homo- interactions can result in a system with a low
parabactin, 26, and L-homofluviabactin, 27, Km at low [S], which converts to a high Km
respectively, Fig. 10.5), (Table 10.2) (59, 66). system at high [Sl (68,691.
Also, the.ferric chelates of (5), (a), and (26) The contributions of molecular dissymme-
(0.5 and 1.0 pit0 inhibited accumulation of try to these distinctive kinetic features are un-
55Fe from ferric (4) by way of simple sub- derscored by the stereospecific differences in
strate-competitive Michaelis-Menten kinetics transport of the ferric catecholamides. The
(59). Conversely, ferric (4) inhibited accumu- similarities in ring size and nature of the che-
1 Introduction

0.3

Ferric D-parabactin
Km = 3.1 pM
Vmax = 125

Figure 10.7. Lineweaver-Burk plot of kinetic data for the transport of [65FelferricL-parabactin (4)
and [55Fe]ferricD-parabactin (25)for 0.1 5 [Sl 5 10a, where [Sl is the concentration of chelate
added to external medium at t = 0. The (25) in this and other experiments can be fitted on a single
regression line (r = 0.999) corresponding to a simple Michaelis-Menten process with comparatively
low affinity (K, = 3.1 + 0.9 a; V, = 125 + 11 pg-atoms of 55Femin-' mg of protein-'). The (4)
data are nonlinear (see Fig. 10.81, but if analyzed separately, the data for 1 < [41 < 10 fit one
line (r = 0.991), suggesting a low affinity system (K, = 3.9 + 1.2 pM; V,, = 495 + 41 pg-atoms of
55Femin-' mg of rotei in-'), whereas the data for 0.1 pkf 5 [41 5 1 pkf fit another line (r = 0.996),
consistent with a high affmity system (K,= 0.24 t 0.06 @; V, = 118 + 19 pg-atoms of 55Femin-'
mg of protein-'). Note that the data in these two concentration ranges are analyzed independently;
that is, the high affinity data are not corrected for contributions made by the low affinity system
operating at [S] < 1 a, nor are the low affinity data corrected for contributions made by the high
affinity system operating at [S] > 1 a. Data are presented as means of four (25) or five (4)
determinations and SDs (bars) for [Sl = 0.1, 0.2, 0.5, 1.0, 2.0, 5.0, and 10.0 a. [Reprinted with
permission from Bergeron and Weimar, J. Bacterial., 172,2650-2657 (1990). Copyright 1990 Arner-
ican Society for Microbiology.]

late donor centers in these catecholamides al- ferric L-parabactin is characteristic of the A
low direct comparison of their circular dichro- chelate enantiomer (59, 73). The CD data also
ism (CD) spectra with those of ferric suggest that other ferric catecholamides that
catecholamide chelates of known configura- contain an oxazoline ring derived from L-thre-
tion (70-72). The positive CD band maxima of onine (i.e., 5,8, and 26) all exist in solution as
492 Iron Chelators and Therapeutic Uses

Table 10.2 Iron Accumulation Kinetic Data


Ferric Chelatea Kinetics Kmb vmac Reference
(4) (5) Bimodal 0.24 + 0.06 f l (high affinity) (r = 0.996) 118 t 19 (59)
3.9 t 1.2 f l (low affinity) (r = 0.991) 495 t 41
(5)(3) Bimodal 0.13 t 0.08 pA4 (high affinity) 46 2 8 (59)
1.6 + 0.8 f l (low affinity) 221 + 18
(6)(3) Bimodal 0.23 2 0.03 f l (high affinity) 129 2 2 (66)
2.2 2 1.6 f l (low affinity) 413 2 149
(8)(3) Bimodal 0.18 t 0.07 f l (high affinity) 72 ? 16 (59)
5.8 t 1.4 f l (low f f i i t y ) 544 t 51
(26) (2) Bimodal 0.26 + 0.10 pit4 (high affinity) 142 t 24 (59)
2.6 -+ 1.3 f l (low affinity) 254 2 31
(27) (3) Bimodal 0.17 + 0.04 pA4 (high affinity) 138 + 19 (66)
1.0 + 0.1 f l (low affinity) 229 t 29
(25) (4) Linear 3.1 + 0.9 f l 125 t 11 (59)
(28) (3) Linear 3.5 2 0.8 f l 96 2 63 (66)
(29) (4) Linear 1.4 2 0.3 f l 324 2 21 (59)
(30) (2) Linear 1.3 + 0.3 f l 330 + 12 (59)
(9) (3) Linear 33 + 9.3 f l 98 + 16 (59)
(11)(2) Linear 26 + 6.1 f l 7.6 2 2.1 (59)
"Number in parentheses refers to the number of assays at each chelate concentration: 0.1,0.2,0.5,1.0,2.0,5.0,and 10 f l
forchelates (41, (ti),(8),(25),and (26);0.1,0.2,0.5,1.0,2.0,and5.0 &for ferric(%), (27),and (28);0.1,0.25,0.5,1.0,2.0,5.0,
and 10 & for ferric (29) and (30);and 0.1, 0.25, 0.5, 1.0,2.0, 5.0, 10, and 20 &for ferric (9) and (11).
bK, 2 SE.
"Picogram-atoms of 55Feper minute per mg of protein (? SE).

the A chelate enantiomers, but that ferric (25) tion from [55Fe]ferric(4) and [55Fe]ferric(8)
is the mirror-image A chelate enantiomer (59). was strongly inhibited to equivalent degrees
Thus, these chelates, such as ferric (4) versus by ferric (29) and ferric (30) (59). These ki-
ferric (25), differ at either three or five chiral netic data indicate a complex inhibition that
centers: the metal center chiral configuration, does not appear to fit the usual simple models
plus the asymmetric carbons in the oxazoline (e.g., competitive, noncompetitive, uncom-
ring(s) derived either from D-(2R,3S)-threo- petitive). Conversely, 55Feaccumulation from
nine in the case of (25) or from L-(2S,3R)-thre- the labeled chelates of both (29) and (30) were
onine in the case of the ligands in the repressed by ferric (41, again, by apparently
L-configuration. complicated kinetics. Accumulation of 55Fe
Interestingly, neither the presence of an L- from [55Fe]ferric (4) and [55Fe]ferric L-para-
oxazoline ring nor a A metal center alone de- bactin A (29) were not diminished by ferric
termines whether a chelate can be used by P. (25), except at relatively high concentrations.
denitrificans (59). First, the bacterium accu- A model consistent with these overall findings
mulated 55Fefrom [55Fe]ferric(25) and [55Fel- entails a stereospecific binding step of high af-
ferric D-fluviabactin(28), although not nearly finity, which requires the L-oxazoline ring, fol-
as efficiently as from [55Fe]ferric(4) or (6). lowed by a nonstereospecific postreceptor pro-
Further, the A-forms of ferric parabactin [fer- cessing involving hydrolysis of the oxazoline
ric L- (29) and D- (30)parabactin A, Fig. 10.51, ring of ferric L-parabactin (4) (E,' = -0.673
in which the oxazoline ring has hydrolyzed to mV, pH 7.0) to the open-chain threonyl struc-
the open-chain threonyl structure, exhibited ture of ferric L-parabactin A (29) [E,' =
linear kinetics, including a relatively high Km -0.400 mV, pH 7.0 (74)], from which iron
and a surprisingly high Vm, (Table 10.2). The might be removed more readily.
CD spectra of ferric (29) and ferric (30) are Although P. denitrificans neither produces
exact mirror images; however, the iron acqui- nor secretes hydroxamate siderophores, both
sition from ferric parabactin A is not ste- labeled ferric chelates of (9) and (11)do facil-
reospecific (Table 10.2). Net 55Fe accumula- itate the transport of iron, apparently by low
1 Introduction

Ferric L-parabactin
high affinity system
([Sl5 1 PM)
Km = 0.24 pM
Vmax= 118

Ferric L-parabactin
low affinity system
(PI2 1 PM)
Km = 3.9 pM
Vmax = 495

Figure 10.8. Enlarged view of [55FelferricL-parabactin of kinetic data presented in Figure 10.7,
emphasizing the bimodal nature of this plot. The data for 0.1 pA4 5 [415 1 and the data for 1 f l
< [4] < 10 pA4 are fitted to regression lines, respectively representing high and low affinity phases of
uptake. Data are presented as means of five determinations and standard deviations (bars) for [Sl =
0.1,0.2,0.5,1.0,2.0,5.0,and 10.0 a. [Reprinted with permission from Bergeron and Weimar, J.
Bacterial., 172,2650-2657(1990).Copyright 1990 American Society for Microbiology.]

affmity, low Vm, transport mechanisms (Ta- smegmatis (82) and Saccharomyces cerevisiae
ble 10.2) (59). Many microorganisms some- (83). The reductase in M. smegmatis has a Km
times produce specific membrane receptors to for ferrimycobactin estimated to be <4 p M
recognize and transport these complexes (6, (82),a value similar to the apparent Kmvalues
60,63, 75-78). for iron acquisition from (29) and (30).One
A nonstereospecific, low affinity system, alternative to a reductase is a ligand-exchange
acting independently of the high affinity mechanism similar to those proposed for my-
L-parabactiniron transport apparatus in P. cobactin-mediated iron transport in Mycobac-
denitrificans, has been reported (79).These terium species (84) and more recently for
uptake systems, which do not appear to be amonabactin transport in Aeromonas hy-
subject to regulation by iron concentration, drophila 495A2 (85). The contribution of
have been characterized in many microorgan- parabactin A to iron transport by a ligand-ex-
isms, both prokaryotic and eukaryotic, in the change mechanism was assessed; the direct
past decade (6, 10,80,81). Many of these sys- metal-ligand interchange seemed insufficient
tems operate by means of a broad-spectrum to account for the V, observed for iron accu-
reductase; systems that use ferrisiderophore mulation from ferric parabactin A (59).On the
reductases have been characterized in diverse basis of kinetic issues, it also seems unlikely
microbial species, including Mycobacterium that free L-parabactin produced by the cell is
494 Iron Chelators and Therapeutic Uses

involved in deferration of ferric hydroxam-


ates, in spite of a formation constant that
strongly favors ferric L-parabactin over both
ferrioxamine B and ferrioxamine E on ther-
modynamic grounds (Table 10.1).
The issue of how the microorganism uses
the iron complex also is observed in the para-
bactinlp. denitrificans system by following the
uptake of [55Felferricparabactin and r3H1fer-
ric parabactin. The uptake of 55Fe by the mi-
crobe increases over time and reaches a pla-
teau; however, no appreciable uptake of
tritiated ligand occurs (57). Furthermore, Figure 10.9. structure of (31), (D,L)-tetramethyl-
whereas the uptake of the tritium label is the parabadin.
same at 4 and 30"C, the difference in the up-
take of [55Felferricparabactin at the two tem-
peratures cannot be ignored. The labeled iron 5 and 10 h by 4/25 without an alteration of the
is taken up rapidly from [55Fe]ferricparabac- rate of growth thereafter. All of the chelators
tin at 30°C; however, there is no measurable affected the growth of S. aureus and C. albi-
transport of 55Fe at 4°C (57). These observa- cans, although the sensitivity was of a some-
tions are consistent with the "iron-taxi" mech- what different nature. The antimicrobial ac-
anism, in which there is only a transient tivity of the iron chelators tested was
association between the siderophore-metal bacteriostatic, rather than bactericidal; fur-
complex and the specific outer membrane re- thermore, no growth suppression of the nor-
ceptor (58). The siderophore remains extracel- mally sensitive organisms was demonstrated
lular at all times, allowing the ligand to deliver in the presence of a 1:l iron:(^,^)-parabactin
iron to the cell membrane repeatedly. complex, further evidence that the effects of
The siderophore receptor and transport the chelators were attributed to iron starva-
system has been exploited by investigators as tion (88).
a novel means of delivering siderophore-anti- The reduced bacteriostatic activity of the
biotic conjugates to microorganisms (86, 87). catecholamides in the cases of P. aeruginosa
Interestingly, however, the ability of the cat- and E. coli can be attributed to these bacteria
echolamide siderophores to bind iron was it- producing their own siderophores in concen-
self postulated to be a means for preventing trations high enough to compete with the ex-
growth of heterologous microorganisms by ogenously introduced siderophore systems.
withholding the necessary iron. Accordingly, The initial period of near-complete growth
selected catecholamide chelators (e.g., 3 and cessation is consistent with this hypothesis.
racemic parabactin 4/25, Fig. 10.2) and their During this period, identification by the or-
methylated analogs [e.g., (D,L)-tetrameth- ganism of iron-starvation conditions induced
ylparabactin, (3111 (Fig. 10.9) were evaluated by the exogenous ligands would lead to the
for their bacteriostatic and fungostatic activ- expression of genes responsible for sid-
ity (88). The methylated analogs (e.g., 31) ex- erophore production and receptor expression
hibited little, if any, effect on the growth of (6,10,81).There is no evidence to suggest that
three pathogenic bacteria (Pseudomonas iron transport in these bacteria is occurring
aeruginosa, E. coli, and Staphylococcus au- through these exogenous chelators. The sid-
reus) and one pathogenic yeast (Candida albi- erophore systems in C. albicans and S. aureus
cans), whereas the nonmethylated ligands appear to be less efficient than those in P.
demonstrated varying bacterio- or fungostatic aeruginosa and E. coli, as evidenced by the
activity against these same microorganisms. sensitivity of C. albicans and S. aureus to all of
In the cases of E. coli and P. aeruginosa, the the catecholamide ligands. Although both of
growth was repressed completely for between these microorganisms produce endogenous
1 Introduction

siderophores (e.g., staphyloferrin from S. au- 66). However, there was not a significant dif-
reus) (89-91), their receptors cannot use the ference in the iron-clearing efficacy of (6) and
catecholamide iron chelators evaluated. (28) in a bile duct-cannulated rodent model
Thus, the repression of proliferation of var- (66); (4) was very efficient at removing iron
ious microorganisms by the spermidine cat- from rodents and primates as well (98). These
echolamide iron chelators (88)seems to be the results underscore the idea that siderophores
result of the ability of the chelators to complex can serve as a platform in the design of thera-
!
I iron; when bacteria that are normally sensi- peutic agents. Stereochemical modification of
tive to the hexacoordinate catecholamide the natural product iron chelators can pro-
iron(II1) chelators were presented with the foundly diminish their capacity to stimulate
iron complexes of these ligands, no antimicro- microbial growth without reducing their iron-
bial activity was observed. Similarly, blocking clearing properties in vivo.
of the catechol coordination sites by methyl- 1.3 lron Dynamics in Humans
ation abrogates the bacterio- or fungostatic ac-
tivity of the catecholamide. These results More complicated organisms, that is, the
should be tempered by an understanding of a higher eukaryotes, have developed large pro-
very serious potential problem with the con- teinaceous molecules to solve the access, stor-
cept of iron deprivation in antimicrobial ther- age, and utilization problems associated with
apy, that is, acceleration of bacterial growth iron. Animals, in particular humans, have
by chelation. The ability of an organism to use evolved a highly efficient mechanism for han-
nonnative chelators (i.e., "prepackaged iron") dling the metal in which two basic "iron-pro-
could produce an extremely dangerous situa- cessing" proteins are involved, one for trans-
tion, as was borne out when the hexacoordi- port, transferrin (Fig. 10.10a), and one for
nate catecholamide chelator enterobactin (1) storage, ferritin (Fig. 10.10b). It is interesting
was tested as a possible deferrating agent in to compare the relative sizes of the molecules
rodents. In this study, a fulminant E. coli in- involved in iron transport in prokaryotes (e.g.,
fection developed in these animals, which ul- -
4, MW 600) and in eukaryotes [e.g., trans-
timately resulted in death from septicemia ferrin, MW 80,000 (24)l. At first, it seems as
(92). Furthermore, the increased susceptibil- though the assembly of transferrin for eukary-
ity of chelation therapy patients using DFO to otic iron transport requirements is very inef-
bacterial infection, particularly by Yersinia ficient. However, because of a mammalian
enterocolitica, is well documented (93, 94); iron storage and transport system that con-
cross-utilization of other siderophores by het- tains elaborate control mechanisms involved
erologous bacterial species in vitro is well es- in the synthesis, cellular uptake, and utiliza-
tablished (10,81, 95, 96). Thus, any chelators tion of its attendant iron transport and stor-
intended for therapeutic use as either an anti- age proteins, such size and complexity are re-
microbial or a deferration agent must be quired.
screened against numerous microorganisms
that might be present in a host. Even though 1.3.1 lron Storage and Transport. The loca-
the ligands need not be active against the bac- tion of the major iron stores in the human
teria or fungi, such chelators must not en- body and a schematic of iron transport are il-
hance their growth. lustrated in Fig. 10.11 (99). The iron metabo-
Strikingly, the Fe(II1) complex of synthetic lism system is quite efficient; little leakage or
enantioenterobactin (derived from D-serine) expansion occurs in healthy humans (23). Hu-
failed to promote the growth of enterobactin- mans both absorb and excrete about 1 mg of
deficient E. coli mutants in vitro (97). Both this transition metal daily. Whereas all tissues
L-parabactin (4) and L-fluviabactin (6) stimu- contain at least some iron, resulting in total
lated the growth of P. denitrificans and pro- iron stores in an average adult human of about
moted iron uptake, whereas their respective 50 mg/kg (slightly less in females), the distri-
D-enantiomers (25 and 28) did neither, in that bution of the metal is not uniform (23). The
the latter compounds were not recognized by hemoglobin in circulating erythrocytes ac-
the bacterial iron-transport apparatus (57, counts for 60-70% of the iron load; ferritin
Iron Chelators and Therapeutic Uses

thelial system, and the bone marrow for red


cell assembly. Most (-80%)of this transitory
iron store (turnover rate, 25-30 md24 - h) is
used for hemoglobin synthesis during ery-
throid development; the rest is distributed to
various tissues (24, 99). The red cells, which
survive for approximately 120 days, are even-
tually processed by the spleen. Phagocytosis of
the senescent cells by reticuloendothelial (RE)
macrophages liberates the iron from hemoglo-
bin to be mobilized by- transferrin. A more de-
tailed examination of the components - of
transport
- (transferrin and the transferrin re-
ceptor) and storage (ferritin) follows.
TransferritvTransferrin Receptor. Transfenin
is a homodimeric globular protein with a MW
of about 80,000 (Fig. 10.10A) (99,100). In hu-
(b) mans, it is synthesized predominantly in the
Serum liver; some s.mthesis
- occurs in brain, testis,
H0L24
and mammary - tissues. Each molecule con-
tains two iron-binding sites. Although trans-
ferrin has very high affinity for iron, this bind-
ing is very sensitive to pH. For example, at
Figure 10.10. The iron transport protein, trans-
physiological pH, the K, of transferrin for
ferrin (a) and the serum isoform of the iron storage
protein, ferritin (b). Serum transferrin (a) is com- iron(II1)is M; in contrast, no significant
posed of two homologous lobes that each bind a sin- chelation occurs at or below pH 4.5 (23, 24).
gle ferric ion. The N-terminal N-lobe and the C- This pH sensitivity is critical to the endosomal
terminal C-lobe each are divided into two dissimilar release of the metal from the protein. Six do-
domains, and the iron-binding site is located within nor groups-two tyrosines, a histidine, an as-
the interdomain cleft. The iron is bound by two ty- partate, and two oxygens from the carbonate
rosines, a histidine, and an aspartic acid residue. anion-are necessary for the ligation of iron
The various isoforms of ferritin (b) found in differ- by transferrin (24, 100). There are a number
ent tissues are composed of different proportions of of iron chelators that bind iron more tightly
heavy (H) and light (L) chains (KL,; x + y = 24);
than does transferrin (e.g., desferrioxamine,
these protein subunits form a shell, surrounding a
core of mineralized ferric iron. The serum isoform K, = lop3' M); these should, on a thermody-
(H,L,,) is shown. [Panel a reproduced with permis- namic basis, be able to remove the metal from
sion from S. Bailey et al., Biochemistry, 27,5804- this protein. However, with rare exceptions,
5812 (1988).Copyright 1988 American Chemical So- for example, the hydroxypyridinones (101),
ciety. Panel b reproduced from P. M. Harrison and this is not the case. Kinetics, rather than sim-
P. Arosio, Biochim. Biophys. Acta, 1275, 161-203 ple thermodynamics, is the issue. Mobilization
-

(1996). Copyright 1996 with permission from of the iron in transferrin can occur with a
Elsevier Science.] number of different chemical agents, includ-
ing ascorbate. The cellular processing of this
sequesters another 20-30%. The remaining iron shuttle protein is a remarkable example
10% serves as a key component of molecules of efficiency. After the delivery of iron to cells,
such as myoglobin, cytochromes, and iron- the protein is recycled for further use (Fig.
containing enzymes (23). Less than 0.1% of 10.12) (99). This process begins with the bind-
the total body iron (-3 mg a t steady state) is ing of transferrin to a cell surface receptor,
associated with transferrin (23,24). This pro- which itself is subject to posttranslational
tein serves as the conduit for the metal be- modification within the endoplasmic reticu-
tween ferritin storage pools, the reticuloendo- lum of the cells in which it is synthesized. Its
1 Introduction

Ferrireductase
Fe(I1)
- 4 Fe(l1l) Heme

1 Plasma

11 1 7
Fe(ll)* Fe(lll) 4 Diferric transferrin

tissues
Bone marrow (Cellular
(Hematopoiesis) metabolic
C
RBC ,Spleen Liver
processes)

hemoglobin (RE system) Ferritin


(Storage)
i +
Excretion Excretion Figure 10.11. Iron absorption and
via urine via bile processing.

level of expression is particularly high in pro- apical region, a protease-like region, and a he-
liferating tissues, although, except for mature lical portion, which is responsible for connect-
erythrocytes, it is present in all of the cells of ing the two monomers (102).
the human body. This receptor is symmetrical The cycle of the transferrin-transferrin re-
and resembles a butterfly (Fig. 10.13) (102). ceptor complex occurs in five steps: (1)binding
The dimer consists of two 90-kDa monomers of transferrin to the receptor, (2) endocytosis,
that are connected by two cysteine disulfide (3) iron removal, (4) return of the apotrans-
bridges. Each of the individual monomers con- ferrin-transferrin receptor complex to the cell
tains three domains. The cytoplasmic tail (61 surface, and (5)release of apotransferrin (Fig.
amino acids in length) is responsible for endo- 10.12). Each transferrin receptor can bind two
cytosis of the receptor-transferrin complex. molecules of transferrin, although the actual
Fatty acid linkages within the 28 amino acid binding affinity varies with the iron status of
transmembrane segment aid in anchoring the transferrin: diferric transferrin >> monofer-
protein within the membrane. The 671 amino ric transferrin > apotransferrin. The K, for
acids at the extracellular carboxy terminus are the diferric transferrin-transferrin receptor
often referred to as the ectodomain. Two mol- complex is around 5 nM (24, 103). Given that
ecules of transferrin bind to lateral clefts the plasma concentration of diferric trans-
within the ectodomain of the dimeric receptor. ferrin is roughly 5 a, it is likely that most
The ectodomain is subdivided further into an cell surface-exposed receptors are saturated.
Iron Chelators and Therapeutic Uses

Figure 10.12. The transferrirdtransferrin receptor cycle. The major steps, depicted clockwise, are:
(a) binding of Fe(II1) (e)to transferrin (0,
Tf), (b) binding of diferric transferrin to the transferrin
receptor (TfR), (c) endocytosis by way of a clathrin-coated pit, (d) iron removal, (e) return of the
apotransferrin-transferrin receptor complex to the cell surface, and (f) release of apotransferrin
(ApoTf).

After transferrin binds to the receptor, the tochondria in immature erythroid cells. Alter-
complex interacts with an adapter protein natively, the metal is stored in ferritin.
within a clathrin-coated pit; this megacom- Ferritin. Ferritin is a large spherical com-
plex is then endocytosed. After the megacom- plex consisting of 24 subunits; the combined
plex reaches the endosome within the cell, the MW is around 450,000 (99, 104). The sphere
iron is released from transferrin; this dissoci- (Fig. 10.10b) manages over 4500 iron atoms
ation is very much dependent on the low en- within its core, thus preventing the metal
dosomal pH, exploiting the poor binding of from causing iron-promoted redox damage;
iron to transferrin at low pH. The apotrans- yet, the protein releases iron on demand for
ferrin, still complexed to the transferrin recep- utilization by other iron-depleted systems.
tor, is exported back to the cell membrane, The sphere is composed of two basic protein
where the apotransferrin is released for fur- chain types, heavy (H) and light (L) chains.
ther complexation. The iron is released from The H chains exhibit ferroxidase activity (99,
the endosome through the transmembrane 104). When these chains are assembled into
portal DMTl (Divalent Metal Transporter 1, the spherical array, the chains create chan-
formerly known as Nrarnp2) and either is de- nels, allowing for access to the iron core. The
livered to other iron-binding molecules (e.g., current thinking is that soluble Fe(I1) is incor-
for incorporation into heme) or is used by mi- porated into the ferritin shell, where it is oxi-

,
1 Introduction

access is the problem. However, iron can be


mobilized from ferritin reductively both in
vitro and in vivo (101, 104). Interestingly, it
seems that the release of iron from ferritin in
Apical the presence of various reducing agents can
occur even when the reducing agent and the
metal are separated by some distance within
the ferritin channel. The implicit danger with
this reductive removal is that the iron released
for chelation, Fe(II), can promote Fenton
chemistry, thus causing significant cellular re-
dox damage, as discussed in detail in Section
1.3.4. Therefore, when the metal is reductively
released from ferritin, a ligand (e.g., trans-
ferrin) must be present in sufficient proximity
to bind and remove it. This concern has been
borne out; a clinical trial was designed to im-
Protease-like prove the efficiency with which desferrioxa-
domain mine, a therapeutic iron chelator, can remove
iron from ferritin by the coadministration of
I
ascorbate. Unfortunately, this trial served to
illustrate how serious the problem of uncon-
Plasma membrane trolled iron removal from ferritin can be, as
discussed in Section 2.1.1.1.
Figure 10.13. Structure of the ectodomain of the
transferrin receptor. (a) Domain organization of the 1.3.2 Molecular Control of Iron Uptake,
transferrin receptor polypeptide chain. The cyto- Processing, and Storage. One of the most in-
plasmic domain is white; the transmembrane seg- teresting discoveries in the area of iron metab-
ment is black; the stalk is gray; and the protease- olism in recent years is the mechanism by
.ike,apical, and helical domains are red, green, and
which the iron storage and transport proteins
fellow,respectively. Numbers indicate domain res-
dues at domain boundaries. (b) Ribbon diagram of
(i.e., ferritin and transferrin) are regulated.
;he transferrin receptor dimer depicted in its likely Whereas the iron-mediated feedback regula-
~rientationwith respect to the plasma membrane. tion mechanisms operate with a number of
h e monomer is colored according to domain (stan- iron-processing proteins, they have been in-
tard coloring as described above), and the other is vestigated most thoroughly for ferritin and for
h e . The stalk region is shown in gray connected to the transferrin receptor. Simply put, the ques-
:he putative membrane-spanning helices. Pink tion is: How does iron concentration control
;pheres indicate the location of Sm3+ ions in the the synthesis of iron-processing proteins? As
:rystalstructure. Arrows show directions of (small) iron levels increase, the cellular storage com-
lisplacements of the apical domain in noncrystallo-
partments (i.e., ferritin levels) must increase
~aphicallyrelated molecules. See color insert. [Re-
~rintedwith permission from C. M. Lawrence, S. also. Conversely, when cellular iron levels are
lay, M. Babyonyshev, R. Galluser, D. W. Borhani, depleted, or there is an increase in iron re-
~ndS. C. Harrison, Science, 286, 779-782 (1999). quirement, as in various proliferative pro-
:opyright 1999 American Association for the Ad- cesses, the efficiency with which iron is incor-
,ancement of Science.] porated by cells must be augmented; that is,
an upregulation of transferrin receptor ex-
iized to Fe(II1) and deposited for mineraliza- pression is required. Both of these aspects of
ion (99, 104, 105). This process of iron* iron processing are handled at the posttran-
xidation, incorporation, and mineralization scriptional level (99).
3 surprisingly slow; it can require hours (104). Both ferritin mRNA and transferrin recep-
Most iron chelators cannot remove iron tor mRNA contain iron-responsive elements
rom ferritin effectively; as with transferrin, (IRES) to which the iron-responsive proteins
500 Iron Chelators and Therapeutic L

Transferrin receptor
-5' AUG

I Increased stability of mRNA

High iron
Aconitase

Ferritin UH
3'

I Efficient translation

Transferrin receptor
5'AUG
I Decreased stability of mRNA
~ndonuclease\L)

Figure 10.14. Posttranscriptional regulation of iron-responsive element-containing gene expres-


sion. Intracellular iron concentration determines binding of iron regulatory proteins (IRP) to iron-
responsive elements (IRE). Binding affinity of iron regulatory proteins to iron-responsive elements
inversely correlates with intracellular iron concentration. For example, a single iron-responsive
element is located in the 5' untranslated region of ferritin mRNA, whereas five iron-responsive
elements are present in the 3' untranslated region of transferrin receptor 1 mRNA. Under conditions
of iron deficiency, iron regulatory proteins bind to the iron-responsive element in the 5' untranslated
region of ferritin mRNA. This binding sterically prevents the recruitment of 43s translation preini-
tiation complex and arrests translation of ferritin. On the other hand, binding of iron regulatory
proteins to the iron-responsive elements in the 3' untranslated region of transfenin receptor 1
mRNA increases mRNA stability and synthesis of transfenin receptor 1. In contrast, under high
concentration of intracellular iron, iron regulatory proteins possess aconitase activity but lack
mRNA-binding activity. In the absence of binding of iron regulatory proteins to iron-responsive
elements, ferritin mRNA is translated efficiently, whereas transferrin receptor 1 mRNA is rapidly
degraded, probably by iron-responsive element-specific endonucleases. [Reproduced from P. T. Lieu,
M. Heiskala, P. A. Peterson, and Y. Yang, Mol. Aspects Med., 22, 1-87 (2001). Copyright 2001 with
permission from Elsevier Science.]

(1and 2; IRP-1 and IRP-2) bind (Fig. 10.14). When cells are replete with iron, an iron-s
The binding of IRP-1 and IRP-2 to the mRNAs fur complex is formed with three cysteine res-
for ferritin and transferrin receptor controls idues within IRP-1 (106); no binding to IR.Es
translation; this binding is dependent on the occurs in this state. However, under low ir'on
iron status. IRP-1 is very similar to mitochon- conditions, the iron-sulfur complex is absent;
drial aconitase (99, 106), the enzyme respon- the protein does not possess aconitase activitY.
sible for the conversion of citrate to isocitrate. This apoprotein accumulates in the cell and
tly to the IREs. The mRNAs for Fe(I1) travels through the cytoplasm and is
the L and H chains of ferritin contain the released through the basolateral membrane
in the 5'-untranslated region; the trans- through a second protein complex. The trans-
IRE is in the 3'-untranslated porter Ferroportin 1 acts in conjunction with
gion of the message. Hephaestin, a multi-copper oxidase that con-
These IREs can serve as either repressors verts Fe(I1) to Fe(II1) for mobilization by
enhancers of translation. The IRP repres- transferrin. Although Fe(II1) uptake is not
sive mode, which occurs in ferritin mRNA, is a particularly efficient, the paraferritin complex
steric issue. Binding of IRPs to the 5' IRE pre- is designed for Fe(II1) uptake and transport
vents initiation of translation of protein syn- (103). This mucin-mediated iron complex con-
thesis by impeding the recruitment of 43s sists of P-integrin, mobilferrin, and flavin
preinitiation complexes. Thus, when cellular mono-oxygenase. Once the iron-paraferritin
iron levels are high, the metal binds to IRP,
complex is transported across the apical cell
preventing its association with the IREs; syn-
membrane, the oxygenase may act to reduce
thesis of ferritin is initiated. The IRE/IRP con-
trol mechanism for the transferrin receptor is Fe(II1) to Fe(I1) for further processing (103).
somewhat different from that described for Finally, heme iron uptake provides the most
ferritin. Binding of IRP to the 3' IREs, rather plentiful source of iron. Hemoglobin is first
than preventing translation, as in ferritin, digested enzymatically; the released heme
enhances the stability of the message by pre- molecule is transported across the apical
venting its degradation, increasing message membrane. The heme is then degraded by
translation. Thus, increases in cellular iron heme oxygenase, resulting in the release of
concentration induce ferritin synthesis, given iron within the cell.
that the ferrated IRPs do not bind to and steri-
cally inhibit translation of ferritin message. 1.3.4 iron-Mediated Damage. In normal,
Conversely, decreases in cellular iron concen- healthy humans, iron is well managed and
tration elicit an increase in transferrin recep- does not serve as a source of oxidative damage.
tor synthesis because deferrated IRPs bind to Although there is always some non-trans-
and stabilize the message. The mRNAs of ferrin-bound iron (NTBI) in the plasma of
other key iron-processing proteins also con- healthy subjects, this amount is negligible;
tain IREs, which control their translation. For most of the metal is associated with heme, is
example, DMT1, an iron portal, has a 3' IRE stored in ferritin, or is utilized in various iron-
that operates in a manner similar to that of containing enzymes. As we have seen, the
the transferrin receptor. metal is shuttled safely between these pools by
the iron-trafficking protein transferrin. How-
1.3.3 iron Absorption. Absorption of iron
ever, there are a number of different disease
in the proximal small intestine is the major
states, discussed briefly below, in which body
source of iron uptake (99, 103). The metal is
iron stores and plasma NTBI levels are suffi-
transported across the brush border and re-
leased to the circulation through the basolat- ciently high to cause significant damage, much
era1 membrane (Fig. 10.11). Heme iron ac- of which is oxidative in nature. To understand
counts for most of the iron absorbed; relatively how oxidative damage unfolds, a description of
small amounts of inorganic Fe(I1) and Fe(II1) the mechanism of iron-mediated damage is in
are taken up also. Although both of these oxi- order.
dation states of iron are presented to the en- As was mentioned, although iron has many
terocytes, the absorption of Fe(I1) is much oxidation states available to it, the most com-
more efficient. In fact, a mucosal ferrireduc- mon oxidation states of iron in the biosphere
tase, which converts Fe(II1) to Fe(II), has been are Fe(I1) and Fe(II1); the Fe(II)/(III) redox
demonstrated as being critical to efficient iron couple is the focus of this discourse. The equi-
absorption (107, 108). Iron(I1) is transported librium shown in Equation 10.8 is very much
dependent on pH and the nature of the ligands
chelating the metal.
Iron Chelators and Therapeutic Uses

Thus, iron can function both as an electron


source (reducing agent) and an electron sink
(oxidizing agent). The interaction of the free
metal with oxygen and with various active ox-
ygen species (e.g., superoxide anion and hy- However, if the hydrogen peroxide concentra-
droperoxide) is the source of iron's toxicity. tion exceeds the K, of these enzymes, or, more
Probably the most significant reaction se- important, if hydrogen peroxide is generated
quence involves the reaction of Fe(I1)with hy- in a milieu in which iron(I1) is present and
drogen peroxide. First, Fe(I1) itself can read these enzymes are absent, damage can unfold.
with molecular oxygen to produce hydrogen In combination with Fe(II), hydrogen perox-
peroxide. Again, the rate of this reaction de- ide is repudiated to be the major source of the
pends on both pH and the nature of the li- tissue damage associated with excess iron.
gands surrounding the metal. The first step in Iron(I1) converts hydrogen peroxide to hy-
this reaction (Equation 10.9) is the production droxyl radical, a very reactive species, and hy-
of superoxide anion: droxide anion, referred to as the Fenton reac-
tion (Equation 10.12) (112-115):

Production of superoxide occurs in a variety of


different cells. For example, during intestinal
injury, neutrophils infiltrate and release su-
peroxide anion (109). Although produced in Note that this Equation neither directly in-
neutrophils by NADPH oxidase (110), super- volves superoxide anion nor results in the lib-
oxide anion is probably most frequently eration of molecular oxygen, as does what has
thought of as a by-product of the conversion of been referred to as the superoxide-driven Fen-
xanthine to uric acid by xanthine oxidase ton reaction or the Haber-Weiss reaction
(111).However produced, superoxide anion (1141, even though hydroxyl radicals are pro-
can dismute to hydrogen peroxide and oxygen duced in both instances. The hydroxyl radical
(Equation 10.10), either spontaneously or reacts very quickly with a variety of cellular
through the action of the copper-dependent constituents and can initiate free-radical
enzyme superoxide dismutase: chain processes. The generation of this radical
by Fe(I1) and its marked reactivity have been
the subject of much controversy over the years
(114, 115). Many investigators have argued
that multivalent forms of iron [e.g., Fe(1V)I or
Hydrogen peroxide is a good oxidizing agent various oxy complexes are the source of iron-
and can be particularly insidious, as it has mediated cellular damage (114). However, ox-
such global cellular access. Because of its idation of aromatic amino acids by pulse radi-
structural similarity to water, it passes olysis-generated hydroxyl radicals occurs at
through membranes very easily. Normally, very different sites than does Fe(1V)-mediated
hydrogen peroxide is managed by either gluta- oxidation (114). In nonchain reactions, one
thione peroxidase or catalase. Glutathione might anticipate a simple reaction between
peroxidase, which is probably the major Fe(I1) and hydrogen peroxide to generate the
"housekeeping" enzyme, reduces hydrogen hydroxyl radical. Unfortunately, the cyclic na-
peroxide to water and simultaneously oxidizes ture of the Fe(II)/Fe(III) redox couple is prob-
glutathione (Equation 10.11A), whereas cata- lematic. The Fe(II1) liberated in Equation
lase converts hydrogen peroxide to water and 10.12 can be reduced back to Fe(I1) through a
molecular oxygen (Equation 10.11B): variety of biological reductants, including
& I O CH3
H

CH3
(33)

HOOC-, n ~ C O O H
N N N
H O O C ~
I COOH
-COOH

(34) (35)

Figure 10.15. Structures of selected synthetic chelators: ethylenediaminetetraacetic acid (EDTA,


32), 1,2-dimethyl-3-hydroxypyridin-4-one(deferiprone; L1; 33; formerly CP20), 2-pyridinecarboxal-
dehyde isonicotinoyl hydrazone (PCIH; 34), and diethylenetriamine pentaacetic acid (DTPA, 35).

ascorbate and superoxide anion. In intestinal example, desferrioxamine binds iron very
injury, the production of superoxide anion by tightly, removing it from the Fenton pool
neutrophils in the colon is the source of acti- (114). This ligand binds Fe(II1) much more
vated oxygen for the generation of hydrogen tightly than it does Fe(I1). Thus, Fe(I1) in the
peroxide (109). Thus, not only does superoxide presence of desferrioxamine is an even better
anion provide hydrogen peroxide, but the an- reducing agent, as the Fe(II)/Fe(III) equilib-
ion also serves as a means of recycling Fe(II1) rium is shifted to the right. However, this
to Fe(I1) for future hydroxyl radical produc- event occurs only once because the iron in the
tion. This net reaction is shown in Equation desferrioxamine/Fe(III) complex is not recy-
cled readily to Fe(I1) for further redox chem-
istry. Other chelators, for example, ethyl-
Fe(II1) + 02112- + Fe(I1)
+ HZOZ (10.13) enediaminetetraacetic acid (EDTA, 32) or
deferiprone (33)(Fig. 10.15),actually promote
Fenton chemistry (116).
This problem is underscored further when one
considers that ascorbate is an effective reduc-
ing agent only in the absence of iron. In fact, a 1.4 iron-Mediated Diseases
mixture of iron salts, ascorbate, and H202is a There are two basic types of iron-related dis-
good source of hydroxyl radicals (114) (Equa- eases, those associated with depleted iron
stores (e.g., anemia) and those associated with
excess iron, also referred to as iron overload.
Fe(II1) + ascorbate + Fe(I1) The latter subset is the focus of the present
(10.14) discussion. As was mentioned in Section 1.3,
+ semidehydroascorbate healthy humans maintain an iron load of
about 50 mg/kg (23, 99). At this level, the
It should be clear, then, that the proteins that body's iron storage and transport can manage
store and transport iron represent a major iron safely. However, in various iron-overload
component of the body's antioxidant defense disorders, the system becomes overwhelmed;
sufficient "unmanaged iron" is available to
Some iron chelators inhibit the Fenton re- cause redox, that is, Fenton chemistry-driven,
action; paradoxically, some stimulate it. For damage. For example, plasma from healthy
Iron Chelators and Therapeutic Uses

human donors does not stimulate free-radical larly interesting example, in that it goes be-
reactions; however, plasma from iron-over- yond the classic iron-driven Fenton chemis-
loaded patients markedly stimulates lipid per- try-mediated damage: an ancillary protein,
oxidation (114). amyloid p-peptide (Ap), derived from amyloid
Two principal kinds of iron-overload dis- precursor protein (APP), is involved. The pre-
ease occur, both of which are genetic disor- dominant thinking is that Ap mediates nerve
ders. The hallmark of primary iron overload cell death in Alzheimer's disease and that iron
(e.g., hemochromatosis)is increased intestinal plays a key role in this process. In cell-free
iron absorption (99,103).Secondary, or trans- systems, Ap and iron interact directly to pro-
fusional, iron overload is associated with mote Fenton chemistry (125). Experiments
chronic transfusion therapy. Such therapy is with neuronal cell lines that are resistant to
reauired in diseases in which anemia caused
A
Ap toxicity consequent to the transcriptional
by aberrant hemoglobin is a problem (e.g., elevation of glutathione peroxidase and cata-
thalassemia, sickle-cell anemia) (117, 1181, or lase have provided quite compelling evidence
in hematopoietic malfunctions (e.g., myelo- for the role of Fenton chemistry in cell death
dysplasia) (119). Depending on the severity of (126). From a mechanistic perspective, the re-
the disease, transfusion is needed up to sev- lationship between AP and iron in neuronal
eral times a month. Each unit of packed red toxicity was best demonstrated in a series of
cells transfused introduces 200 mg of iron into experiments that used B12 cells, a neuronal
the closed metabolic pathway. Eventually, the cell line, and primary cultures of cerebral cor-
transfused cells are processed through the re- tex neurons. In these experiments Fe(II1) sub-
ticuloendothelial (RE) system as occurs in stantially enhanced the toxic effects of AP.
healthy subjects; the added iron is mobilized The iron chelator DFO ameliorated FelAP-in-
by transferrin. Unfortunately, because there duced neuronal cell damage in both B12 cells
is no effective mechanism for iron excretion, and in primary cortical cells; this prevention
these transfused patients continue to accumu- was dose dependent. Furthermore, the most
late the metal. Patients with primary hemo- efficiently transported form of the drug, the
chromatosis can be managed adequately by Ga(II1)-DFOcomplex, was the most effective.
phlebotomy; the frequency of bleeding de- Finally, the cell damage was minimized signif-
pends on the extent of the iron overload. How- icantly when iron was chelated, even in the
ever, patients with transfusional iron overload presence of added hydrogen peroxide (127).
must be managed by chelation therapy. Thus, although it is unlikely that Ap/F'elH,O,-
Although primary hemochromatosis and induced cell toxicity is the only source of neu-
transfusional iron overload represent the ma- ronal injury seen in Alzheimer's patients, this
jor diseases in this category, there has been mechanism could well be a principal source of
substantial interest in recent -years in the role the problem.
that iron plays in diverse disorders, including As discussed in detail above, regulation of
neurodegenerative disorders such as Alzhei- the expression of ferritin and other proteins
mer's disease (120),Parkinson's disease (121), involved in iron metabolism occurs by interac-
and Friedreich's ataxia (122-124). In each of tions of IRPs with IRES contained in the
these diseases there is an increased iron con- mRNA transcripts. Deletion of the gene en-
tent of affected regions of the brain. These fo- coding IRP2 causes misregulation of iron me-
cal increases in iron concentration have been tabolism and neurodegeneration in mice
associated with the expected oxidative stress (128). Independently reported lines of evi-
issues, extending from membrane damage to dence suggest that iron may play a similar reg-
mitochondrial dysfunction and cell death. ulatory role in Alzheimer's disease. Affected
In Alzheimer's disease there is an excess individuals from two familial Alzheimer's ped-
accumulation of iron in those regions of the igrees were found to contain a mutation that
brain associated with the characteristic defi- disrupts a stem-loop in APP mRNA contain-
cits in neurological function: the hippocam- ing the consensus sequence CAGUGA charac-
pus, amygdala, nucleus basalis, and cerebral teristic of an IRE (129). There are, in fact,
cortex (120). Alzheimer's disease is a particu- significant overlaps in the regulation of the
2 Clinical Use of Chelating Agents

APP and ferritin genes (130). These observa- of penetrating the mitochondrial membrane,
tions suggest that the synthesis of the AP pre- have been proposed to treat the mitochondrial
cursor APP may itself be shut off with an iron iron overload in Friedreich's ataxia (144).
chelator in a manner similar to the control of Ultimately, the solution in each of these
the synthesis of ferritin. Clearly, removal of disease states is the same: identify a method of
excess iron would ameliorate APliron-medi- opening up the closed iron metabolic loop.
ated Fenton pathophysiology, and perhaps Generally, this translates into designing che-
would suppress AP production as well. lators that will selectively sequester this tran-
The marked increase of iron in the Parkin- sition metal and render it excretable. There-
sonian substantia nigra underscores the role fore, the remainder of this chapter focuses on
of the metal in this disease (121). It has been current methods of chelation therapy and ap-
suggested that this iron is responsible for the proaches to the design, synthesis, and assess-
oxidative stress seen in the degeneration of ment of new iron chelators.
the dopaminergic neurons in the substantia
nigra. The autooxidation and oxidative deami-
nation of dopamine by monoamine oxidase in 2 CLINICAL USE OF CHELATING AGENTS
the metabolically active basal ganglia result in
the accumulation of neuromelanin, the black The pharmacological properties that define an
pigment for which the substantia nigra is ideal iron chelator are very much dependent
named, and the production of hydrogen perox- on the iron-mediated disease being treated.
ide (131, 132). In Parkinson's the normal One of the major considerations is whether
mechanisms that manage peroxide (e.g., per- the treatment is of a long- or short-term na-
oxidase, catalase, glutathione, ascorbate) are ture. This issue has a particular impact on the
defective (133-138). In addition, neuromela- toxicity profile requirements of the ligand. For
nin itself binds iron in a Fenton-reactive form example, the lifelong treatment either of a ge-
(131, 139-141). Thus, the conditions are per- netic disorder, such as severe hereditary
fect for the iron-catalyzed production of hy- hemochromatosis, or of transfusional iron
droxyl radicals and the ensuing damage. The overload, as can occur in the management of
pathophysiological role of iron in Parkinson's sickle-cell anemia (118) or thalassemia, dic-
disease is substantiated further by the finding tates the use of a ligand that can maintain
that removal of iron from the brain by intra- patients in negative iron balance, that is, be
cerebral-ventricular injection of DFO amelio- able to clear 250-400 kg of iron per kg of body
rated 6-hydroxydopamine-induced damage to weight per day (145), and do so with minimal
nigrostriatal dopamine neurons (142,143).Al- toxicity over an extended period of time. How-
though the protection conferred by the chela- ever, the relatively brief application of an iron
tor was not complete, it was quite real, provid- chelator either to control iron-induced cardiac
ing additional support for the concept of iron reperfusion damage during cardiac surgery
chelators as therapeutic platforms in neurode- (146) or to curb the global effects of acute iron
generative maladies. Desferrioxamine does poisoning (147) allows for chelators with dif-
not penetrate cells well at all; ligands with bet- ferent toxicity profiles. In the former situa-
ter transport and iron-clearance properties tions, chronic iron overload adds another di-
would be superior candidates for the treat- mension to take into account, patient
ment of such disorders. compliance (94, 117). These issues demand
Friedreich's ataxia is an autosomal reces- that consideration be given to the mode of ad-
sive mutation of the gene encoding frataxin, a ministration required, that is, p a r e n t e d ver-
protein involved in mitochondrial iron ho- sus oral dosing.
meostasis. A deficiency in frataxin in Fried- There are two levels of toxicity that define a
reich's ataxia leads to an accumulation of iron therapeutic agent: that which is implicit in the
in mitochondria, oxidative stress, and neuro- structure and unrelated to its therapeutic tar-
degeneration (122-124). 2-Pyridinecarboxal- get and that which is associated with the pur-
dehyde isonicotinoyl hydrazone (34; Fig. pose for which the molecule is designed. For
10.15) and its analogs, iron chelators capable example, the aminoglycoside antibiotics elicit
506 Iron Chelators and Therapeutic Uses

Table 10.3 Formation Constants of Selected Ligands with Iron and Other Metals
Formation Constant for Metal Chelate
(K, M-9"
Ligand Fe(II1) Ga(II1) Al(II1) Zn(I1) References
Desferrioxamine Bb 1030.99 1028.66
1024.5 1010.04c.d (22, 148, 151)
DTPAe lo2a 1025.54f 1018.7 l01s.zs d (15, 150)
(L1Ig
1,2-Dimethyl-3-hydroxypyridin-4-one 1035.92 1035.76 1032.62 1013.53 (18, 19)
HBED b 1039.68 1039.57 1024.78 1018.37d (20, 152, 153)
EDTA~ 1025.0 1020.3f 1016.5 1016.44d (15, 150)
"Unless otherwise indicated, all constants were measured at 25"C, ionic strength 0.1 M.
bK = [Fe(III)-HL+]/[Fe(III)1[HL2].
"Ionic strength = 0.2 M.
the case of DFO, the aminopolycarboxylates, and HBED with Zn(II), the equilibrium is reported as K = [Zn(II)-LII
[Zn(II)I[Ll.
"K= [Fe(III)-L2-I/[Fe(III)I[L6-I.
fAt 20°C.
gP3 = KIK&; for Zn(II), P2 = K,K2.
hK = [Fe(III)-L-]/[Fe(III)I[L4-I.

nephrotoxicity, which is unrelated to their in- ble 10.3) (150-153). Nevertheless, the metal
tended target. On the other hand, antihyper- selectivity issue is one that must be deliber-
tensives can manifest not only toxicity implicit ated very carefully in the design and testing of
in their structures, but also adverse effects as- new chelators.
sociated with their intended function. An Comparisons of Chelators. As was apparent
overdose of an antihypertensive medication in the discussion of metal complex formation
can cause a lethal drop in blood pressure. In in Section 1.2.1, it is important to have a scale
the case of deferrating agents, there is the is- that compares the binding of different ligands
sue of the toxicity implicit by the very defini- at the same molarity, pH, and iron concentra-
tion of iron chelation; that is. these molecules tion (i.e., pM values). Likewise, it is critical to
are designed to remove a metal essential for
apply a consistent scale to evaluate the chela-
life. Thus, parameters that reflect iron stores
tors on a physiological level; this scaling is re-
must be monitored to ensure that too much
ferred to as efficiency. The efficiency of an iron
iron is not removed from the patient (117).
Although unchecked iron removal is rarely, if chelator (Ec,,) is defined as the chelator-in-
ever, a problem clinically, it can be of major duced (net) iron clearance (c,,CL), divided by
significance in designing preclinical animal the theoretical iron clearance (TCL, i.e., total
toxicity trials in the course of evaluating new iron-binding capacity of chelator adminis-
iron chelators. Finally, there is the issue of tered). The chelator-induced (net) iron clear-
metal selectivity. For example, there are sev- ance (c,,CL) is the total urinary and fecal iron
eral metals that have coordination chemis- excretion measured in the presence of the li-
tries similar to that of FeUII), for example, gand (E c,,L) less the total iron clearance in
Al(III), Ga(III), and Cr(II1); all are bound the absence of the ligand (E c,,). Thus, the
tightly by hexadentate ligands. Even natural efficiency (Ec,,) is expressed as a percentage,
product siderophores, for example, DFO, can as shown in Equation 10.15:
bind these metals; fortunately, the iron com-
plexes are thermodynamically more stable (2cFeL - E
EcFe= X 100 (10.15)
than the corresponding Ga(II1) and AUIII) TCL
chelates (Table 10.3) (148). However, DFO
can be used to clear Al(II1) from dialysis pa- The TCL depends on how one defines the stoi-
tients (149). In the case of hexacoordinate sid- chiometry and the stability of the metal com-
erophores, the selectivity is greater than that plexes. An example of this is seen with DFO
for ligands of lower denticity, such as L1 (Ta- (9).This hexacoordinate ligand forms a very
Market

Agent in the
(Proprietary United
~&e) Structure States?

r-
n n Yes

'NH

Ferriprox 0
HO- -N

~~d
I
b o
Nob

DTPA

\
COOH

tight 1:l complex with Fe(II1) at pH 7.4; the (Fig. 10.31,a siderophore produced by S. pilosus
formation constant (K,) is approximately lo3' (35). Two synthetic chelators are available out-
M - I (21, 22,48). Thus, at concentrations of 1 side the United States as alternatives for those
pM ligand and 1 iron, virtually all of the who cannot tolerate DFO, the aminopolycar-
iron is in the form of the DFO chelate, or fer- boxylic acid diethylenetriamine pentaacetic acid
rioxamine. If a test animal were administered (DTPA; 35) (Fig. 10.15) and the pyridinone 1,2-
a 10 pmol/kg body weight dose of DFO, in the- dimethyl-3-hydroxypyridin-4-one (trade name
ory, a total of 10 pmoVkg of the iron chelate Ferriprox, also known as deferiprone and L1;
should be excreted, if (a) the iron were avail- formerly referred to as CP20; 33) (Fig. 10.15)
able for chelation and (b) the efficiency of the (Table 10.4).
chelator were 100%. Unfortunately, as de-
scribed in detail below, the actual efficiency is 2.1 .I Desferrioxamine (DFO, 9). In addi-
only a small fraction of this. tion to DFO, S. pilosus assembles eight more
hydroxamate ligands, two of which are cyclic
2.1 Iron Chelators on the Market
(36, 37, 154); two of these (10 and 11) are
The treatment of choice for iron chelation is shown in Fig. 10.3. On the surface, this fact
desferrioxamine B (trade name Desferal, de- would suggest that the microorganism wastes
feroxamine, DFO, 9, N-[5-[3-[(5-aminopenty1)- considerable energy by assembling the "extra"
hydroxycarbamoyl]propionamido]pentyl]-3- ligands. Although a definitive answer has not
[[5-(N-hydroxyacetarnido)pentyl]-carbamoyll- yet been reached, it seems unlikely that such a
propionohydroxamic acid methanesulfonate) waste is indeed the case. As described in the
508 Iron Chelators and Therapeutic Uses

Table 10.4 (Continued)

Who Markets
USP or Nonproprietary the Nongeneric Efficacy1 Route of
Name Substance? Potency Administrationa Dose
Desferrioxamine B, Novartis - 5% S .C. s.c.: 550 mg!kgIday
methanesulfonate salt efficiency i.v. i.v.: 515 mg/kg/h for
the first 1000 mg
and 125 mg/h
thereafter

1,2-Dimethyl-3-hydroxy- Apotex - 2.5% p.0. 75-100 mgflrg/day,


pyridin-4-one; efficiency divided
deferiprone; L1

DTPA BioTest - 5% S.C. 3 glday of the Ca


(Astrapin efficiency complex as its
Pharma) trisodium salt

"s.c.,subcutaneously; i.v., intravenously; p.o.,per os (oral).


bLicensedin Europe, Australia, and India, still considered experimental in the United States.
'Not licensed in the United States for the indication of iron overload, although it is approved as a chelating agent for use
in cases of poisoning with plutonium or other transuranic metals.

example above, DFO is a hexacoordinate che- ously or intravenously, has served as the drug
lator, which contains three bidentate hydrox- of choice for iron overload disorders (117,157,
amate fragments (Fig. 10.3). As mentioned be- 158).Its oral absorption is very poor; thus, the
fore, the Fe(II1) complex is very stable (14,21, drug is not given by this route (159). A few
22, 48). For comparison, Table 10.1 lists the general comments are of interest regarding
formation constants for a variety of Fe(II1) dosing, side effects, and drug interactions. Ac-
complexes. For pharmaceutical use, the mole- cording to the Novartis package insert, the
cule is prepared by fermentation of the micro- LD,, is 287 mg/kg when given intravenously
organism, isolation of the compound as its hy- to mice; this figure is 329 mg/kg in rats. Des-
drochloride salt, and processing through ion feral is made up in sterile water to a concen-
exchange to the methanesulfonate salt (Des- tration of 10% w/v; this solution is isotonic.
feral, MW 657) to improve its solubility. The preferred method of administration for
Initially, DFO was used to treat patients the treatment of chronic iron overload is at a
suffering from primary and secondary iron dose of 20-40 mg/kgJday by slow subcutane-
overload (155, 156) as well as pediatric pa- ous infusion over a period of 8-24 h; the rec-
tients suffering from acute iron poisoning ommended total daily dose is between 1000
(147). Since these early trials, desferrioxa- and 2000 mg. Intravenous administration is
mine B mesylate, whether given subcutane- suggested only in cases of cardiovascular col-
Clinical Use of Chelating Agents

lapse secondary to acute iron intoxication; un- patients undergoing such a regimen (173).
der these conditions, doses are not to exceed The ascorbate problem is related to the discus-
15 mgikgh for the first 1000 mg. Subsequent sion of iron chemistry in Section 1.3.4. Ascor-
i.v, administration, if needed, must be at a bate reduces Fe(II1) to Fe(I1). Recall (Section
slower rate not to exceed 125 mg/h. 1.3.4) that Fe(I1) is the major contributor to
2.1.1.1 Side Effects of Desferrioxamine, Fenton chemistry, which reduces hydrogen
The cautionary restrictions mentioned above peroxide to hydroxide ion and the hydroxyl
are likely related to the profound impact that radical. Furthermore, DFO ligates Fe(III), not
i.v.-administered DFO can have on blood pres- Fe(I1). However, the Fe(II)/Fe(III) couple is
sure (i.e., hypotension) and cardiac function driven as Fe(II1) is sequestered by DFO. This
(147). In addition, intravenous infusions of increases the reducing capacity of Fe(II), but
DFO at doses higher than the recommended only on a one-time basis. Thus, patients re-
15 mgikgh for greater than 24 h for therapy of ceiving DFO are cautioned to use ascorbate
acute iron poisoning have resulted in several only under specific conditions (117, 172). Un-
instances of acute respiratory distress syn- less ample DFO is administered such that the
drome (160,161). The syndrome also has been excess iron that is released reductively from
reported in a child who had been treated with transferrin and ferritin can be sequestered,
intravenous DFO according to the current ascorbate should not be given to iron-over-
guidelines (162). Other side effects observed loaded patients. It would seem, then, that
with prolonged use of DFO include both ocular treatment of iron-overloaded patients with
and auditory problems (163-165), pulmonary ascorbate is, in and of itself, a potential prob-
toxicity (1601, bone changes (166),and growth lem. This is in keeping with the observations
retardation (167). These effects have been re- in iron-overloaded, scorbutic Bantus (174,
ported as being reversible on cessation of che- 175).
lation therapy with DFO (168) and have Finally, one of the most common com-
diminished in frequency as management plaints among patients who receive DFO sub-
strategies have improved (117). A particularly cutaneously is the discomfort at the site(s) of
interesting cautionary note is related to the infusion. Although some anaphylactoid symp-
rare, but nevertheless documented, increased toms have been reported, this is very uncom-
susceptibility of patients using DFO to infec- mon (176, 177); however, local erythema is
tion by Yersinia enterocolitica and Y.pseudo- typical (178, 179). In an effort to identify the
tuberculosis (93, 169). The suggested reason nature of the allergic response, investigators
for this augmented susceptibility is that ferri- examined the effect of DFO on human ba-
oxamine formed within treated patients pro- sophils and rodent mast cells in vitro and in
vides prepackaged Fe(II1) for these microor- human skin (177). Incubation of human ba-
ganisms (93). sophils with DFO did not induce histamine
At the level of drug interaction, two partic- release from these cells. Furthermore, prein-
ular phenomena, the distortion of imaging cubation of basophils with DFO had no impact
; measurements and ascorbate-induced cardiac on histamine release from cells that were pre-
effects, are interesting because of their mech- viously stimulated with either anti-human
anisms. First, imaging with 67Gain patients IgE or with f-met peptide. However, when
using Desferal has been shown to be con- seven healthy patients ndive to the drug were
founded (170). The reason, as pointed out ear- given subcutaneous injections of DFO, all of
lier, is that Ga(II1) is ligated by DFO and is them developed a significant wheal-and-flare
excreted quickly through the kidneys. Second, response, which was dose dependent and
some patients who received concomitant reached its maximum after 15 min. Treatment
treatment with Desferal and high ( S O 0 mg with the HI antagonist ceterizine elicited a
daily in an adult) doses of vitamin C experi- substantial reduction in the wheal-and-flare
enced cardiac impairment (117,171,172);sev- response, indicating that the wheal-and-flare
eral deaths were reported in the mid-1970s in response was associated with local mast cells.
510 Iron Chelators and Therapeutic Uses

deamination

HO
CH3

OH OH

0 0
(36)

Figure 10.16. DFO (9)and its major, deaminated metabolite, Metabolite B (36).

Finally, DFO also activated rat peritoneal tracted intravenous or subcutaneous infu-
mast cells, a type of connective tissue cell; the sions of DFO were quite effective in promoting
histamine release was dependent on the dose iron excretion (147, 157, 159); thus, it is pos-
of DFO. These findings suggest that the DFO- sible to maintain patients in negative iron bal-
mediated allergic response is an IgE-indepen- ance when DFO is administered by either of
dent, nonimmunologic stimulatory effect on these two routes.
cutaneous mast cells. The investigators The requirement for slow infusion to real-
opined that DFO could be used as a positive ize DFO's maximum efficacy is probably re-
control in cutaneous allergy testing. This lated to the ligand's short half-life (5-10 min)
should underscore the profound discomfort in human plasma (180). Reports have varied
that patients have with the drug. The anaphy- regarding the proportion of the iron excreted
lactoid problem certainly is related to other through the stool versus the urine; the range
issues. The possibility does exist that, because is from 50-70% in the stool (181, 182). The
this drug is a fermentation product, small, un- urinary iron is likely derived from plasma
detectable amounts of protein contaminant(s) NTBI and the RE system, whereas the stool
are not removed during the purification pro- iron arises from hepatocytes (159,183). Upon
cess; these trace impurities may be responsi- subcutaneous administration of the drug, the
ble for the observed sensitivity. plasma concentrations reach a steady state at
2.1.1.2 Pharmacology of Desferrioxamine. 4 h; a rapid decrease in concentration occurs
Originally, DFO was administered intramus- on cessation of infusion (184). Besides the
cularly (155, 156). A substantial increase in metal chelate, the major metabolite of DFO
urinary iron output, which appeared to be re- (36, Fig. 10.16), which has been found in both
lated directly to the extent of the patient's iron the plasma (185) and the urine (185, 186) of
overload, was observed. However, daily intra- patients treated with DFO, results from oxida-
muscular injections of DFO were unable to re- tive deamination at the amino terminus of the
move sufficient iron from thalassemia pa- molecule, parallel to the metabolism of lysine
tients; the use of intramuscular injections (185). The pharmacokinetic properties of the
could not keep up with the iron loading from metal chelate, ferrioxamine, are somewhat
the transfusion therapy. Nevertheless, pro- different from those of the parent drug (180,
2 Clinical Use of Chelating Agents

184, 187). As expected, the concentration of tients given the ligand suffer pain at the site of
ferrioxamine rises slowly during DFO admin- injection, nausea, diarrhea, and sore throat
istration, but declines more slowly than the and mouth (191, 192). In one trial (192), the
concentration of the parent drug when the in- patients' plasma zinc levels decreased from
fusion is stopped. It appears as though any the normal range of 11.5-17.5 to 5 pmol/L af-
chelate formed extracellularly (i.e., by chela- ter 5 days of subcutaneous infusions with
tion of plasma NTBI) is restricted to the extra- DTPA as its calcium complex. At the same
cellular space (184). Desferrioxamine-induced time, this treatment exerted little effect on
fecal iron excretion derives from hepatocyte other divalent cations, such as Cu, Ca, and Mg,
iron: plasma DFO is taken up by the liver, the in the plasma. Generally, the side effects were
iron is cleared from the hepatocytes, and the ameliorated within 10 days after the treat-
ferrioxamine is excreted in the bile (159,184). ment was discontinued. In the course of this
As with most drugs, with increasing dose, same investigation, the attempt was made to
the DFO plasma level increases and subse- overcome the side effects associated with zinc
quently plateaus (180,184,187). The response depletion by administering the DTPA as its
also can be quantitated by following the li- zinc complex. Unfortunately, the Fe(III)/
gands' efficiency upon increasing doses. Aug- Zn(I1) exchange was ineffective in these pa-
menting the chelator dose results in an in- tients; there was little, if any, iron clearance.
creased iron clearance up to a plateau; beyond However, when patients were given oral zinc
this plateau, the efficiency of the ligand de- sulfate supplements ("effervescent zinc") sev-
creases (157). eral times daily during the courses of subcuta-
As alluded to above, ascorbic acid can have neous DTPA administration, the side effects
a profound impact on DFO-promoted urinary were minimized (192, 193).
iron excretion (173, 188, 189). When patients 2.1.2.2 Pharmacology of DTPA. Investiga-
receiving chelation therapy with DFO were tors have found that the iron balance realized
given oral doses of vitamin C (500 mg three upon administration with DFO or DTPA is
times daily), urinary iron excretion increased comparable (190); the efficiencies are similar
between 25 and 200%, although alterations in when 24-h subcutaneous infusions of 4 g of
fecal iron excretion were not as consistent DFO and 3 g of Ca-DTPA are compared di-
(189). However, because of concerns about rectly (192). However, the proportions of the
cardiotoxicity as mentioned earlier, this com- metal excreted in the stool and urine are dif-
bination therapy may be problematic. Only ferent. Whereas DFO induces excretion of iron
under conditions of tissue ascorbate deficiency by way of both urine and stool (Section
is repletion with ascorbate (100 mg daily) rec- 2.1.1.2), DTPA promotes urinary excretion
ommended (117). only (192). When both drugs were given simul-
taneously, the results were additive, not syn-
2.1.2 Diethylenetriamine Pentaacetic Acid ergistic. Furthermore, unlike the situation
(DTPA, 35). The use of the aminopolycarbox- with DFO, ascorbate supplementation during
ylate diethylenetriamine pentaacetic acid chelation therapy with DTPA did not enhance
(DTPA, 35) (Fig. 10.15) as a therapeutic iron iron excretion (192). Thus, DTPA has served
chelator was initiated at about the same time as one alternative to DFO, but only when pa-
as the assessments of DFO (190, 191). As tients cannot tolerate the latter (194).
shown in Table 10.1, (35)forms a 1:1 complex Although the overwhelming proportion of
with Fe(II1); the formation constant and pM an orally administered dose of 14C-DTPA
value are lo2' M-' and 23.8, respectively (15). passes through the gastrointestinal tract un-
However, the salient difference between these absorbed, intravenously administered radiola-
two agents is their metal selectivity. DFO, a beled ligand is excreted quantitatively in the
siderophore, is far more selective for iron than urine (195, 196). This is consistent with the
is DTPA. pattern of iron excretion described above. The
2.1.2.1 Side Effects of DTPA. In fact, the predominant thinking is that DTPA is ex-
toxic effects associated with DTPA have been creted by glomerular filtration; the t,,, for ex-
connected with its ability to chelate zinc. Pa- change between the plasma and extracellular
Iron Chelators and Therapeutic Uses

fluid, the return to the plasma, and the clear- studies by the prevalence of hepatitis C infec-
ance from the plasma into the urine are all tions, attributed to the frequency of blood
brief, 2.5, 6.3, and 19 min, respectively (196). transfusions in the thalassemic patient popu-
DTPA is metabolized only minimally by the lation. Unfortunately for both patients and in-
body (197); of this small amount, release of vestigators, hepatitis C itself can elicit liver
acetate groups has been observed as expired fibrosis (208).Further clouding the issue is the
C02 in rats (198). possibility that incompletely complexed iron
[i.e., 1:l and 2:l L1-Fe(II1) complexes] can
2.1.3 1,2-Dimethyl-3-hydroxypyridin-4-one participate in Fenton chemistry (Section
(Deferiprone, 11; 33). Another alternative, 1.3.4); L1 promotes Fenton chemistry in sev-
which has been licensed for sale in India since eral in vitro systems (116). It is possible that
1994, in Europe since 1999, and in Australia this augmentation of free-radical formation
since 2001, is the orally active, bidentate hy- could contribute to the observed hepatic in-
droxypyridinone 1,2-dimethyl-3-hydroxypyri- jury; such damage has been noted in hepatic
din-4-one (trade name Ferriprox, deferiprone, cell culture (209). However, these issues re-
L1,33) (Fig. 10.15). This ligandforms 1:1,2:1, quire further investigation. What is more im-
and 3:l complexes with Fe(II1) (Table 10.1), portant is that investigators not condemn the
depending on the availability of the chelator entire hydroxypyridinone class if L1 is found
(15, 18, 19). The total formation constant (P, conclusively to be problematic at the level of
is 1035.92 M- 1. b
= K1K&3) , ecause of the stoi- toxicity.
chiometry, the pM value is 18.3, much less 2.1.3.2 Pharmacology of L 1. Surprisingly,
than that of DFO (Table 10.1). Species distri- the toxicity issues surrounding L1 have ob-
bution plots for the L1-Fe(II1) complexes [i.e., scured the facts regarding the drug's efficacy
fraction of (Ll),-Fe(III), (Ll),-Fe(III), and as a deferration agent. Early findings sug-
(Ll),-Fe(1II)I show that at physiological pH, gested that L1 was the long-anticipated orally
an L1 concentration of 3 x M, and an effective iron chelator. Most of the iron excre-
Fe(II1) concentration of 1 x lo-, M, the tion induced by L1 is urinary, although some
(Ll),-Fe(II1) complex is by far the major spe- increase in fecal iron has been noted (205,
cies. However, at 1 x M Fe(II1) and 3 x 210). Pharmacokinetic analyses indicated
lop6 M L1, complete (Ll)3-Fe(III) complex that, like DFO, the kinetics varied with the
formation does not occur until pH 10 (15). iron status of the individual (211-213).Gener-
2.1.3.1 Side Effects of L 1. The oral activity ally, the pharmacokinetic parameters are the
of L1 has made it very attractive for use in same, regardless of whether L1 is adminis-
clinical settings. However, two issues that de- tered during feeding or after fasting (212). Af-
tract from its widespread use in the United ter rapid absorption from the stomach, the
States and elsewhere are concerns about its terminal elimination half-life is 1.5-2 h, al-
toxicity profile and continuing questions re- though there are wide variations, especially
garding the efficacy of the chelator. The over- among thalassemics (211, 213, 214). Most of
whelming majority of the controversy over L1 the dose is excreted uncomplexed as the gluc-
has centered on the former topic, rather than uronide conjugate (37, Fig. 10.17); smaller
the latter. proportions are eliminated as the free ligand,
There is agreement among investigators the iron complex, and the 3-0-methylated con-
regarding some of the side effects of de- jugate (38, Fig. 10.17) (211, 215,216).
feriprone, that is, nausea, vomiting, agranulo- Although the widespread use of L1 for the
cytosis, increased alanine transaminase lev- treatment of transfusional iron overload has
els, arthropathy, diminished plasma zinc some support (217), over a decade after the
levels, and some immunologic changes (199- initial human studies, many investigators be-
207). The debate surrounding L1 is its possible lieve that L1 is not an efficient enough chela-
role in the induction of liver fibrosis; the dis- tor to maintain thalassemic patients in nega-
agreement seems to derive from (a) small pa- tive iron balance when administered alone
tient populations in the trials and, more im- (117, 204, 206, 218). Some reports indicate
portant, (b)the confounds introduced in these that, although it is effective initially in pro-
:al Use of Chelating Agents

KH3
I CH3
Figure 0.17. L1 (33),
uronide 1conjugate (371,its
andglue
3-0-methylated conjugate (38).
its

Most of an L1 dose is excreted as


CH3
(37);(38)accounts for 1% or less
(38) of an administered dose.

moting iron clearance in those patients with of DFO (15) and are sufficiently labile for
significant iron overload, it is not useful in in- transfer of the metal to DFO. Upon abstrac-
dividuals who have lower, but still dangerous, tion from L1 by DFO, the metal can no longer
hepatic iron deposits (207, 218, 219). Most of participate in Fenton chemistry. Different
the trials were conducted using daily doses of modes of combining DFO and L1 have been
575 mg/kg (205-207, 218). Furthermore, on attempted, including simultaneous [i.e., L1 is
the basis of some of the human clinical trials given orally during the DFO infusion (221,
(206, 218) and of preliminary reports in ani- 223)] and sequential [i.e., L1 is given during
mals (220), the suggestion has been made that the day and DFO at night (223) or L1 is given
L1 can actually promote iron absorption, pre- orally for 4 weekdays and DFO is infused over
sumably of low molecular weight complexes 2 weekend days (222)l. The sequential therapy
(208). resulted in an additive effect; no adverse
More recently, investigators have assessed events occurred in either of these two groups
the efficacy of L1 and DFO in a combined reg- of patients (222, 223). However, the simulta-
imen (221-223). The rationale for using such a neous administration of the two ligands pro-
combination is based on the fact that L1 can duced a synergistic impact on iron excretion
remove iron from transferrin and can cross without any apparent toxicity (221,223). This
cell membranes, thus acting as a "shuttle," synergism was particularly noteworthy under
whereas DFO can do neither particularly well, the regimen undertaken by three patients in
but can function as a "sink" in the blood- which L1 was given at a daily dose of 100
stream, promoting the ultimate excretion of mg/kg in three divided doses that coincided
the metal (224). Even though DFO can bind with t = 0,4, and 8 h of the DFO infusion at a
iron more tightly than does transferrin, the subcutaneous dose of 40 mg/kg (223). Because
kinetics of transferrin deferration by DFO are of the small numbers of patients who have re-
too slow to be of therapeutic value, yet L1 can ceived any one regimen, seven at the most
remove iron from transferrin (101, 217). In (222), it is unclear whether such combination
addition, recall that the (L1)-Fe(II1) and therapy will prove to be safe and effective
(Ll),-Fe(II1) complexes have smaller forma- when extended to larger numbers of patients
tion constants than does the Fe(II1) complex and carried out for longer time periods. Ulti-
Iron Chelators and Therapeutic Uses

mately, the issues surrounding the toxicity of pounds as antibiotics (230), investigations of
L1 may prove to be moot; it is unlikely that L1 the antagonists continued. In the early 1960s,
will serve adequately as a single orally effec- several reports were published concerning the
tive deferration agent. isolation and structural characterization of
these iron-containing antagonists, the ferriox-
amines, which contained two or three hydrox-
3 HISTORY OF CHELATION THERAPY; amate moieties, separated by varying spacers
DISCOVERY OF AGENTS W I T H IRON- (3537). One of these, ferrioxamine B (35),
CHELATING ACTIVITY was assessed as an iron donor in a patient with
iron-deficiency anemia; the intent was to mea-
The use of phlebotomy had been regarded as sure the pharmacokinetics of, and the fate of
the treatment of choice for primary hemochro- the iron derived from, ferrioxamine. The fer-
matosis since the mid-1940s (225); however, rioxamine was excreted quickly and quantita-
clinicians were well aware that such treat- tively in the urine without any loss of the iron
ment was not necessarily practical in all situ- to the tissues. This was surprising at the time
ations. Thus, the alternative of chelation ther- because the entire iron content of other par-
apy was examined beginning in the early enterally administered iron preparations was
1950s. Although any number of compounds retained by the body (230).
bind iron in vitro, the specific removal of iron The investigators considered the following:
from an animal or human is another matter, for the iron in ferrioxamine to be excreted
as was found in the early trials of chelating quantitatively, it must be bound very tightly
agents. The first studies with iron-chelating under physiological conditions. If this were
agents in animals and humans used ethyl- the case, a deferrated ferrioxamine should re-
enediaminetetraacetate (EDTA) (32) (Fig. move iron from stores in the body, rendering
10.151, an agent that was known to chelate the now chelated iron excretable in the urine
iron as well as other metals; the results were (230). Accordingly, an iron-free ferrioxamine,
disappointing (226, 227). A structurally simi- desferri-ferrioxamine B, (desferrioxamine B,
lar compound, DTPA (351, appeared to be a or DFO for short) as the hydrochloride salt
better choice after it was found that this li- was prepared and evaluated. Experiments in
gand did not surrender iron to transferrin in rabbits and dogs revealed that DFO did re-
vitro (228). The compound was assessed in move iron from animals; the compound was
both animals and humans and found to be most effective in those animals that had been
more effective in removing iron in vivo than previously loaded with iron (156, 231). The
was EDTA (155, 191). However, as discussed metal specificity of DFO was assessed in rab-
in Section 2.1.2, the use of DTPA is not wide- bits and compared with that of the known che-
spread because of its side effects. lator, EDTA (231, 232). The specificity of the
Meanwhile. an unrelated search for sub- siderophore for iron in vivo was confirmed by
stances with antibiotic activity was under measurements of the stability constants of the
way. The goal was to test such materials se- two ligands in vitro (48). Not only was the sta-
creted by strains of Streptomyces as potential bility constant of the iron-DFO complex
antibiotics (229). In the course of attempts to greater than those of the complexes with other
optimize the yield of what would be known as divalent and trivalent ions, much more spe-
ferrimycin from the fermentation process, it cific than EDTA, but the stability constant of
was found that the effect of this compound the DFO-iron complex was much stronger
was diminished by- culture filtrates from other than that of the EDTA-iron complex. The in-
Streptomyces species. Further isolation of this teraction of DFO with ferritin, transferrin,
antagonist was carried out in a n effort to both and hemoglobin was measured; the investiga-
characterize the antagonist itself and gain in- tors were satisfied that DFO chelates iron
sight into the mechanism of action of ferrimy- from none of these iron transport or storage
cin. Although it was found that bacteria proteins (156, 186). After the initial trial in a
quickly became resistant to the ferrimycins, patient with severe hemochromatosis (156),
ending the development of this class of com- the requirement of high doses dictated a form
4 Recent Developments

of DFO that was more soluble in water than CI-


the hydrochloride; the methanesulfonate form
of the compound was derived and is known -02% <c02H
today by the trade name Desferal, still the
treatment of choice for transfusional iron
overload and acute iron poisoning.
Desferal was discovered and subsequently
developed in a corporate laboratory. However,
i because of the relatively limited market for
iron-chelatingagents (and their resulting des-
ignation as "orphan" drugs), many of the po-
t e n t i a l alternatives, including deferiprone,
' were discovered in academic laboratories most
'
frequently sponsored by the U.S. National In-
stitutes of Health, rather in than corporate
settings.

4 RECENT DEVELOPMENTS

It should be clear that the ideal chelator for


the treatment of iron overload disease is not
yet available. However, investigators have
sought chelators with improved efficiency (i.e., Figure 10.18. Structure of N,N'-bis(2-hydroxy-
greater Ec,, values), regardless of the mode of benzy1)ethylenediamine-N,N'-diacetic acid (HBED)
administration. This section covers com- as the monohydrochloride dihydrate (39a) and as
pounds that, in recent years, have shown great the monosodium salt (NaHBED, 39b).
promise in animal studies.
seemed imminent. Unfortunately, the rodent
4.1 N,N1-Bis(2-hydroxybenzyl)- findings were not replicated in higher animals.
ethylenediamine-N,Nr-diacetic In the iron-overloaded Cebus apella model, an
Acid (HBED, 39a) excellent predictor of how a chelator will per-
The polyanionic amine N,N'-bis(2-hydroxy- form when administered to humans (Section
benzy1)ethylenediamine-N,N1-diacetic acid 5.2.3), the iron-clearing efficiency of DFO
(HBED, 39a, Fig. 10.18) is a synthetic given subcutaneously at a dose of 150 pmollkg
hexadentate ligand (20). Like DFO, HBED was 5.5 +- 0.9% (235); however, the efficiency
forms a 1:l complex with iron with high affin- of orally administered HBED, also dosed at
ity and selectivity; the formation constant and 150 pmolkg, was significantly lower, 0.5 2
pM value for the complex are 1039.6sM-' and 0.5% (236). Not surprisingly, HBED was also a
26.74, respectively (Table 10.1) (15). Because disappointment when given orally to patients
HBED is a synthetic product, problems of local (182, 237); the limited iron excretion that re-
reactions to potential fermentation by-prod- sulted is insufficient for use of this agent in the
ucts not removed during purification would be treatment of transfusional iron overload.
absent. Also, for patients allergic to DFO, However, when given parenterally to rodents
HBED, a member of a different family of che- or primates, HBED performed very well (238-
lators, would be unlikely to provoke a similar 241).
response. HBED has been investigated thor-
oughly in rodents (233,234). The drug looked 4.1 .I Parenteral Administration to Rodents.
very promising in this model when adminis- Early evaluations of HBED were carried out in
tered orally and, not unexpectedly, these find- the hypertransfused rat using intravenous ad-
ings generated a great deal of excitement. The ministration; these demonstrated a two- to
availability of an orally active iron chelator threefold greater excretion using HBED ver-
-
Iron Chelators and Therapeutic Uses

-
r
DFO (s.c.)
HBED (p.0.)
HBED (s.c.)

Time (hours)
Figure 10.19. Time course of mean biliary iron excretion in normal rats after administration of
DFO by subcutaneous injection and after administration of HBED by gavage or by subcutaneous
injection. Both chelators were given at a dose of 150 pmoUkg body weight. The peak amounts of iron
excreted with subcutaneous HBED (asterisks) were more than twofold greater than the peak iron
excretion after either subcutaneous DFO or HBED given by gavage (P< 0.05 at t = 3 h and P < 0.01
at t = 6 h). [From R. J. Bergeron, J. Wiegand, and G. M.Brittenham, Blood, 91,1446-1452 (1998).
Copyright American Society of Hematology, used by permission.]

sus the reference chelator, DFO (238, 239). tion of 436 176 pg Fe/kg body weight (P<
Subcutaneous administration of HBED was 0.02); the efficiency was 5.2 f 2.1%. HBED
examined in the bile duct-cannulated rodent given to the rodents by subcutaneous injection
model, which has normal iron stores (240). was more than three times as effective in in-
Groups of rats were given either DFO by sub- ducing iron excretion as DFO administered
cutaneous injection, or HBED as the monohy- subcutaneously (P < 0.001), inducing the ex-
drochloride dihydrate (39~1,Fig. 10.18) orally cretion of 679 t 8 pg Fe/kg body weight at an
by gavage or by subcutaneous injection at a overall efficiency of 8.1 ? 0.1%.
dose of 150 pmol/kg of body weight. Fig. 10.19
shows the time course of the mean biliary iron 4.1.2 Parenteral Administration to Primates.
excretion induced bv " the chelators in each These studies were carried out in iron-over-
group of rats, expressed as pg Fetkg body loaded C. apella primates (242). In brief, the
weight. The peak amounts of iron excreted reference chelator DFO was administered
with subcutaneous HBED were more than subcutaneously at three different doses or as a
twofold greater than the peak iron excretion 20-min intravenous infusion at two different
after either HBED given by gavage or subcu- doses (Table 10.5). Because of the poor solubil-
taneous DFO (P < 0.05 at 3 h and P < 0.01 at ity of the HBED monohydrochloridedihydrate
6 h). The iron excretion induced by the chela- that was used in the rodent studies and the
tors in each group of rats is shown in Fig. fact that the necessary manipulations to pre-
10.20, expressed as the net mean amount of pare the material for injection could be prob-
iron excreted in the urine and in the bile (pg lematic in a clinical setting, the HBED
Fe/kg body weight) and as the efficiency of iron monosodium salt (NaHBED, 39b, Fig. 10.18)
chelation. Subcutaneous DFO induced the ex- was prepared and evaluated. The salt is much
cretion of 209 ? 59 pg Fekg body weight and more soluble than the monohydrochloride di-
was found to have an efficiency of 2.5 ? 0.7%. hydrate (39a). The solubility is in excess of
Compared to subcutaneous DFO, HBED given 30% (w/v)in water; when the drug is dissolved
orally resulted in a twofold greater iron excre- in saline the unadjusted pH of the resulting
4 Recent Developments 517

N Urine
Bile

DFO (s.c.) HBED (p.0.) HBED (s.c.)

Figure 10.20. Mean net iron excretion in normal rats after administration of DFO by subcutaneous
injection and after administration of HBED by gavage or by subcutaneous injection. Excretion is
shown as pg F e k g body weight on the scale of the left vertical axis and as efficiency of chelation on the
right vertical axis. Both chelators were given a t a dose of 150 pmoltkg body weight. [From R. J.
Bergeron, J. Wiegand, and G. M. Brittenham, Blood,91, 1446-1452 (1998). Copyright American
Society of Hematology, used by permission.]

solution is 7.3 (241). Several methods of ad- monohydrochloride dihydrate in buffer or in


ministration of NaHBED were investigated: Cremophor, 9.9 + 2.1% (P > 0.2) and 14.9 +
subcutaneous bolus at doses of 75 and 150 5.2% (P > 0.7), respectively (240). In addition,
pmol/kg, as well as a three-dose regimen of 75 NaHBED has been administered at a dose of
pmolkg every other day for three doses; intra- 75 pmolkg every other day for three doses
venous bolus at doses of 50 and 75 pmoVkg, (Fig. 10.21), for a total dose of 225 pmolkg.
and 20-min intravenous infusion at doses of The first dose of the drug induced the excre-
150 and 225 pmol/kg (Table 10.5). tion of half as much iron as did the 150
4.1.2.1 Subcutaneous Administration. Des- pmolkg dose, 597 + 91 pg/kg; the 24-h effi-
ferrioxamine administered subcutaneously in ciency was 14.2 + 2.2% (Table 10.5) (P > 0.3
aqueous solution at a dose of 75 pmolkg in- versus 150 pmolkg single dose). The second
duced the excretion of 213 ? 112 pgkg of iron; and third injections of the drug stimulated the
the efficiency was 5.0 + 2.6% (243). A dose of excretion of comparable amounts of iron; the
150 pmol/kg was found to have a similar effi- 24-h efficiencies were very similar to each
ciency and induced the excretion of approxi- other. The total iron excreted as a result of the
mately twice the amount of iron, 435 ? 115 pg three injections was 1837 ? 301 pgkg of iron,
Fekg body weight (240) (Table 10.5; P > 0.4 and the overall efficiency was 14.6 + 2.4% (Ta-
rersus 75 pmol/kg dose). Increasing the dose ble 10.5). These data are virtually identical to
;o 300 pmoVkg induced the excretion of 716 2 the iron excretion induced after a single sub-
144 pg/kg of iron and had an efficiency of 4.2 2 cutaneous injection of HBED in a buffer given
.4% (241). at a dose of 81 pmolkg, 608 ? 175 pglkg and
In contrast, when NaHBED was given to an efficiency of 13.0 ? 4.6% (241) (P > 0.6, P >
he primates at a single dose of 150 pmolkg, it 0.5, and P > 0.5 for doses 1-3, respectively).
iduced the excretion of 1139 + 383 pg/kg of To measure the degree of iron balance
,on and had an efficiency of 13.6 ? 4.5% (Ta- achieved by the chelators, the total amount of
le 10.5) (241). The observed efficiency is well iron intake was compared with the total
ithin error of that observed after the subcu- amount of iron excreted [i.e., net iron balance
ineous injection of 162 pmolkg of the HBED = dietary iron intake - (urinary + fecal iron
Table 10.5 Comparison of Efficiencies and Net Iron Balance of Intravenous and Subcutaneous HBED (39b)versus Those of Intravenous
and Subcutaneous DFO (9) in C.apella Primates
Dose
Induced Fe Fe Balance
a
Drug Route (pmoukg) (mglkg) N Efficiency (%) (~glkg) (c~~/kg)~ Reference
DFO s.c. bolus 75
DFO S.C. bolus 150
DFO s.c. bolus 300
DFO 20 min i.v. inf 75
DFO 20 min i.v. inf 150
01 HBED s.c. bolus 75 (day 1)c
d
m HBED s.c. bolus 150
HBED s.c. bolus 225 (75 X 3
doses)d
HBED i.v. bolus 50
HBED i.v. bolus 75
HBED 20 min i.v. inf 150
HBED 20 min i.v. inf 225
"s.c., subcutaneous; i.v., intravenous; i d , infusion.
bNetiron balance = dietary iron intake - (urinary iron + fecal iron). Animals in a negative iron balance are excreting more iron than they are absorbing. To maintain iron balance,
250-400 pg of iron/kg/day = 1750 to 2800 pg of Fewweek must be cleared (145).
"Day + I to day +2 versus day -3 to day 0 iron balance figures are shown.
dCumulativeafter the three injections.
Recent Developments 519

a 800 t E j Urine
H Feces
T T T

Figure 10.21. Urinary and fecal iron excretion (pg/kg) induced by the subcutaneous administration
of HBED monosodium salt, 75 pmolkg for three doses (225 pmolkg total). Drug was administered on
days 0,2, and 4. Note the prompt return to baseline levels within 24 h of each dose; baseline iron levels
in the urine and stool have not been subtracted. [From R. J. Bergeron, J. Wiegand, and G. M.
Brittenham, Blood, 93,370-375 (1999). Copyright American Society of Hematology, used by permis-
sion.]

excretion)]; animals in a negative iron balance 4.1.2.2 Intravenous Administration. Although


are excreting more iron than they are absorb- slow intravenous infusions of DFO (<lo mg/
ing. Monkeys treated subcutaneously with kg/h) have been well tolerated in patients with
DFI0 at doses of either 75,150, or 300 pmolikg chronic iron overload (244-246), rapid intra-
we1m e held in negative iron balance (Table venous administration for the treatment of
10.15); the excreted iron was 60 t 135, 278 2 acute iron poisoning can produce grave side
185, and 711 ? 230 pgJkg, respectively, more effects, as described in detail in section 2.1.1.1
than they absorbed (240,241, 243). The sub- (147, 160-162). The manufacturer recom-
timeous administration of NaHBED was mends that the total daily parenteral dose of
abh? to hold the monkeys in a negative iron DFO should not exceed 6 g. Because 1 g of
ballm e (Table 10.5) (241). Monkeys treated DFO binds only about 90 mg of elemental iron,
wit1n NaHBED at a single dose of 150 pmolikg and a single tablet for the treatment of iron
excireted 899 2 365 p@g of iron more than deficiency in adults may contain as much as 65
the: ;r absorbed. Also, NaHBED given subcuta- mg of elemental iron, 6 g of DFO given paren-
neo.usly every other day for three doses (75 terally may not be adequate in cases of severe
W'd/kg/dose) resulted in a negative iron bal- iron poisoning. Considering the findings with
anccz (Table 10.5). The iron excreted during a subcutaneously administered NaHBED in
7-dliy period after the administration of the light of these limitations of DFO, it became of
first; dose amounted to 1578 ? 345 p&g more interest to find whether intravenous adminis-
thar1 they absorbed. As was observed with the tration of this ligand would (a)be an effective
winary and fecal iron-clearance data, animals means of deferration of iron-loaded animals
treated subcutaneously with NaHBED consis- and (b) elicit hypotension in normal animals.
tently have a negative iron balance that is 2-3 The latter subject is addressed in Section
time:s greater than that observed with DFO. 4.1.3.
The results of these studies (240, 241, 243) Intravenous administration of DFO to the
clesurly indicate that both subcutaneously ad- iron-loaded primates at a dose of 75 pmolikg
miniistered DFO and NaHBED can hold the induced the excretion of 237 2 67 &kg of iron
monkeys in a negative iron balance (Table and had an efficiency of 5.6 2 1.6%. Increasing
10.51. the dose to 150 pmolikg resulted in the excre-
Iron Chelators and Therapeutic Uses

tion of 332 + 66 pg/kg of iron and an efficiency level of 300 mg Felkg, served as saline-treated
of 3.9 5 0.8% (Table 10.5) (P< 0.04). Interest- controls. Upon necropsy, the most significant
ingly, although the efficiency of DFO given ei- finding was the accumulation of hemosiderin
ther subcutaneously or intravenously at a in the macrophages of the liver, spleen, and
dose of 75 pmol/kg was similar, 5.0 ? 2.6% lymph nodes of both test and control animals.
versus 5.6 + 1.6% (P > 0.3), this was not the There was no systemic toxicity that could be
case when the dose was increased to 150 pmoV attributed to the NaHBED under this intrave-
kg. DFO given subcutaneously at 150 pmollkg nous regimen. Another systemic toxicity trial
resulted in an iron-clearing efficiency of 5.1 5 was carried out in dogs using subcutaneous
1.3% (Section 4.1.2.1), but the same dose given administration of NaHBED. These non-iron-
as an intravenous infusion resulted in an effi- overloaded dogs were given NaHBED at grad-
ciency of 3.9 2 0.8% (P < 0.02). The efficien- uated doses of up to 300 pmol/kg/day. The
cies of NaHBED administered to the iron- drug was injected as a subcutaneous bolus ev-
loaded primates as an intravenous bolus at ery other day into one of two sites on a rotating
doses of 50 and 75 pmollkg were also similar to basis. Upon necropsy, histopathological anal-
those of the drug administered subcutane- ysis did not reveal any drug-related abnormal-
ously (Section 4.1.2.1), 12.1 ? 2.5% and 11.5 ? ities beyond those in the skin. The descrip-
1.3% (P > 0.3); the corresponding iron excre- tions of the reactions in the skin at the sites
tions were 338 t 68 and 482 + 54 pg/kg of that were injected with NaHBED ranged from
iron. The efficiency of NaHBED given subcu- early, focally extensive fibroplasia and mild in-
taneously at a dose of 75 pmolkg was greater flammation in the superficial subcutis to pan-
than the same dose given as an intravenous niculitis, which was subacute and focally ex-
bolus, 14.2 ? 2.2% versus 11.5 + 1.3%,respec- tensive, and moderate to severe inflammation
tively (Table 10.5) (P< 0.05). When given as a in the deep subcutis. The descriptions of the
20-min intravenous infusion, an increase in skin from the sites injected with saline in-
the dose of NaHBED from 150 to 225 pmolkg cluded early fibroplasia, which ranged from
resulted in the excretion of more iron, but a diffuse, moderate, and superficial to focally
decline in efficiency (Table 10.5). extensive in the deep subcutis; one site pre-
sented with panniculitis. The drug was admin-
4.1.3 Preclinical Toxicity Trials. Acute tox- istered to the dogs subcutaneously at a con-
icity assessments of HBED using p a r e n t e d centration of 25% (w/v)and injection volumes
administration in mice indicated an LD,, in of up to 5.2 mLI10 kg for the 300 pmollkg
excess of 800 mg/kg; no drug-related effects doses. In addition, there did appear to be
were observed in mice given the drug intra- somewhat of a dose response, with the animals
peritoneally at doses up to 200 mg/kg for 10 in the higher volume groups having more local
weeks (233). At subcutaneous doses of up to irritation than those in the lower volume
300 pmollkg every other day for 14 days (7 groups. This finding of local irritation at the
doses), no toxicity was noted in rats given injection sites led to the use of a rodent model
NaHBED (6% w/v) (241). In addition, no ery- to determine the cause.
thema was noted at any of the injection sites, The results in dogs and preliminary exper-
either grossly or histologically. At necropsy, iments in rodents implied that the hypertonic-
neither macroscopic examination nor histo- ity of the 25% (w/v) solution used might be
logical evaluation of tissues revealed abnor- responsible for the local irritation observed.
malities in tissues that were attributable to Accordingly, groups of four rodents were given
the drug (241). a 100-pLsubcutaneous bolus of isotonic saline
Systemic toxicity trials of NaHBED have or NaHBED at varying concentrations in dis-
been carried out in dogs (243). Four beagles tilled H,O. Animals were administered the
were iron loaded to a level of 300 mg Felkg and same drug concentrations in the same volume
were subsequently given an intravenous dose as a 5-h subcutaneous infusion as well. A 300-g
of 75 pmolkg NaHBED in 50 mL isotonic sa- rat receiving 100 pL of the drug solution
line as a 20-min infusion once daily for 14 would be receiving a volume of drug solution
days. Two additional dogs, also iron loaded to a roughly comparable to administration of 20
:ent Developments

200 r

t 1 Time (rnin.)
t tTime 'mi")
Saliine i.v. bolus DFO i.v. bolus Saline i.v. bolus DFO i.v. bolus

Time (min.)

Saliine i.v. bolus HBED i.v. bolus


f
Saline i.v. bolus
t
HBED i.v. bolus

Figwe 10.22. Effect of intravenous bolus administration of DFO (a and b) and NaHBED (c and d)
(300 ~mol/kg)on the blood pressure (mmHg, a and c) and heart rate (beatslmin, b and d) of normo-
tensive rats (n= 5 for each chelator). a: P < 0.001 fort = 5.5 to 15 min; P < 0.005 fort = 25 min. b:
P <: 0.001 fort = 5.5 to 35 min. [From R. J. Bergeron, J. Wiegand, and G. M. Brittenham, Blood, 99,
301.9-3026 (2002). Copyright American Society of Hematology, used by permission.]

mL of 1;he drug solution to a 60-kgperson. The doses of DFO cannot be administered intrave-
histopi thol logical descriptors for both bolus nously for the treatment of acute iron poison-
and inf 'usion saline controls necropsied at 48 h ing; this can result in inadequate chelation
and 7 d ays postdosing included endothelial hy- and subsequent acute iron-induced cardiac
pertro~ ~ h y minimal
, inflammation, and scat- damage, a significant problem. Recent experi-
tered nnast cells in the subcutis. With the ex- ments suggest that intravenous infusion of
ceptionI of the rats treated with 20% NaHBED NaHBED for the treatment of acute iron poi-
as a su bcutaneous bolus, in which mild pan- soning will not be subject to these restrictions
niculitis was noted in one each of the rodents (243). When rodents were given 300 pmol/kg
necrop!3ied 48 h or 1 week postdosing, all of the DFO in a 0.5-mL volume as an intravenous
test anlimals presented with essentially the bolus, there was a 25% decrease in blood pres-
same histopathology as that of the control an- sure that did not return to baseline levels until
I imals. Therefore, it is possible to prevent 35 rnin postdrug (n= 5, P < 0.001 fort = 5.5 to
NaHBICD-related irritation by either giving 15 min and P < 0.005 for t = 25 min, Fig.
the drugas a slow subcutaneous infusion or as 10.22a). The heart rate in these animals also
a subciutaneous bolus at concentrations of increased by 16% and likewise did not return
515% 1N/V (243). to predrug levels until beyond 35 min postdos-
Bec~ use of the risk of hypotension, large ing (Fig. 10.22b). In contrast, there was no
522 Iron Chelators and Therapeutic Uses

effect on either blood pressure or heart rate in 5.1 Synthetic Approaches


rats given the same dose of NaHBED as an
intravenous bolus (Fig. 10.22c, d). However, 5.1 .I Catecholamides. Total syntheses of
when either chelator was administered at the the catecholamide chelators N1,Ns-bis(2,3-di-
same dose and volume as a 20-min intrave- hydroxybenzoy1)spermidine (compound 11, 3)
nous infusion, no effect on either blood pres- (247-256), L-parabactin (4) (257-260), L-ago-
sure or heart rate was recorded. Thus, bactin (5) (261), L-fluviabactin (6) (66), and
L-vibriobactin (8) (262) (Fig. 10.2) have been
NaHBED could be administered much more
rapidly and in greater amounts than could reported. During the assembly of these com-
DFO in the treatment of acute iron poisoning. pounds, care must be taken to avoid air oxida-
These results provide evidence for the tion of the 2,3-dihydroxybenzamide functions,
especially at higher pH values (263), and con-
safety and tolerability of NaHBED when ad-
tamination by ferric salts. Moreover, sid-
ministered either in the manner that would be
erophores (4-8) contain one or two acid-sen-
used chronically, for the treatment of iron sitive oxazoline rings, which must be formed
overload, or acutely, for the treatment of iron stereospecifically. The final synthetic chal-
poisoning. Comparative studies of iron excre- lenge is the regiospecific placement of the acyl
tion in the iron-loaded C. apella monkey have groups onto the triamine backbones.
shown that NaHBED is two to three times The triamine backbones of chelators (3-6)
more efficient as an iron chelator than DFO are symmetrically substituted; 2,3-dihydroxy-
after either subcutaneous or intravenous ad- benzoyl groups are on the terminal nitrogens.
ministration. Further, intravenous infusion of Iron-specific ligands (4-6) also possess an ox-
large doses of NaHBED for the treatment of azoline ring, which is derived from L-threo-
acute iron poisoning should not carry the nine and salicylic acid for L-parabactin (4) or
same risk of hypotension as does similar ad- 2,3-dihydroxybenzoicacid for L-agrobactin (5)
ministration of DFO. Overall, the need for and L-fluviabactin (6), on the internal poly-
prompt completion of the preclinical evalua- amine nitrogen. Reagents have been devel-
tion of p a r e n t e d NaHBED in preparation for oped that block the primary amines (264) or
studies of iron balance in human volunteers is secondary amine (265) of the triamines nor-
indicated. Thus, NaHBED may provide an al- spermidine, spermidine, and homospermi-
ternative to DFO for the treatment of both dine. Thus, the syntheses of L-parabactin (4)
chronic transfusional iron overload and of (257) and L-agrobactin (5) (261) began with
acute iron poisoning. attachment of 2,3-dihydroxybenzoic acid
equivalents to the unprotected amino groups
of N4-ben~~lspermidine. Alternatively, sper-
midine and norspermidine have been acylated
5 THINGS TO COME efficiently at the terminal nitrogens in the pro-
duction of compound I1 (3) (252-256) and L-
This final section focuses on approaches that fluviabactin (6) (66), respectively; a suffi-
are being used currently in the course of the ciently bulky acylating agent must be chosen
search for the ideal iron chelator for the treat- for such a high degree of selectivity.
ment of iron overload. We will (a) delineate In the norspermidine-based catechol-
synthetic methods that allow access to hydrox- amides L-vulnibactin (7)and L-vibriobactin (8)
amate, catecholamide, desferrithiocin-related, there is no such symmetry with respect to the
and citric acid-based chelators; (b) describe acyl groups. In the case of these natural prod-
animal models used for evaluating the efficacy ucts, it is clear that selective acylation of one
of the ligands as deferration agents in vivo; primary and one secondary nitrogen of nor-
and (c) review structure-activity studies with spermidine with the oxazoline unit is an un-
desferrithiocin and its analogs to illustrate reasonable ex~ectationin the absence of
how the design, synthesis, and testing of iron protecting groups. To access this more chal-
chelators are integrated to produce candidates lenging system, a more versatile triamine re-
for clinical evaluation. agent was developed with three different
5 Things to Come 523

..
0 0
(44) R = BOC

,=r
(45) R = H

J OH
H

N
N
-N
- NH

0 2HBr 0 \ EtO

Figme 10.23. Total synthesis of L-vibriobactin(8). (a) TFAA/NEt$CH,Cl, (92%); (b) 2,3-dime-
tho:xybenzoyl chloride/NEt$CH,Cl, (99%); (c) TFA, then H,/PdC1,/12 M HCl/CH,OH (93%); (d)
N-(itert-butoxycarbonyl)-~-threonineN-hydroxysuccinimide ester/DMF (99%);(e) TFA, then N+CO,
(93'%); (f) BBr3/CH2C12(63%); ( g ) CH30H/refluX/33h (58%).

x-oceccinggroups: tert-butoxycarbonyl, triflu- zylamine and acrylonitrile were transformed


roacetyl, and benzyl (266). These functional- by efficient steps into N4-benzyl-N1-(tert-bu-
ties are orthogonal, that is, each amino group toxycarbony1)norspermidine (40); protection
:an be unmasked separately (267), permit- of the remaining terminal amine with triflu-
;ing attachment of three different groups to oroacetic anhydride gave reagent (41) (266)
;he polyamine backbone in any order. Ben- (Fig. 10.23).
Iron Chelators and Therapeutic Uses

The synthesis of L-vibriobactin (8, Fig. 5.1.2 Hydroxamates. Numerous naturally


10.2), a hexacoordinate chelator isolated from occurring hydroxamate chelators and their
Vibrio cholerae (31), began by acylation of (40) analogs have been synthesized (87),including
with 2,3-dimethoxybenzoyl chloride, provid- arthrobactin (13)(270),aerobactin (14) (271),
ing trisubstituted norspermidine (42) in high nannochelin A (16) (272-2741, schizokinen
yield (262). The tert-butoxycarbonyl group of (19) (270), and mycobactin S, in which R is
(42) was removed by brief exposure to triflu- pentadecyl (15) (275) (Fig. 10.3). Of primary
4
oroacetic acid, and the N -benzyl group was importance to the synthesis of hydroxamate
cleaved by hydrogenolysis over palladium siderophores and their synthetic analogs is the
chloride in methanolic HC1, furnishing mono- availability of N-alkylated hydroxylamines,
acylated norspermidine dihydrochloride (43) which upon treatment with a stoichiometric
in 93% yield over two steps. After generating amount of an acylating agent form predomi-
the free diamine of (43) with aqueous base, nantly N-hydroxy amides (i.e., hydroxamic
acylation with the N-hydroxysuccinimide es- acids) (276). Primary amines have been con-
ter of L-N-(tert-butoxycarbony1)threonine(2.2 verted to the corresponding N-hydroxylamines
equivalents) in DMF gave intermediate (44) in (277-280) or N-(benzoy1oxy)hydroxylamines
99% yield. The BOC groups of (44) were (281) using methods that avoid further oxida-
cleanly removed by TFA, and N1,N4-bis[~- tion. In the latter compounds, acylation was di-
threonyll-N'-(2,3-dimethoxybenzoy1)norsper- rected exclusively onto nitrogen (282). These O-
midine (45) was obtained in 93% yield after protected hydroxamates could be deprotected by
neutralization of the salt. The 0-methyl pro- mild base, for example, generating the first syn-
tecting groups were cleaved using BBr,, af- thetic sample of nannochelin A (16),a dimethyl
fording N1,N4-bis[~-threonyll-N7-(2,3-dihy-ester (272). N-Substituted hydroxylamines also
droxybenzoy1)norspermidine dihydrobromide can be obtained from aldehydes through partial
(46) in 63% yield. reduction of their oximes (283,284).Saturation
As in the syntheses of L-parabactin (4) of an 0-protected oxime using borane-pyridine
(2571, L-agrobactin (5) (2611, and L-fluviabac- complex without cleaving the nitrogen-oxygen
tin (6) (66),the final step in the L-vibriobactin bond was a pivotal step in the most recent syn-
(8) synthesis required stereospecific forma- thesis of dihydroxamic acid (16) (274). Finally,
tion of the acid-sensitive oxazoline rings. although direct alkylation of hydroxylamine
Ethyl 2-hydroxybenzimidate, derived from leads to a mixture of products (2851, many N,O-
the treatment of 2-cyanophenol with HC1 (g) diprotected hydroxylamines have been alky-
in CH,CH,OH (268), was used to effect this lated efficiently at nitrogen (286). For example,
transformation to chelator (4). Unfortunately, this transformation was effected by treatment
2,3-dihydroxybenzonitrilewas unreactive to of N,O-bis(tert-butoxycarbony1)hydroxylamine
the same reagents, even under forcing condi- with alkyl bromides (287) or by direct coupling
tions; thus, imidate (47) for final products (5), of a series of bis(alkoxycarbony1)hydro~l-
(6), and (8) was accessed by another route amines with both primary and secondary alco-
(261). Reacting 2,3-bis(benzy1oxy)benzoylchlo- hols under Mitsunobu conditions (288),followed
ride (269) with concentrated NH40H/CH,C1, by removal of both protective groups.
gave 2,3-bis(benzy1oxy)benzamide (92% yield). The syntheses of desferrioxamine B (Des-
0-Alkylation of the amide with triethyloxonium feral is its methanesulfonate salt) (9)and des-
hexafluorophosphate in CH,Cl, provided the ferrioxamine E (nocardamine, 111, two of the
catechol-dibenzylated imidate ester in 81% siderophores isolated from Streptomyces pilo-
yield. Hydrogenolysis of the benzyls under mild sus (35, 37) (Fig. 10.3),are described here. To
conditions (10%Pd-C, CH,CH,OH, 1 atm) gave demonstrate the general utility of the syn-
crystalline ethyl 2,3-dihydroxybenzimidate(47) thetic methods employed, the preparation of
in 73%yield. Heating bis(vic-aminoalcohol) (46) two analogs of DFO is also presented. Hexaco-
with excess imino ether (47) in refluxing meth- ordinate ligands DFO B (linear) and DFO E
anol under a nitrogen atmosphere using iron- (macrocyclic)are predicated on two fundamen-
free glassware furnished L-vibriobactin (8). tal synthons: 1-amino-5-(N-hydr0xyamino)pen-
and succinic acid, and the key to the effi- amic acid siderophores and their analogs. Be-
of hydroxamate iron chelators cause N-hydroxydiamine containing three to
specific condensation of these five carbons is a fundamental, repeating unit
of many such chelators, a method of differen-
thesis of desferrioxamine B tiating its two nitrogens and protecting its
started with 1-amino-5-nitropentane (48) oxygen was sought. Thus, an N-hydroxy-1,5-
(Fig. 10.24)(289),which was in turn accessed diaminohydroxypentane (cadaverine) equiva-
in a two-step sequence from N-(5-bromopen- lent, 0-benzyl-N-(tert-butoxycarbony1)-N-(4-
ty1)phthalimidein 47%yield (290). Amine (48) cyanobuty1)hydroxylamine (62) was synthesized
was protected with a carbobenzyloxy (CBZ) (293). In this reagent the primary m i n e is
group, generating (49).The nitro group of (49) masked as a nitrile; the hydroxylamine is N-
was reduced with zinc to hydroxyarnino com- tert-butoxycarbonyl, O-benzyl diprotected
pound (50), which was N-acylated with either (Fig. 10.25). The synthesis of reagent (62) be-
succinic anhydride, providing (53), or with gan with the conversion of O-benzylhydroxy-
acetic anhydride, giving (54). The geometry of lamine hydrochloride to its stable, crystalline
carboxylic acid (53) allowed intramolecular N-(tert-butoxycarbonyl) derivative (61), em-
activation as the ester by ring closure to 0-acyl ploying di-tert-butyl dicarbonate in 97% yield
hydroxamate (55) in 84% yield. The CBZ pro- (294). Carbamate (61),which is now available
tecting group of (54) was removed by hydrog- commercially, was N-alkylated with 5-chlo-
enolysis. Primary m i n e (56) was condensed rovaleronitrile to give the versatile reagent
with (55), and the carbanate (57) was con- (62) in 87%yield. Homologs of the 1,5-diamin-
verted to terminal amine (58),which was acy- opentane reagent could be generated by N-al-
lated with active ester (551, giving (59). Cata- kylation of hydroxylamine equivalent (61)
lytic reduction of (59)furnished desfenioxamine with other w-haloalkanenitriles. Brief expo-
B (9) as its hydrochloride. sure of N-(tert-butoxycarbony1)nitrile (62) to
Recently, an alternative synthesis of trifluoroacetic acid resulted in 0-benzyl-N-(4-
N-(benzyloxycarbony1)-N'-hydroxy-1,5-dia- cyanobutyl)hydroxylamine (63) and volatile
minopentane (50) has been reported (291). Pi- by-products in 75% yield. Alternatively, the
peridine (51)was oxidized with calcium hypo- nitrile group of (62) was selectively hydroge-
chlorite to its imine, which was converted to nated over Raney nickel catalyst in methan-
the N-CBZ enamine. Ring opening of the car- olic ammonia, giving N-(5-aminopenty1)-0-
bamate and trapping of the aldehyde with hy- benzyl-N-(tert-butoxy-carbony1)hydroxyl-
droxylamine gave CBZ amino oxime (52), amine (64) in 83% yield. The hydroxylamine
which was reduced to N-hydroxyamine (50) oxygen in (62) could remain protected by the
using borane-pyridine complex. Thus, N-hy- benzyl moiety until catalytic unmasking of a
droxycadaverine equivalent (50) was accessed hydroxamic acid chelator (295).
from reagent (51) in greater than twice the 0-Benzyl-N-(4-cyanobutyl)hydrorrylamine
yield of the prior route and was carried (63) was alternately elaborated with succinic
through to Desferal in 24% overall yield (291). anhydride andgem-disubstituted diaminopen-
The use of protecting groups in Fig. 10.24 tane (64) to a DFO skeleton in which the pri-
was minimal; the CBZ on the terminal nitro- mary m i n e and the hydroxamic acids re-
gen was removed easily. However, attachment mained masked as the nitrile and 0-benzyl
Z
kI of the acetyl group early in the route restricts hydroxamates, respectively (296). Hydrogena-
the utility of the scheme. A second synthesis tion generated DFO B hydrochloride (9) in
gave DFO B in 45% overall yield, and the 44% overall yield from hydroxylamine reagent
acetyl group was also affixed near the begin- (61) (296). Cleavage of the benzyl groups oc-
ning of the synthetic sequence (292). This curred more rapidly than did saturation of the
route began with 4-cyanobutanal, which is un- cyano group (297).
stable and, like 1-amino-5-nitropentane (48), The most recent synthesis of DFO (9) is
difficult to access. both direct and versatile; construction of the
The effective use of protecting groups is linear molecule is streamlined by capitalizing
pivotal to the systematic synthesis of hydrox- on the electronic differences between a pri-
CBZ,
N -NHOH
H

..
0
(57) R = CBZ
(58) R = H

.. ..
0 0
(59) R = CBZ
(9)R=H HCI

Figure 10.24. Total synthesis of desferrioxamineB (9).(a) CBZ-CU3 N NaOH (86%);(b) Zn/NH,Cl
(aq)/EtOH (74%); (c) Ca(OCl)&TBE/AcOH (aq)/CH,OH; (d) CBZ-CVKOH/MTBE/CH,OH; (e)
NH,OH-HCI/CH,OH/pyridine (75%); (f) pyridine.BHflC1 (aq)/CH,OH (90%); (g) succinic anhy-
dride/pyridine/KOH (aq) (91%);(h) Ac,O/ pyridine (96%);(i) Ac20/100"C (84%);( j) H,/Pd-C/CH,OH
(quantitative); (k)THF (80%);(1) j; (m) (55)ITHF (66%);(n) H#d-C/CH,OH/O.l M HC1(90%).
5 Things to Come

OBn OBn

NC /\/\/ NH
OBn
I b
NC o N \ B
I
O c
- C
H2N
N
-
I
' BOC

Figure 10.25. Synthesis of the triprotected N-hydroxycadaverine reagent (62). (a) NaH/NaI/DMF/
80"C/4 h (87%);(b) TFA (75%);(c) H, (58 psi)/Raney nickelhonc NH,OH/methanolic NH, (83%).

mary amine and an N-(benzy1oxy)amine (Fig. addition to providing DFO (9) in three trans-
10.26) (297). This route also facilitates modi- formations, (70) is a versatile DFO reagent
fication of either terminus of chelator (9) by that allows replacement of the acetyl terminus
(a) protecting the m i n e with tert-butoxycar- of DFO by any acyl group.
bony1 (BOC), a group that is orthogonal to the Reaction of (70) with acetyl chloride pro-
0-benzyls; and (b) attachment of the acetyl duced tetraprotected DFO (71). The benzyl
function near the end of the sequence. groups of (71) were cleaved by hydrogen under
Successive exposure of N-(benzy1oxy)-N- mild conditions to give N-(tert-butoxycar-
(tert-butoxycarbony1)-1,5-pentanediamine bony1)DFO (72). Catalytic unmasking of the
(64) to TFA, aqueous base, and HC1 provided hydroxamate esters of (71) was accomplished
N-benzyloxy-1,5-pentanediaminedihydrochlo- rapidly and with no detectable nitrogen-oxy-
ride (65), also available by alkylation of O-
gen bond cleavage to amide by-product. Brief
benzylhydroxylamine with N-(5-bromopen-
exposure of N-(tert-butoxycarbony1)DFO(72)
tyl)phthalimide, followed by hydrazinolysis
(298).The free diamine of (65) was combined to TFA resulted in the formation of DFO (9) as
with 2-(tert-butoxycarbony1oxyimino)-2-phe- its trifluoroacetate salt in 45% overall yield
nylacetonitrile (BOC-ON, 1 equivalent), from known diamine (65). The high field pro-
cleanly providing N-(benzy1oxy)-N'-(tert-bu- ton NMR spectrum of the synthetic chelator
toxycarbony1)-1,5-pentanediamine(66). Both was identical to that of Desferal, except for the
steric (299) and electronic (300) factors ex- latter's methanesulfonate singlet.
plain the highly regioselective acylation of the Trihydroxamate reagent (70) facilitates al-
diamine. teration of DFO at either end of the molecule,
Reacting (benzy1oxy)amine (66) with suc- thus providing more flexibility in accessing
cinic anhydride in pyridine gave carboxylic DFO analogs than did prior routes. After cou-
acid (67). Treatment of acid (67) with 1,l'- pling the N-(benzyloxy)terminus with an acy-
carbonyldiimidazole (CDI) in CH,Cl, gener- lating agent and removal of the BOC protect-
ated the N-acyl imidazole, which was treated ing group, the primary m i n e could also be
with (65) as the free diamine, resulting in N- derivatized. Unblocking of the hydroxamates
(benzy1oxy)amine(68). As was the case in the would yield a panel of DFO analogs for deter-
protection of diamine (65) by BOC, acylation mining structure-activity relationships in the
occurred at the primary amine end of (65) search for a chelator with a longer clearance
with a high degree of selectivity (253,270,301, time and even oral bioavailability.
302). The last two steps were repeated; that is, The syntheses of the macrocyclic hydrox-
N-(benzy1oxy)amine (68) was converted to amate siderophores bisucaberin (12) (2931, al-
acid (69) with succinic anhydride. Next, regio- caligin (20) (301),and nocardamine (11)(303)
specific acylation of diamine (65) with acid (Fig. 10.3) have been accomplished begin-
(69) gave tris[N-(benzy1oxy)aminel (70). In ning with N-(tert-butoxycarbony1)-0-benzyl-
528 Iron Chelators and Therapeutic Uses

(64) - a,b
RHN
N
-
OBn
I
R
''
c
BOCHN -NLf I 0
(65)R=R'=H HCI OBn

/
(66)R=BOC R'=H (67)

BOCHN -/-NL./yi-
I
NHOBn

OBn 0
(68)

(70) R=BOC, R'=Bn, R = H


(71) R = BOC, R' = Bn, R = Ac
(72) R=BOC, R'=H, R = A c
(9) R = H TFA, R'=H, R = A c

Figure 10.26. Total synthesis of desferrioxamine B (9). (a) TFA/CH,Cl,, 1 N NaOH, conc HCU
CH3CH20H (82%); (b) 1 N NaOH/Et,O, BOC-ONITHF (quantitative); (c) succinic anhydridelpyri-
dine/80°C/90 min (88%); (d) 1,11-carbonyldiimidazole/(65) (free diamine)/CH,Cl, (quantitative); (e)
c/81°C/2 h (81%); (f) d (90%); (g) AcCl/EX,N/CH,Cl, (87%); (h) HJ10% Pd-C/CH,OH (81%); (i) TFA
(quantitative). [Reprinted with permission from R. J. Bergeron, J. S. McManis, 0.Phanstiel, N,and
J. R. T. Vinson, J. Org. Chem., 60, 109-114 (1995). Copyright 1995 American Chemical Society.]

hydroxylamine (61) (294). 0-BenzyLN44-cya- (74) in 78% yield (303). Subjecting (74) to hy-
nobuty1)hydroxylamine (63) was elaborated drogen over Raney nickel reduced only the ni-
with succinic anhydride and N-(5-aminopen- trile group, furnishing w-amino acid (75). Di-
ty1)-0-benzyl-N-(tert-butoxycarbony1)hydroxyl- phenylphosphoryl azide, the Yamada reagent,
arnine (64) to form DFO E intermediate (73) was added to acyclic precursor (75) (2.6 mM in
(Fig. 10.27) (293). DMF), and the reaction mixture was stirred
Addition of the third succinate unit of DFO for 4 days at 0% to produce O,Or,0-tribenzyl-
E to benzyloxyamine (73) gave w-cyano acid nocardamine (76) in 54% yield. The genera-
Figure 10.27. Total synthesis of desferrioxamine E (Nocardamhe, 11). (a) succinic anhydride1
pyridine/lOl°C/l10 min (78%);(b) H2 (60 psi)/Raney nickeVconc NH,OH/methanolic NH, (21%);(c)
(PhO),PON3/DMF/O"C/4days (54%);(d) H2/10%Pd-C/CH30H.

tion of a 33-membered macrocycle using rou- An efficient synthesis of the trihydroxa-


tine reaction conditions further illustrates the mate danoxamine has been achieved (304) be-
utility of this lactamization method (293,301). ginning with N-benzyloxy-1,5-pentanediamine
Catalytic removal of the 0-benzyl protecting dihydrochloride (65)(293,298). The structure
groups of (76) generated nocardamine (11) of the final product, which is the iron-chelat-
(303). ing component of the antibacterials Salmycins
Iron Chelators and Therapeutic Uses

A and B (305), matches that of desferrioxam- overloaded bile duct-cannulated rat model.
ine G (lo), except for an alcohol group in place When the chelators were administered subcu-
of the amine. taneously, analog (88) was nearly 3 times as
The short half-life of DFO in the body and effective as DFO in promoting iron clearance;
the fact that patients must be continuously (89)was 2.5 times as effective as (9) (296).
infused led investigators to prepare and test A hybrid hexacoodinate iron chelator that
analogs of DFO as potential therapeutic iron contains catecholamides and an N-hydroxy
chelators. Because replacement of the termi- amide moiety has been assembled. Specifi-
nal 5-aminopentyl unit of desferrioxamine B cally, the central nitrogen of compound I1 (3)
with a heptyl group rendered the molecule was elaborated to an N-methylhydroxamic
highly insoluble in a variety of vehicles (2921, acid using a succinic acid spacer, resulting in
chelators (88) and (89),which are polyether the analog spermexatol(308).
analogs of DFO, were prepared to enhance the
siderophore's overall solubility (Fig. 10.28). 5.1.3 Desferrithiocin and its Analogs. The
Specifically, in DFO polyether analog (88), the siderophore desferrithiocin [(S)-4,5-dihydro-
charged aminoalkyl chain was replaced with a 2-(3-hydroxy-2-pyridinyl)-4-methyl-4-thia-
neutral triether chain, and in bidtriether) zolecarboxylic acid, DFT, (24), Fig. 10.41 from
(89), the acetyl of DFO was substituted, as Streptomyces antibioticus (54) has been syn-
well, by a triether acyl group (296). thesized (309) by the cyclocondensation of D-a-
The monomethyl ether of triethylene glycol methyl cysteine (90) (Fig. 10.29) with 3-hy-
was converted to its tosylate (77) (306),which droxy-2-cyanopyridine (310). The unusual
was used to alkylate N-(tert-butoxycarbony1)- amino acid (90) is available from the hydroly-
0-benzylhydroxylamine (61), resulting in sis of DFT (6 M HC1/90"C/90 min) (54) or by
triether (78) (Fig. 10.28) (296). The nitrogen stereoselective a-methylation of a D-cysteine
of alkylated product (78) was deprotected with derivative (309). Because the DFT skeleton is
TFA, giving (79), which was acylated with suc- a useful pharmacophore on which to predicate
cinic anhydride to afford carboxylic acid (80). the design of orally effective iron chelators
Efficient coupling of (80)and N-(5-aminopen- (311), a large number of DFT analogs have
ty1)-0-benzyl-N-(tert-butoxycarbony1)-hydrox- been prepared. The structure-activity studies
ylamine (64) was effected with diphenylphos- were focused on identifying those fragments of
phoryl azide to make tetracoordinate equiva- DFT responsible for its oral iron-clearing
lent (81). The protected hexacoordinate re- properties in an effort to construct a less toxic
agent (84) was obtained from (81) by
analog (311-315).
repeating the three conversions: unmasking
The majority of these DFTs have been
to amine (82),four carbon elaboration to (83),
made by reaction of an aromatic nitrile with
and elongation to (84). Acidic cleavage of the
tert-butoxycarbonyl group in (84) furnished cysteine or its analogs. The production of (5')-
benzyloxyamine (85),which was acylated with desazadesferrithiocin (DADFT, 93) was ac-
acetic anhydride to produce (86) or with 3,6,9- complished by cyclocondensation of 2-cyano-
trioxadecanoyl chloride (307) to afford (87). phenol (91) with (S)-a-methyl cysteine (90)
Catalytic hydrogenation of the 0-benzyls of (Fig. 10.29) (315). The high field NMR spectra
(86) and (87) gave hexacoordinate ligands (88) of synthetic (93) and the siderophore 4-meth-
and (89), respectively, in good overall yield. ylaeruginoic acid, which has been isolated
Both of these neutral DFO analogs are soluble from the Streptomyces species KCTC 9303,
in chloroform and in water, and thus enhance were virtually identical; however, the C-4 ste-
the lipophilicity of the DFO molecule while reochemistry of the natural product was not
maintaining its hydrophilicity. specified (316). Therefore, a CD measurement
This change in solubility properties ren- of (93) was run; wavelengths of the maximum
dered both drugs more effective than desferri- and two minima of the CD of (93) matched
oxamine at removing iron in the non-iron- closely with literature values of the natural
(78) R = BOC OBn
(79) R = H
(80) R = CO(CH2)2C02H
id,e,f
OBn
I
H3CO~0/\/0-
I 0
OBn
(81) R = BOC

XH
(82) R = H
(83) R = CO(CH2)2C02H

H3C0-O~0- I igh N
/N
-B
a
) H
I3

I
OBn N
- OBn

0 0

XH
(84) R = BOC

H ~ C O ~ ~ / \ / O - I
OBn
0 (85) R = H

N
-
iiorj !qjlNr.MN
K
0

H
I
OBn
R

0 0
(86) R = CH3
(87) R = CH20(CH2)20(CH2)20CH3

Figure 10.28. Synthesis of desferrioxamine analogs with ether linkages (88and 89). (a) NaHDMFI
72"C/18 h (77%); (b) TFA/CH,Cl, (91%); (c) succinic anhydride/pyridine/9O"C/2h (92%); (d) (64)1
(PhO),PON&t,NDMF (94%); (e) b (87%);(f) c (95%);(g) d (78%); (h) b (quantitative); (i) Ac,O/
pyridine (91%);(j) 3,6,9-trioxadecanoyl chloride/Et,N/CH,Cl, (78%);(k)HJ10% Pd-C/CH30H(86%
for 88; 73% for 89). [Reprinted with permission from R. J. Bergeron, J. Wiegand, J. S. McManis, and
P. T. Perumal, J. Med. Chem., 34,3182-3187 (1991). Copyright 1991 American Chemical Society.]
Iron Chelators and Therapeutic Uses

Figure 10.29. Synthesis of (S)-4,5-dihydro-2-(2-hydroxyphenyl)-4-methyl-4-thiolecbolic acid


(93), (S)-4,5-dihydro-2-(2,4-dihydr0xyphenyl)-4-methyl-4-tolebolic acid (941, and (S)-4,5-
dihydro-2-(2-hydroxy-l-naphthalenyl)-4-thiazolbolic acid (96). (a) phosphate buffer (pH 6)/
CH30H/34"C/5days; (b) citric acid (32%);(c) phosphate buffer (pH 6)/NaHCO,/CH,OH/7l0C/3.5
days; (d) dilute HCI (57%);(e) ~-cysteine/CH~OH/reflux/2
days.

chelator (316). Thus, the first total synthesis overall yield of 42%, was heated with D-cys-
of the natural product 4-methylaeruginoic teine in methanol, generating naphthyl chela-
acid proves that, like DFT, it possesses the tor (96).
(5')-configuration.
The preparation of crystalline (S)-4,5- 5.1.4 Rhizoferrin. Rhizoferrin was first
dihydro-2-(2,4-dihydroxypheny1)-4-methyl-4- isolated from Rhizopus microsporus var. rhi-
thiazolecarboxylic acid (94), the 4'-hydroxy zopodiformis, an organism associated with
analog of DADFT, also began with amino acid mucormycosis seen in dialysis patients (50),
(go), which was condensed with 2,4-dihy- and occurs in several Zygomycetes strains of
droxybenzonitrile (92) under weakly acidic fungi (317). Structure determination of rhizo-
conditions (Fig. 10.29). ferrin (22) (Fig. 10.4) revealed a putrescine
For sterically crowded DFT systems, for ex- center with each nitrogen acylated by citric
ample, (S)-4,5-dihydro-2-(2-hydroxy-1-naph-acid at its 1-carboxylate (50). Thus, although
thaleny1)-4-thiazolecarboxylic acid (961, an rhizoferrin contains a polyamine backbone, it
imidate ester was required to generate the is neither a catecholamide nor a hydroxamic
thiazoline ring (Fig. 10.29) (313). Ethyl 2-hy- acid. Unlike the hydroxamates arthrobactin
droxy-1-naphthimidate (95), available in six (13) (39) and schizokinen (19) (45), in which
steps from 2-hydroxy-1-naphthaldehyde in an citric acid is symmetrically 1,3-disubstituted,
Things to Come

. COOH

.COOH

Figure 10.30. Total synthesis of rhizoferrin (22). (a) NaOH (aq)/CH,OH; dilute HCl (39%); (b)
(-)-brucine; fractional crystallization; HCl; (c) (PhO),PON,/triethylamine/DMF(26%);(d) a (77%);
(e) Li/NHJI'HF (64%).[Reprinted from R. J. Bergeron, M. G. Xin, R. E. Smith, M. Wollenweber, J. S.
McManis, C. Ludin, and K. Abboud, Tetrahedron, 53,427-434 (1997). Copyright 1996 with permis-
sion from Elsevier Science.]

the central carbon of each citric acid unit in generating the diamide (101) in 26% yield.
rhizoferrin is asymmetric. These two sites of The methyl esters of (101) were hydrolyzed
the molecule are in the (R)-configuration ac- using sodium hydroxide to give N,N'-dibenzyl
cording to CD spectroscopy compared with rhizoferrin (102) in 77% yield.
those of natural tartaric acid (318). The prin- Because N-benzyl amides are resistant to
cipal challenge of the synthesis of rhizoferrin hydrogenolysis (3221, dissolving metal reduc-
(22) was to access a citrate synthon of correct tion conditions were employed. Treatment of
configuration for coupling to both termini of tetraacid (102) with excess lithium metal in
putrescine to confirm the absolute configura- liquid ammonia and protonation of the salts
tion of the siderophore. on a cation exchange resin column furnished
The synthesis (319) of rhizoferrin began rhizoferrin (22). The high field NMR and high
with trimethyl citrate (97), which was con- resolution mass spectrum of the synthetic
verted to 1,2-dimethylcitrate (98) in 39%yield compound were essentially identical to the
by a sterically controlled saponification (320) published spectra of the natural product (50).
(Fig. 10.30). The enantiomers of carboxylic The absolute configurations of the synthetic
acid (98) were separated by forming their (-1- sample and the natural material were identi-
brucine salts and crystallization from water. cal (R,R),given that both exhibited a negative
Single-crystal X-ray diffraction of the solid re- Cotton effect at the same wavelength (318).
vealed 1,2-dimethylcitrate in the R-configura- Moreover, a siderophore was isolated from
tion. Acidification of the salt furnished (R)- Ralstonia pickettii DSM 6297 and character-
1,2-dimethylcitrate (99). ized as (8,s)-, thus, enantio-rhizoferrin, in
N1,N4-Dibenzyl-l,4-diaminobutane(100) that it exhibited a positive Cotton effect (323).
(321) was acylated with chiral acid (99) (2 The total synthesis of rhizoferrin (22) is the
equivalents) using diphenylphosphoryl azide, first example of the conversion of a chiral citric
lron Chelators and Therapeutic Uses

acid fragment, as confirmed by X-ray diffrac- rouloux, anisocytosis, and reticulocytosis;


tion and high field NMR analyses, to a chela- splenomegaly; and increased intestinal iron
tor. absorption (325, 328, 329). One gene-target-
ing approach created an accurate murine ho-
5.2 Animal Models Employed in lron molog of the most severe form of a-thalasse-
Metabolism and lron Chelator Studies mia, Bart's hydrops fetalis, in which all four
copies of the a-globin gene are inactivated; the
To better define both the impact of defective
iron metabolism and the effect of potential condition could, at least in part, be rescued by
therapeutic iron chelators in uiuo, several an- addition of two copies of a normal a-globin
imal models have been developed. Modern mo- gene (330). Another transgenic mouse model
lecular biology has accelerated the develop- of this disorder was generated by way of an
ment and the molecular characterization of insertional mutation into either the 6 or the a1
mutant animals that carry a particular genetic gene locus (329). Studies in this model re-
defect in iron transport; these animals are ex- vealed that (a) inactivation of the embryonic
cellent model systems for studying the impact 6-globin gene effectively downregulated the a1
of misdirected iron transport and utilization gene locus, consequently inducing a-thalasse-
in mammals and may, in the future, be useful mia as effectively as did direct mutagenesis of
for assessing therapeutic ligands. Although the a1 gene; and (b) inactivation of the 6-glo-
the early animal experiments with DFO often bin gene did not yield a lethal phenotype
involved rabbits (156) or dogs (147),the meth- universally.
ods developed and used since the mid-1980s Further work in this model system indi-
for the measurement of the efficacy and toxic- cated that different genetic backgrounds, par-
ity of candidates for chelation therapy employ ticularly variants in the p-globin gene, appear
relatively common wild-type rodents or pri- to modify the thalassemia phenotype (331).
mates. However, no such compensatory mechanism
appears to exist in the murine models of
5.2.1 Genetically Characterized Rodents. p-thalassemia that have been studied to date
The genetic alterations of iron transport have (327,332-335). The phenotype of the mice car-
been obtained either by breeding/spontaneous rying a deletion of the b l gene (Hbbth-'/
mutation [e.g., Belgrade rat (324), a-thalasse- Hbbth-I)was relatively mild, given that these
mic mouse (325)l or by direct genetic manipu- mice survived to adulthood and were fertile
lation to generate mice with a defined muta- (327). The abnormalities included hemolytic
tion [e.g., transferrin receptor 1-deficient anemia, microcytosis, anisocytosis, spleno-
mouse (3261, Irp2 knockout mouse (128)l. megaly, and disproportionate levels of a-glo-
Clearly, future studies using many of these bin (327). Surprisingly, in the presence of in-
models will be instrumental in determining creased iron absorption, no increase in cardiac
the interrelationships of the components of iron was noted, although iron deposition in
mammalian iron transport systems; however, the spleen, liver, and kidney was prominent
a detailed description of each of these is out- (332).The observed hematological parameters
side the scope of this chapter. Therefore, the more closely resemble those of human
focus of this segment will be on models of the p-thalassemics who have undergone splenec-
hemoglobinopathies. tomy, rather than those of unsplenectomized
Although spontaneous mutant a-thalasse- thalassemics (327). In stark contrast, either
mic (325) and p-thalassemic (327) mice were disruption of the b l locus (Hbbth-2/Hbbth-')
studied in the mid-1980s, the development of (333) or deletion of the b l and b2 loci (Hbbth-3/
transgenic mice has been a key advance in Hbbth-3) (334, 335) yielded homozygotes,
probing the hemoglobinopathies on a molecu- which died either in utero or within hours of
lar level. The a-thalassemic mice exhibit, sim- birth. Hbbth-3heterozygotes in one of the dou-
ilar to the human disorder, defective a-chain ble-deletion models demonstrated a patho-
synthesis, mild hemolytic anemia, including physiology comparable to that of patients with
microcytosis, poikilocytosis with extensive p-thalassemia intermedia (335), whereas
5 Things to Come 535

Table 10.6 Iron-Overloaded Rodent Models Employed in Efficacy Studies of Iron Chelators
Route'of Iron Selected
Species Administrationa Iron Composition References
Rattmouse i.p. Iron dextran
Rat i.p.1i.v. Iron polymaltose
Mouse i.p.1i.v. Iron dextran + 59Felactoferrin
Ratlmouse p.0. Ferrocene
Rat/mouse i.v. Hypertransfusion with or
without radiolabeling
Ratlmouse p.0. Carbonyl iron
Rathamster i.v. 59Feferritin
Guinea pig p.0. Carbonyl iron
Guinea pig i.p. Iron dextran
Gerbil S.C. Iron dextran
?.p., intraperitoneal; i.v., intravenous; p.o., per os (oral); s.c., subcutaneous.

those in another presented a more severe phe- assess the potential efficacy of candidate iron
notype than that seen in human p-thalasse- chelators. The rodents used, among others,
mia heterozygotes (334). have included mice, rats, guinea pigs, harn-
Perhaps the most extensive studies of a sters, and gerbils. Animals with normal iron
murine model of a hemoglobinopathy have stores as well as iron-overloaded animals (Ta-
been in any one of several models of sickle-cell ble 10.6) have been used.
anemia (336-343), the treatment of which en- 5.2.2.1 Non-Iron Overloaded Bile Duct-
tails transfusion therapy at an increasing rate Cannulated Rat. The non-iron overloaded bile
(118,344).The later-generation models mimic duct-cannulated rat model (240,311,312,314,
the molecular events that occur in the course 346) has been used as a primary screen for
of development much more closely; the switch candidate iron chelators before their testing in
from fetal hemoglobin to adult hemoglobin an iron-loaded Cebus apella monkey model
production occurs in addition to the sickling (98,235,236,242,347). Briefly, male Sprague-
red cell phenotype and its attendant morbidity Dawley rats averaging 400 g were anesthe-
(338, 340, 341). However, striking progress tized using sodium pentobarbital given
has been made in earlier models (339, 342, intraperitoneally (i.p.). The bile d u d was can-
343); both an experimental drug treatment nulated using 22-gauge polyethylene tubing,
(345) and a genetic rescue protocol (337) have about 1 cm from the duodenum. The rats were
been attempted in this model with a measure housed in plastic metabolic cages during the
of success. Clearly, mice carrying a defined he- experimental period and were given free ac-
moglobinopathy will be useful in probing the cess to water. Bile samples were collected at
molecular basis of these disorders as well as in 3-h intervals, and urine samples were taken
the evaluation of therapeutic agents, includ- every 24 h.
ing iron chelators (332). The iron-clearing efficiency (Ec,,; see dis-
cussion in Section 2) is calculated by subtract-
5.2.2 Wild-type Rodents. Although rodents ing the iron excretion of control animals from
represent a viable first-line animal screen, the iron excretion of the treated animals. This
~ ~

providing a rapid way to identify and discard number is then divided by the theoretical out-
chelators that are ineffective in vivo, there is put to obtain the efficiency. Although iron
no strict correspondence between the effec- clearance may be overestimated due to a lack
tiveness of a chelator in rodents and that in of enterohepatic recirculation, nevertheless,
primates. Nevertheless, rodents often provide the model allows for the rapid screening of
a "go or no-go" answer for further develop- new chelators and identifies compounds to
ment of a potential chelator. Over the years, a take forward to the iron loaded Cebus primate
number of rodent models have been used to model.
Iron Chelators and Therapeutic Uses

5.2.2.2 Iron-Overloaded Rodent Models. trast, 100% of the radioactivity associated


Rodents have been iron overloaded by a num- with the administration of the 59Fe-DRBCwas
ber of different methods (183, 348-380) to in the RE cells and 0% in the hepatic paren-
provide an animal model for the study of po- chymal cells. Once radiolabeling is completed,
tential iron chelators. Some of these methods candidate chekators are administered; the re-
are included in Table 10.6. Although a thor- duction of RE and parenchymal radioactivity
ough description of all of these models is be- is compared to that of control animals.
yond the scope of this chapter, some of the 5.2.2.2.3 Gerbils. The Mongolian gerbil
models used more commonly are discussed in treated with subcutaneous iron dextran has
greater detail. been develo~edas the first rodent model of
A

5.2.2.2: I Mice. Traditionally, mice have iron overload that seems to reproduce both the
been used to assess the acute and chronic tox- cardiac and hepatic toxicity found in chronic
icity (LD,,) of new drugs. In addition to this iron overload in humans (377-380). Gerbils
role, investigators have utilized mice exten- treated with weekly doses of subcutaneous
sively to assess candidate iron chelators. In iron dextran develop concentrations of hepatic
many laboratories (352-357), the animals iron that are in the range of those found in
have been iron overloaded with the intraperi- patients with transfusional iron overload and
toneal injection of iron dextran, 2 mg of iron present with hepatic fibrosis within 1-3
per week for 4 weeks. After a 2-week equilibra- months after stopping the iron dextran injec-
tion period, the mice were given 59Felactofer- tions (379). In addition, cardiotoxic effects of
rin intravenously through the tail vein. This iron in the gerbil similar to those of patients
regimen delivers iron preferentially to the with iron-induced cardiomyopathy have also
liver. The 59Feinjection was allowed to stabi- been demonstrated between 2 and 3 months
lize for an additional 2-3 weeks. Then, mice post iron dextran. The pathology of the af-
were given the drug of interest either orally or fected tissues is similar to that which occurs in
parenterally; the urine and feces were col- end-stage disease in humans.
lected and assaved" for ,'Fe excretion. Com-
pounds that showed an increase in 59Feexcre- 5.2.3 Primate Models. Although the rat is
tion relative to untreated controls could then useful as a primary screen of efficacy, activity
undergo further testing in additional animal of a chelator in this species is poorly predictive
models. of that in humans. Consequently, two second-
5.2.2.2.2 Rats, Selective Radioiron Probes. A ary primate models have been developed: the
number of methods have been used to over- iron-overloaded C. apella monkey model and
load rats for the testing of iron chelators (Ta- the iron-overloaded marmoset (Callithrixjac-
ble 10.6). One such method involves using se- chus) model.
lective radioiron probes to label the two major 5.2.3.1 Cebus apella. The approach for set-
storage pools available for chelation. This dis- ting up a nonhuman primate model was based
tinction between the storage compartments, on a collaboration in the late 1980s. However,
the hepatic parenchyma or the RE system, is this initial work suggested problems with
important. Whereas parenchymal siderosis is background noise; as a result, iron-clearance
responsible for serious organ dysfunction, levels were far in excess of theoretical possibil-
iron in RE cells is relatively innocuous. ity (381). It was obvious that the system
In this model (183, 362-366), rats are hy- needed substantial refinement. Drawing on
pertransfused by injections of packed red the experience gained from this work, some
blood cells. Heat-damaged erythrocytes (59Fe- decisive improvements were implemented (98,
DRBC) and ,'Fe-ferritin are prepared and ad- 235,236,242,347): the use of larger groups of
ministered to the rats intravenously. Greater both normal and iron-loaded animals; the de-
than 80% of soluble ferritin and heat-damaged velopment of improved metabolic cages and
RBCs are located in the liver and spleen within handling procedures for the experimental an-
1 h of intravenous injection. In the case of the imals; the validation of all procedures for the
59Fe-ferritin,97-100% is located in the paren- trace metal analysis of samples of a controlled
chymal cells and 0-3% in the RE cells. In con- low iron diet, urine, and feces; the perfor-
5 Things to Come 537

mance of iron balance studies; and a complete monitored. Three days before drug adminis-
set of clinical analyses before and after the tration, days -2 to 0, baseline iron intake and
administration of each drug sample. output values were measured. This same mea-
5.2.3.l . 1 C. apella Iron Loading. After in- surement was made for days + l through +3.
trarnuscular anesthesia with ketamine, an in- The total amount of iron intake was compared
travenous infusion was started in a leg vein. with the total iron output. Net iron balance =
Iron dextran was added to sterile normal sa- dietary iron intake - (urinary iron + fecal
1 line and administered to the animals at a dose iron). -Animals in a negative iron balance are
of 200-300 mg/kg. The iron solution was in- excreting more iron than they are absorbing.
fused over 45-60 min. Two to three infusions, 5.2.3.1.4 C. apella Hematological Screen.
separated by 10-14 days, were necessary to Blood samples were taken from the monkeys
load the monkeys to a level of 500 mglkg of before their being transferred to the metabolic
iron. This brought the serum transferrin iron cages, and extensive parameters were evalu-
saturation to 70-80%. The serum half-life of ated (346). The blood samples were always
iron dextran in humans is 2.5-3 days (382). drawn at the same time of day because of the
Twenty half-lives, 60 days, elapsed before any diurnal variability in some of the measure-
of the animals were used in iron-clearing ments (e.g., UIBC, plasma iron). The assays
experiments. performed permit the assessment of the
The iron loading produced by the infusion health of the animals going into the experi-
of iron dextran in the C. apella monkey at this ment and can identify subtle changes that a
time resembles that found in patients with test drug may induce in an animal. A postdrug
transfusional iron overload and supports the blood sample was taken from each animal on
suitability of this model for the evaluation of the last day of the experiment.
the efficacy of candidate iron-chelating agents 5.2.3.1.5 C. apella Fecal and Urine Sample
(R. J. Bergeron, G . M. Brittenham, H. Fujioka, Collection and Analysis; Efficiency Calcula-
W. R. Weimar, and J. Wiegand, unpublished tions. Fecal and urine samples were collected
observations). The hepatic iron concentration at 24-h intervals and assayed (346). Iron con-
was about 10-fold higher than that in the con- centrations were measured by flame atomic
trol animal and was well above the threshold absorption. The efficiency of each ligand was
for cardiac disease and early " death found in calculated as described earlier.
studies of patients with thalassemia major and The iron-overloaded C. apella monkey
transfusional iron overload (117, 383). Light model has been employed successfully for over
microscopy of liver samples using Prussian a decade. The ability of the primate model to
blue stain revealed that the iron load was ex- predict how a drug will perform in humans is
tensive and large siderosomes were clear; elec- illustrated by a comparison of data from stud-
ton micrographs demonstrated prominent ies of (a) DFO (9) administered subcutane-
iron deposition both in hepatocytes and in ously, (b) the bidentate L1 (33) given orally,
Kupffer cells. This pattern mirrors that found and (c) the hexadentate chelator HBED (39)
in chronically transfused patients. given orally or subcutaneously, in rodents, pri-
5.2.3.1.2 C. apella Metabolic Cages. Dur- mates, and humans, as shown in Fig. 10.31.
ing the evaluation of various iron chelators, The ligands were administered at the same
the animals were moved from normal -primate iron-binding equivalence. Experiments in the
cages to specially constructed, metal-free met- rodents and the monkeys were carried out at
abolic cages (347).The animals were housed in doses of 150 pmol/kg for DFO and HBED and
these cages for 7 days before exposure to the at 450 pmol/kg for L1. The data for human
chelator of interest and throughout the course studies of DFO, orally administered HBED,
of the experiment. and L1 were derived from earlier clinical trials
5.2.3.1.3 C. apella iron-Balance Studies. (182,210). Note that the human data for DFO
Animals were maintained on a low iron liquid are not comparable to those for the monkey or
diet (346) for 7 days before drug administra- the rat in the strictest sense because the che-
tion. The animals were given food according to lator was administered at a dose of 92 pmollkg
their body weight. Intake was very carefully as a subcutaneous infusion over 8 h. This
lron Chelators and Therapeutic Uses

T T 13Urine

Rat Cebus Human

Figure 10.31. Chelator-induced iron excretion in rats, monkeys, and humans. In the animals, the
ligands were administered at the same iron binding equivalence. Experiments in the rodents and the
monkeys were carried out at doses of 150 pmol/kg for DFO and HBED (both oral and subcutaneous
administration) and at 450 pmol/kg for L1.

mode of administration is likely the reason for chelator will perform in humans and suggests
the apparently greater efficiency of DFO in the that subcutaneous HBED should be 2-3 times
human study. Patients were administered as efficient as DFO (240,241) in the patients.
HBED orally at doses of 103 or 206 pmolkg, 5.2.3.2 Marmosets (Callithrix jacchus).
although the iron outputs at either dose were Since the development of the C. apella model,
within experimental error of each other and a similar methodology has been adopted by
are plotted accordingly in Fig. 10.31. Patients another chelator program (384-386), substi-
received L1 at a daily dose of approximately tuting marmosets (Callithrix jacchus) for the
540 pmolkg. C. apella monkey. The overall experimental
As shown in Fig. 10.31, data from the stud- strategy with the marmoset is similar to that
ies in rats would have predicted HBED (given used with the Cebus monkeys.
orally or subcutaneously) to be far superior to 5.2.3.2.7 Marmoset lron Loading. The mar-
either DFO or L1 in humans. By contrast, the mosets were iron overloaded by the intraperi-
results from the studies in primates suggested toned injection of iron(II1) hydroxide polyiso-
that both orally administered HBED and L1 maltose (386). The iron was injected at 14-day
should not have performed as well as DFO. intervals. The iron pools were allowed to equil-
The relative efficiencies of DFO, orally admin- ibrate for approximately 60 days.
istered HBED, and L1 in the C. apella model 5.2.3.2.2 Marmoset Metabolic Cages. The
were almost identical with those seen in hu- animals were placed in specially designed
mans, even to the mode of iron excretion (bil- acrylic glass metabolic cages 48 h before drug
iary versus urinary). Therefore, the C. apella administration and for 48 h thereafter (386).
primate is an excellent predictive tool of how a The cages were constructed such that urine
Things

E' 2500
CO
- Marmoset Cebus
%, 2000 -
Y

-
;?, 1500 - Urine
-= 1000 -

o n
.-0 Faeces
500 -
v
IL"
0
150 300 450 300 150 300 450 300
S.C. S.C. S.C. p.0. S.C. S.C. S.C. p.0.
Dose [prnol/kg]and route
Figure 10.32. Dose-dependent urinary and fecal iron excretion in iron-overloaded marmosets and
Cebus monkeys induced by DFO; each bar represents the mean of four to six animals. [Reprinted with
permission from T. Sergejew, P. Forgiarini, and H.-P. Schnebli, Br. J. Haematol., 110, 985-992
(2001). Copyright 2001 Blackwell Science Ltd.]

and feces could be collected separately. Be- iron-clearing efficiency when iron was still be-
cause of animal welfare concerns, the animals ing excreted beyond the allowed Sday post-
were not allowed to be in the metabolic cages dosing collection period.
for longer than 48 h postdrug (385). When iron-clearance data from the two pri-
5.2.3.2.3 Marmoset Fecal and Urine Samples. mate models are compared, the results are
The animals were switched to a low iron diet fairly similar (386). DFO and HBED are active
(386) 7 days before the administration of the upon subcutaneous administration to both
test drug and for 48 h thereafter. Urine and species and ineffective when administered
feces samples were collected at 24-h intervals orally, the same situation that is seen in hu-
for 2 days before drug administration and 2 mans. In addition, when looking at three hy-
days postdrug (385). Urinary iron excretion droxypyridinones, L1 (33), CP94, and CP102,
was determined colorimetrically using a both models predict that L1 would have a low
bathophenanthroline method. Fecal samples activity, which is also the case in patients, and
were weighed and tested for occult blood. Iron that the order of effectiveness is CP102 >
content was determined by flame atomic ab- CP94 > L1 (386). However, there are also
sorption spectrometry after wet-ashing each some instances where the two models are
24-h sample. quite different.
5.2.3.3 Comparison of the C. apella and Although DFO is active in both animal
Marmoset (C. jacchus) Models. The marmoset models when administered subcutaneously,
model has been used extensively as a means to the mode of iron excretion is different (Fig.
identifjr active iron chelators. The major ad- 10.32). In the marmoset, the majority of the
vantage of the marmoset model over the estab- induced iron is excreted in the feces, which is
lished C. apella model is the animals' small also the case when this drug is given subcuta-
size, 400 g versus 4 kg for the C. apella. This neously to rats. In C. apella, as in humans,
smaller size makes it possible to dose the ani- approximately 50% of the induced iron is ex-
mals without the need for anesthesia. In addi- creted in the urine and the remainder in the
tion, the amount of drug needed is no more feces. In addition, DFO is much less effective
than that needed for a rat. A major disadvan- at lower doses in the marmoset than in the
tage of the data derived in this protocol is that Cebus (Fig. 10.32). This may be as a result of
the marmosets were kept in the metabolic the more rapid metabolism of DFO in marmo-
cages for only 2 days before and 2 days after set plasma than in C. apella.
drug administration (385). This shorter dura- When the stability of DFO was assessed in
tion of sample collection made it difficult to the plasma of several different species, the
determine both baseline iron excretion and drug was found to be rapidly metabolized in
Iron Chelators and Therapeutic Uses

Figure 10.33. The stability of des-


ferrioxamine in plasma from differ-
ent species over 3 h. All incubations
were performed on pooled plasma
samples from two or more animals
and averages were taken from dupli-
cate analyses. [Reprinted with per-
mission from A. Steward, I. Williarn-
son, T. Madigan, A. Bretnd, and
I. F. Hassan, Br. J. Haematol., 95,
654-659 (1996). Copyright 1996
Blackwell Science Ltd.] Time (h)

the plasma of the rat, mouse, rabbit, and mar- 5.3 Integration of Design, Synthesis, and
moset (374). In fact, the rat and the marmoset Testing: Desferrithiocin Analogs
had no detectable drug remaining after only In this summary of several studies of the
1h (Fig. 10.33). However, the hamster, guinea structure-activity relationships (SARs) of the
pig, dog, and C. apella plasma stability results DFTs, the primary measure of activity is the
were similar to what is seen in humans, that efficiency of the ligand, as assessed in both
is, >SO% of the parent drug remained after rodent and primate models and compared
incubating at 37°C for 3 h (374). Finally, al- with subcutaneously administered DFO. In
though dimethyl N,Nf-bis(2-hydroxybenzyl)- the case of the DFT analogs, the efficiency cal-
ethylenediamine-N,N1-diacetate(dmHBED) culation is based on the formation of a 2:l
is very active when given orally or subcutane- complex with a formation constant assumed to
ously to the marmosets, this compound has be similar to that of the parent compound, 4 X
low activity when administered to C. apella lo2' M - I (56). Figures 10.35-10.42 were con-
(386) (Fig. 10.34) and to humans. This lack of structed to demonstrate how alterations in the
activity in the C. apella model is attributed to structure of DFT changes its iron-clearing ef-
the inability of C. apella to hydrolyze the di- ficiency (Fig. 10.35, Figs. 10.37-10.41) or tox-
ester to the active compound. icity (Figs. 10.36 and 10.42). Figures 10.35 and
To date, the iron-loaded C. apella monkeys 10.37-10.41 also include the (primate dose),
were able to qualitatively predict the effective- (mode of administration), and the fraction of
ness of the chelators in the human clinical iron excreted in the [bile or stool and urine].
studies in every case. The primates' responses From the perspective of simplifying the
were similar to the human data with regard to SAR studies (Fig. 10.35), the two most impor-
both the mode of iron excretion and to a com- tant manipulations on the parent ligand (24)
pound's efficacy, thus rendering the C. apella were removal of the aromatic nitrogen to yield
monkey as an excellent secondary screen for (S)-4,5-dihydro-2-(2-hydroxyphenyl)-4-meth-
evaluating iron chelators before initiating yl-4-thiazolecarboxylic acid [(SbdesazaDFT,
costly human clinical trials. (93)l and removal of the thiazoline methyl
5 Things to Come

DFO s.c. L1 CP94 CP102

Figure 10.34. Comparison of


total iron excretion in iron-
overloaded marmosets (black
columns) and Cebus monkeys
(white columns). All iron chela-
tors indicated were applied at
150 pmol iron-binding equiva-
lentskg; each bar represents
the mean of three to six ani-
mals. [Reprinted with permis-
sion from T. Sergejew, P. For-
giarini, and H.-P. Schnebli,
Br. J. Haematol., 110, 985-992
(2001). Copyright 2001 Black-
HBED HBED S.C. drnHBED drnHBEB S.C. well Science Ltd.]

to produce (S)-4,5-dihydro-2-(3-hydroxy-2- The effects of these small structural alter-


pyridiny1)-4-thiazolecarboxylic acid [(S)-des- ations on the toxicity profile of the parent com-
methylDFT, (10311. Both compounds per- pound (24) were truly remarkable (Fig.
formed well in the primate model when given 10.36). The natural product, (24), was a very
orally, better than DFO administered subcu- effective iron chelator when given orally to
taneously at the same iron-binding equiva- rats or monkeys. However, the compound also
lence (235, 315). Surprisingly, (93) was an elicited significant nephrotoxicity when ad-
exceptional deferrating agent when adminis- ministered chronically to rats. At a dose of 384
tered subcutaneously (Fig. 10.35). This was pmoVkg/day, five animals were dead by day 5
consistent with the idea that neither the aro- (Fig. 10.36). Abstraction of the methyl group
matic nitrogen nor the thiazoline methyl was from the thiazoline ring of (24) to yield (103)
requisite for iron clearance. The next modifi- resulted in a compound that was far less toxic
cation, further underscoring the minor role than (24) (235).When administered orally at a
played by the aromatic nitrogen and the thia- dose of 384 pmol/kg/day for 10 days, (103) was
zoline methyl, was formally removing the well tolerated, no deaths were observed, and
thiazoline methyl from (93) or the aromatic extensive histological evaluation revealed no
nitrogen from (103) to produce (S)-4,5-dihy- drug-related abnormalities.
dro-2-(2-hydroxypheny1)-4-thiazolecarboxylic Continuing with the toxicity SAR, it was
acid [(S)-desazadesmethylDFT, (104)l. This found that removal of the aromatic nitrogen
tridentate ligand was very active when admin- from (24) to produce (93) resulted in an analog
istered orally to primates (Fig. 10.35) (235). that induced severe gastrointestinal (GI) tox-
542 Iron Chelators and Therapeutic Uses

(93) (24)
Rat: 2.7 f 0.5 (PO) Rat: 5.5 f3.2 (PO) Rat: 2.4 f 0.6 (po)
[loo bile, 0 urine] [93 bile, 7 urine] [82 bile, 18 urine]
3.2 f1.8 (sc) Monkey (150 pmollkg): Monkey (150 pmollkg):
[98 bile, 2 urine] 16.1 f8.5 (PO) 4.8 k 2.7 (PO)
Monkey (75 pmollkg): [78 stool, 22 urine] [48 stool, 52 urine]
*
21.5 12 (PO) (300 pmollkg):
[76 stool, 24 urinel 8.0 + 2.5 (PO)
(300 pmollkg): 142 stool, 58 urine]
13.1 f 4.0 (PO)
[86 stool, 14 urinel
(300 pmollkg):
43.3 k 8.8 (sc)
[96 stool, 4 urine] .Rat:1.4 f 0.6 (po)
[I 00 bile, 0 urine]
Monkey (300 pmollkg):
*
12.4 7.6 (PO) v
Thiazoline ring
[go stool, 10 urine]
modifications,
series I I A
Addition of

Addition of
electron-
donating
group, series
VB
Alteration of
distance between
chelating centers,
series I I
Thiazoline ring
modifications,
series I I B
electron-donating &
-withdrawing groups,
series V A
Fusion of aromatic
rings, series IV

C-4 configuration,
series Ill

Figure 10.35. SARs of the DFTs and iron clearance. The dose of DFT or analog in the rats is 150
pmoVkg; the dose in the monkeys is as shown in parentheses for each ligand. The mode of adminis-
tration is shown in parentheses next to the efficiency (%, f SD). The fraction of iron excreted in the
bile or stool and urine is shown in brackets. Series I-V can be found in sections 5.3.1.1-5.3.1.5 (235,

icity in rodents (315). The same was true with the animals (314). Thus, although removing
removal of both the aromatic nitrogen and the the aromatic nitrogen and thiazoline methyl
thiazoline methyl group to generate (104). from the DFT framework (104) may be com-
When (104) was administered at a daily dose patible with iron clearance, the profile of the
of 384 pmolkg, dosing of this ligand was system completely shifts from renal to GI tox-
stopped after the fifth dose because of the rap- icity.
idly deteriorating condition of the animals. All Because of its structural simplicity the tri-
of the animals were dead by day 6. At nec- dentate ligand (104) lent itself to further SAR
ropsy, the stomachs of all of the animals were analyses. This framework contains neither
hemorrhagic and grossly distended with gas the picolinic acid fragment nor the unusual
and fluid; the stomach walls were translucent. amino acid (S)-@-methylcysteine (90). Conse-
The intestines were also hemorrhagic, and quently, these systems are much easier to as-
pressure necrosis of the spleen, from the semble than (24), (93), or (103). Five types of
grossly distended stomach, was noted in two of structural modifications were performed on
OH OH

C02H

(93) (103)
Rats: (po) All animals Rats: All animals dead Rats: 10-day:
dead by day 5: severe by day 5: severe well-tolerated; all
GI toxicity. nephrotoxicity. histopathologies
(sc) All animals normal.
dead by day 5: severe GI 30-day: well-tolerated; all
toxicity. histopathologies normal.

(104)
Rats: All animals dead
by day 6: severe GI
toxicity.

Figure 10.36. Structure-activity relationship of the DFTs and toxicity. Unless otherwise indicated
in parentheses, the ligands were administered orally at a dose of 384 pmol/kg/day, equivalent to 100
mg/kg/day of the sodium salt of DFT (235,314,315).
Iron Chelators and Therapeutic Uses

( 3 7 3 - C 0 2 H

(105) (106) (107)


Rat: 10.5 (po) Rat: s 0.5 (po) Rat:10.5 (po)
5 0.5 (sc)

Figure 10.37. Series I: Alteration of distances between chelating centers. The efficiency and mode
of administration (in parentheses) are shown.

(104), and the effect of these changes on che- at C-4 (Section 5.3.1.3, compounds 115-117
lator-induced iron excretion andlor toxicity versus 103, 104, and 118, Fig. 10.391, benz-
was evaluated. These included alterations in fusion of the aromatic rings (Section 5.3.1.4,
the distances between chelating (donor) cen- compounds 96 and 119-123, Fig. 10.40), and
ters (Section 5.3.1.1, compounds 105-107, addition of electron-donating and -withdraw-
Fig. 10.37), thiazoline ring modification ing groups to the aromatic ring (Section
(Section 5.3.1.2, compounds 108-114, Fig. 5.3.1.5, compounds 94, 118, and 124-126,
10.38),configurational [(R)-and (S)-]changes Fig. 10.41).

(a) Of 103

RyJjtOH

(108) (109)
Rat: 5 0.5 (po) Rat: 5 0.5 (po)
(b) Of 104

(110)
Rat:10.5 (po) Rat: 10.5 (po)
Monkey (300 pmol/kg):
10.5 (PO)

(113) (114)
Rat: 10.5 (po) Rat: 10.5 (po)
5 0.5 (sc) 1 0.5 (sc)

Figure 10.38. Series 11: Thiazoline ring modifications. The efficiency and mode of administration
(in parentheses) are shown.
Monkey (at 300) < 0.002

(103)
Rat: 3.9 f 1.8 (po) Monkey (300 pmoVkg): Rat:2.4 f0.6 (po) Monkey (150 pmol/kg):
[82 bile, 18 urine] 4.8 + 2.7 (PO)
154 stool, 46 urine] [48 stool, 52 urine]
(300 pmollkg):
8.0 f2.5 (PO)
[42 stool, 58 urine]

Monkey > 0.44


C02H

(116) (104)
Rat: 4.2 + 1.6 (po) Monkey (300 pmollkg): Rat: 1.4 f 0.6 (po) Monkey (300 ~mollkg):
[96 bile, 4 urine] 8.2 f 3.2 (PO) [I00 bile, 0 urine] 12.4 f 7.6 (PO)
[80 stool, 20 urine] [90 stool, 10 urine]

HO

Monkey (at 150) < 0.03


. IC02H
a C02H

(117) (118)
Rat: <- 0.5 (po) Monkey (150 pmollkg): Rat: 2.4 f 0.9 (po)
1.7 f 0.8 (PO) [I 00 bile, 0 urine]
[76 stool, 24 urine] Monkey (150 pmollkg): (300 pmollkg):
4.2 -+ 1.4 (po) 5.3 1.7 (PO)
_+

[70 stool, 30 urine] [90stool, 10 urine]

Figure 10.39. Series 111: C-4 stereochemistry. The efficiency, mode of administration (in parenthe-
ses), and fraction of iron excreted in the bile or stool and urine (in brackets) are shown.

5.3.1 IronClearance Evaluations from the phenolic donor (105) or separating


5.3.1.1 Changes in the Distances Between the phenol hydroxyl-thiazoline nitrogen bi-
the Ligating Centers (Series I, Compounds 105- dentate fragment from the carboxyl donor
107). Although earlier studies indicated that (106 and 107) (Fig. 10.37). The former elon-
the iron-clearing efficacy of desferrioxamine gation derived from insertion of a methylene
analogs was changed when the nature of the bridge between the aromatic and thiazoline
tether between ligating centers was modified rings, the latter one from insertion of one
significantly (346), relatively small alterations (106) or two (107) methylenes between the
in the distance between centers should have thiazoline ring and carboxy terminus. In every
little impact on the entropy of iron binding. case, these tridentate ligands (105-107) were
Extension of the distance between the ligating no longer orally active iron chelators; even
centers involved either separating the thiazo- subcutaneous administration of (105) was a
line nitrogen-carboxyl bidentate fragment failure (314).
Iron Chelators and Therapeutic Uses

(96) (119)
Rat: 5 0.5 (po) Rat: 10.5 (po)
Monkey (300 pmollkg): 5 0.5 (sc)
10.5 (PO) Monkey (300 ~mollkg):
0 0.5 (PO)
10.5 (sc)

(120)
Rat: 3.7 f 1.l (po) Rat: 2.9 f 1.3 (po)
[95 bile, 5 urine] [lo0 bile, 0 urine]
Monkey (300 pmollkg): Monkey (300 pmollkg):
*
2.1 0.7 (PO) 0.7 f 0.3 (PO)
[67 stool, 33 urine] [50 stool, 50 urine]
0.4 0.8 (sc) 2 0.5 (sc)

C02H
- 10.40. Series IV:
Figure
Fusion of aromatic rings. ,(122)
~ --, (123)
The efficiency, mode of ad-
ministration (in parenthe- Rat: 5.9 f 3.2 (po) Rat: 12.3 k 3.2 (po)
ses), and fraction of iron [90 bile, 10 urine] [I 00 bile, 0 urine]
excreted in the bile or stool Monkey (150 pmollkg): Monkey (75 pmollkg):
and urine (in brackets) are 3.5 2 1.8 (PO) 10.5 (PO)
shown. [68 stool, 32 urine]

5.3.1.2 Thiazoline Ring Modification (Series clearing iron in rodents, regardless of the
11, Compounds 108-114). Expansion of the mode of administration; nor was analog (112)
five-membered thiazoline of (103) to a six- active in primates (Fig. 10.38). Interestingly,
membered A2-thiazine (108) voided iron- compound (111)was absorbed quite well in
clearing activity (Fig. 10.38) (311). The same primates (387), but did not promote iron
phenomenon was observed when the thiazo- excretion.
line of (103)was oxidized to the corresponding The lack of activity of (111)was particu-
thiazole (109)(314) or the thiazoline of (104) larly noteworthy, in view of the fact that this
was reduced to a thiazolidine (110)(311). Fur- donor group is part of parabactin (4, Fig. 10.2).
thermore, when the sulfur atom in (104) was Interestingly, pharmacokinetic analyses of
replaced with an oxygen (111and 112) (311),a (111)in primates showed it to be well ab-
nitrogen (113) (314), or a methylene (114) sorbed orally, achieving significant plasma
(314), the compounds were not effective at levels that remained high even at 8 h, consid-
Things

(a)To 104

(124) (125) (126)


Rat: 1 0.5 (po) Rat: 0.9 f 0.3 (po) Rat: 10.5 (po)
1 0.5 (sc) [lo0 bile, 0 urine] 1 0.5 (sc)
*
0.5 0.9 (sc)
[96 bile, 4 urine]

Monkey (150 pmol/kg): (300 pmol/kg):


4.2 f 1.4 (po) 5.3 f 1.7 (PO)
[70 stool, 30 urine] [90 stool, 10 urine]
(118) (150 pmollkg (in H20)): (300 pmollkg (in H20)):
Rat: 2.4 f 0.9 (po) 5.6 k 0.9 (sc) 4.8 f 1.4 (po)
[I 00 bile, 0 urine] [92 stool, 8 urine] [85 stool, 15 urine]

(b) To 93

HO

q. S
C02H
Monkey (75 pmollkg):
*
17.7 3.9 (PO)
[79 stool, 21 urine]
(150 pmollkg):
*
13.4 5.8 (PO)
[86 stool, 14 urine]
(94)
Rat: 1 0.5 (po)

Figure 10.41. Series V: Addition of electron-donating and -withdrawing groups. The efficiency,
mode of administration (in parentheses), and fraction of iron excreted in the bile or stool and urine (in
brackets) are shown.

erably greater than those of (103).A terminal be that this ligand is promoting the excretion
elimination phase was difficult to discern from of iron very slowly, at a level below the limits
these 0- to 8-h experiments. It was also strik- of detection.
ing that the renal clearance of (111)was very 5.3.1.3 Configurational [(R)- and (S)-I
poor, less than 1/40 that observed for (103) Changes at C-4 (Series 111, Compound 115 ver-
(387). There was a considerably diminished sus 103, Compound 11 6 versus 104, Compound
fraction of ligand (111)unbound to human 117 versus 118). The effect of changing the
serum albumin versus (103). A high level of stereochemistry at C-4 on the iron-clearance
albumin binding may account for the lack of properties of the DFT analogs is model depen-
activity (because protein-bound ligand is un- dent (Fig. 10.39). Although (R)-desmethyl-
available for iron binding), the prolonged DFT (115) and (R)-desazadesmethylDFT
plasma residence time, and the poor renal (116) perform better in .therodent model than
clearance (387). Alternatively, it may simply their corresponding (S)-enantiomers(103 and
Iron Chelators and Therapeutic Uses

(93) (104)
Rats: All animals dead by Rats: All animals dead by
day 5: severe GI toxicity. day 6: severe GI toxicity.

Figure 10.42. Effect of add-


ing electron-donating groups
on the toxicity of (93) and
(104). The ligands were ad-
ministered orally at a dose of
384 pmol/kg/day, equivalent Rats: 10-day: well-tolerated; Rats: 10-day:
to 100 mg/kg/day of the so- histopathologies normal well-tolerated;all
dium salt of DFT. except for mild nephrotoxicity. histopathologies normal.

104, respectively), in the primate model, the to levels approaching the limits of detection,
(5')-enantiomer (103) was more efficient than whereas substantial plasma concentrations of
its (R)- counterpart. However, the difference (115)still remained. Perhaps of special sigmf-
between (116) and (104) is not statistically icance is that in every instance Fe(III)[1151,
significant (315). Thus, it seemed as though in the plasma exceeded 25 mg/L (50 p M ) for
(S)-enantiomer (104), beyond the ease of syn- several hours and remained above 10 mg/L (20
thesis of its analogs (i.e., the absence of the a at 8 h. The ratios for AUCo-JAUCo-, are
picolinic acid fragment), was the best pharma- 0.89 and 0.81 for (103) and Fe(III)[10312,re-
cophore from which to launch additional SAR spectively, compared to 0.50 and 0.44 for (115)
studies. and Fe(III)[115],, reflecting the prolonged
This choice was underscored further by a residence times of the latter enantiomer.
finding in primates during a pharmacokinetic The most remarkable and revealing finding
study of (R)- and (S)-desmethylDFT (115 and from the pharmacokinetic experiments was
103, respectively) (388). Surprisingly, one out the marked enantioselectivity of the renal
of four primates given the (R)-enantiomer clearance observed with (103), a clearance
(115)at a dose of 300 pmolkg died; adminis- that was 3.5 times greater than that of (1151,
tration of the (S)-enantiomer (103) did not and a Fe(III)[103], clearance that was 6.8
elicit side effects even at a dose of 450 pmolkg times greater than that of Fe(III)[1151, (388).
(235). Inspection of the plasma concentration- Thus, in the case of (115), unlike the situation
time curves for (115) versus (103) and for with (103), a substantial amount of ferric ion
Fe(III)[11512versus Fe(III)[103], reveals sev- is mobilized into the plasma, where it persists
eral striking features. The peak plasma con- at very high concentrations over a prolonged
centrations as well as areas under the curves period of time. Although a complete explana-
(AUCs) of (115) and Fe(III)[11512 curves tion of the mechanism of chiral recognition of
equaled or exceeded those of the correspond- DFT analogs is not readily apparent based on
ing (103) and Fe(III)[10312 curves. Eight these experiments, the plasma pharrnacoki-
hours posttreatment, the plasma concentra- netic results do provide a basis to explain the
tions of (103) and Fe(III)[103], had declined selective toxicity of (115)in the primates. The
choice of (S)-over (R)-enantiomerswas under- 118,124-126 , Fig. 10.41). Initially, the elec-
scored further when the performance of (117) tronic properties of the substituents were con-
and (118)was compared in the primates (Fig. sidered relative to the thiazoline ring.
10.39). Again the (S)-enantiomer, (118)in this Introduction of a second hydroxyl group at
set, turned out to be the more active ligand position 3 of the aromatic ring of (104) to gen-
(315). erate (124) resulted in an inactive chelator in
5.3.1.4 Benz-Fusion (Series IV, Compounds rodents. However, hydroxylation at position 4
96,119-123). Evaluation of the (S)-and (R)- [4'-hydroxy-(S)-desazadesmethylDFT,(118)l
pairs of naphthyl analogs 4,5-dihydro-2- was a maneuver compatible with iron clear-
(2-hydroxy-1-naphthaleny1)-4-thiazolecar- ance. A methoxy was introduced in the 3 posi-
boxylic acid (96 and (119), Fig. 10.40) and tion (125) and a carboxy in the 4 position
4,5-dihydro-2-(3-hydroxy-2-naphthalenyl)-4- (126). In the rodent model, (125) was margin-
thiazolecarboxylic acid (120 and 121, Fig. ally effective; (126) was ineffective. A compar-
10.40) demonstrated that, although the ison of the 3-hydroxylated ligand (124) with
1-(2-hydroxynaphthy1)-enantiomers(96) and the 3-methoxylated analog (125) was carried
(119) were not effective iron-clearing agents out to examine possible confounding effects by
after oral administration to the rats, their po- the potential auxiliary metal coordination site
sitional isomers, the 2-(3-hydroxynaphthy1)- in (124). In both cases, the groups are elec-
compounds (120) and (121), were (313). None tron-withdrawing by induction from the thia-
of the naphthyl DFTs investigated was an ef- zoline nitrogen; however, the methoxyl group
fective iron chelator in the primates, even on cannot serve as a ligand donor. When the hy-
subcutaneous administration. The benz-fused droxyl group is in the 4 position (118),it do-
ligands (S)-and (R)-4,5-dihydro-243-hydroxy- nates electrons to the nitrogen, whereas a car-
2-quinoliny1)-4-thiazolecarboxylicacid (122 boxyl group at this carbon (126) withdraws
and 123, respectively, Fig. 10.40) were synthe- electrons from the nitrogen (314). Finally, 4'-
sized and evaluated in an attempt to restore hydroxylation was also carried out on (93)
activity in the primate model (314). The drugs (Fig. 10.41) to yield 4'-hydroxy-(Sbdesaza-
were both active in rodents upon oral admin- DFT (94), which was also a very active iron
istration. However, only the (S)-enantiomer chelator when administered orally to pri-
(122) was efficacious in primates, and then mates, although it was unexpectedly inactive
only marginally so. in rodents (315). Thus, the complete story on
5.3.1.5 Addition of Electron-Donating and the efficacy of the hydroxylated and methoxy-
-Withdrawing Groups to the Aromatic Ring (of lated ligands must await primate trials.
104, Series V A, Compounds 1 18, 124-126; of
93, Series V B, Compound 94). The above SAR 5.3.2 Toxicity. Once the alterations in the
provided a framework, (S)-desazadesmethyl- redox potential of the aromatic ring of (93)or
DFT (104), that could be manipulated with (104) that were compatible with iron clear-
ease. A second SAR focused on ameliorating ance were delineated, the second SAR focused
toxicity. Because of the ease of synthesis of the on toxicity was performed. The compounds
desazadesmethylDFTs, their iron-clearing tested to date that induced iron excretion in
properties, and the facility with which toxicity the primate model, 4'-hydroxy-(S)-desazades-
can be monitored, (104) was an excellent plat- methylDFT (118)and 4'-hydroxy-(S)-desaza-
form from which to explore the construction of DFT (94),were examined and compared to des-
a nontoxic DFT analog. The impact of altering azademethylDFT (104) and desazaDFT (931,
the redox properties of the aromatic ring of respectively. When an electron-donating hy-
(104) on the iron-clearing properties of the droxyl group is added to position 4 of the aro-
compounds was assessed before any evalua- matic ring of (104) as in (118),the gastroin-
tions of toxicity or metabolic profiles. The re- testinal toxicity problem found in (104) was
dox properties were modified by introducing absent: no deaths occurred when this com-
electron-donating or -withdrawing groups pound was given at a dose of 384 pmoVkg/day
into the aromatic ring of (104) (compounds for 10 days (Fig. 10.42) or at a dose of 250 pmol
Iron Chelators and Therapeutic Uses

Figure 10.43. p,p-Dimethyl


analog of (118)and (127)and
the 5'-(OH) metabolite of
(104) and (128).

per kg/dose over 30 days. Histopathological with (104) was conducted. The putative me-
analysis of tissues (including stomach, small tabolite (128) was synthesized by condensa-
intestine, large intestine, kidney, and liver) re- tion of 2,5-dihydroxybenzonitrilewith D-cys-
vealed no drug-related abnormalities in any of teine. Application of HPLC methodologies
these animals (314). (388) for quantitation of DFTs in tissue and
When the same manipulation was per- fluids worked well for this metabolite. Analy-
formed on the aromatic ring of (93) to give sis of 24-h urine specimens from animals
(94), the gastrointestinal toxicity problem treated with (104) indicated that this ligand
found with (93) was absent again; no deaths was indeed 5'-hydroxylated; furthermore, this
occurred when (94) was given orally at a dose metabolite represents about 18% of the total
of 384 hmol/kg/day for 10 days, greater than
concentration of drug and metabolites in the
twice the time period for (93) (Fig. 10.42). Al-
urine. Neither bile nor other tissues have been
though macroscopic inspection of organs l day
examined. Interestingly, when the synthetic
after the final dose revealed no drug-related
abnormalities, histopathological analysis metabolite (128) was given to rodents in an
showed that compound (94) was associated iron-clearance experiment, it showed no ob-
with some very mild nephrotoxicity, which servable clearance activity; thus, 5'-hydrox-
was considered to be reversible, but nothing ylation inactivates the drug (R.J.B., J.M.,
comparable to what was observed with the W.R.W., and J.W., unpublished observations).
natural product (24) (315). This raises several very interesting questions
A hydroxyl group was appended to the 4 regarding other DFT analogs. Is oxidative de-
position of the aromatic ring of a P,P-dimethyl activation occurring with these ligands, and to
cysteine derivative, which was known to elicit what extent? Does this deactivation correlate
gastrointestinal toxicity (235).This manipula- with iron-clearing efficiency? If so, will block-
tion was effective in ameliorating the toxic ef- ing this position by introduction of a different
fects. Although rats given this hydroxylated atom or functional group at this position in-
analog (127, Fig. 10.43) at a dose of 384 crease the chelators' efficiency? Furthermore,
hmoVkg for 10 days demonstrated renal toxic- what role does oxidative deactivation play in
ity, when analog (127) was given to both iron- primates, if any? The toxicity of the metabo-
loaded rats and rats with normal iron stores at lites remains to be evaluated thoroughly. If
a dose of 130 pmollkg for 30 days, no drug- these metabolites are more toxic than the par-
related changes in tissue pathologies were ent drug, this makes protection of desaza-
noted. DFTs from oxidative deactivation even more
attractive.
5.3.3 Metabolism of Desazadesferrithiocins. Drug design in the arena of iron chelation
One of the principal metabolites of aspirin, therapy presents unique challenges because of
gentisic acid, derives from hydroxylation at the necessity of striking a balance between ef-
the 5 position of the aromatic ring (389,390). ficacy and toxicity resulting from excessive
To determine whether this was the fate of iron removal. However, the lessons taken
(104) and how this might affect its activity, a from nature's chelator design strategies, mod-
search for 5'-hydroxydesazadesmethylDFT ern synthetic techniques, and the use of ap-
(128, Fig. 10.43) in the urine of rats treated propriate animal models should bring about
References

improved therapeutic agents for the treat- 7 ACKNOWLEDGMENTS


ment of both global and focal iron overload in
the years to come. The research carried out in the Bergeron lab-
oratories has been supported generously over
6 WEB SITE ADDRESSES A N D the years by both the corporate sector and the
RECOMMENDED READING FOR FURTHER National Institutes of Health (currently by
INFORMATION grant R01-DK49108).We express our sincere
appreciation to Dr. David G. Badman of the
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CHAPTER ELEVEN

j Thyroid Hormones and

DENISE. RYONO
Discovery Chemistry

GARY J. GROVER
Metabolic Diseases Biology
Bristol-Myers Squibb
Princeton, New Jersey

KARIN MELLSTROM
Cell Biology
Karo Bio AB
Huddinge, Sweden

Contents
1 Introduction, 564
2 Molecular Mechanism and Physiology of Thyroid
Hormone Action, 564
2.1 Introduction, 564
2.2 Molecular Mechanism of Thyroid Hormone
Action, 565
2.2.1 Background, 565
2.2.2 Domain Structure of the Thyroid
Hormone Receptors, 566
2.2.3 TR Receptor Isoforms, 566
2.2.4 Transcriptional Regulation, 566
2.2.4.1 Thyroid Hormone Response
Elements, 566
2.2.4.2RXR Heterodimers, 566
2.2.4.3Role of Coactivators and
Corepressors, 567
2.3 Physiology of Thyroid Hormone Action, 568
2.3.1 Regulation of Thyroid Hormones, 568
2.3.2 Thyroid Hormone Biosynthesis, 568
2.3.3 Thyroid Hormone Transport, Cellular
Uptake, and Metabolism, 569
2.3.3.1 Thyroid Hormone Transport In
Blood, 569
Burger's Medicinal Chemistry and Drug Discovery 2.3.3.2 Uptake and Metabolism of T,,
Sixth Edition, Volume 3: CardiovascularAgents and 569
Endocrines 2.4 Biologic Actions of Thyroid Hormones, 570
Edited by Donald J. Abraham 2.4.1 Metabolic Rate, 570
ISBN 0-471-37029-0 O 2003 John Wiley & Sons, Inc. 2.4.2 Lipid Metabolism, 571
Thyroid Hormones and Thyromimetics

2.4.3 Carbohydrate Metabolism, 571 4.1 Introduction, 576


2.4.4 Protein Metabolism, 571 4.2 Active Hormone Conformation
2.5 Thyroid Hormone Effects and Fkceptor-Ligand Structures, 576
on Specific Tissues, 571 4.3 Receptor Binding of Thyroid
2.5.1 Heart and Cardiovascular System, 571 Hormone Analogs, 577
2.5.2 Liver, 572 4.3.1 Background, 577
2.5.3 Bone and Skeletal Muscle, 572 4.3.2 Summary of Basic Thyromimetic SAR,
2.5.4 Receptor Isoform Knockouts, 573 578
2.6 Thyroid Diseases, 574 4.4 Selected Biologically Active Thyroid
2.6.1 Hypothyroidism, 574
Hormone Analogs, 580
2.6.2 Hyperthyroidism and Thyrotoxicosis,
4.4.1 Background, 580
574
3 Therapeutic Potential 4.4.2 L-T, (Liothyronine), L-T,
of Thyroid Hormone Analogs, 575 (Levothyroxine),and Triac, 580
3.1 Introduction, 575 4.4.3 SKF-94901, 581
3.2 Lipid Lowering, 575 4.4.4 Oxamic Acids, 582
3.3 Obesity, 575 4.4.5 GC-1 and KB-141,584
3.4 Cardiac Indications, 575 5 Recent Developments and Things to Come, 585
3.5 Hypothyroidism, 576 5.1 Antagonists, 585
3.6 Other Disorders, 576 5.2 Drug Design, 586
4 Structure-Activity Relationships and Biologic 5.3 Future Drug Development, 587
Activities of Thyroid Hormones and Analogs, 576

1 INTRODUCTION (R, and R,) adjacent to the linker atom has


been termed the "inner" or "non-prime" ring.
Thyroid hormones and their analogs act by This ring also possesses the amino acid side-
binding to their cognate receptors in the nu- chain at R, that is biosynthetically derived
cleus and regulating the expression of diverse from tyrosine. The second aromatic ring, the
(6
genes throughout the body. Consequently, outer" or "prime" ring, contains the remain-
such compounds represent significant prom- ing iodine substitution positions at R,' and
ise for the development of new therapy. Unfor- R,', as well as a critical phenolic hydroxyl
tunately, the potential for this class of drugs group at R,'. Thus, T, (L-3,5,3'-triiodothyro-
has historically been limited by concerns for nine) has the structure shown by (1)and T,
mechanism-related side effects. However im- (L-3,5,3',5'-tetraiodothyronineor L-thyrox-
proved assays using purified receptors, ine) has the structure indicated by (2).
greater knowledge of receptor-ligand interac-
tions, and importantly, new advances in un- 2 MOLECULAR MECHANISM
derstanding the molecular biology of nuclear AND PHYSIOLOGY OF THYROID
hormone receptors, including the thyroid hor- HORMONE ACTION
mone receptors (TRs), may now point the way
to developing new thyroid hormone drugs
2.1 Introduction
with selective and consequently safer profiles
of biologic activity. The normal thyroid gland is the largest endo-
Essentially all naturally occurring thyroid crine gland in the body. The name thyroid is
hormones and their synthetic analogs (thyro- derived from its proximity to the laryngeal
mimetics) are structurally characterized by a thyroid cartilage, which resembles a Greek
basic construct of two aromatic rings joined by shield. Our earliest knowledge of the thyroid
a single linker atom (X) as depicted below. The dates back thousands of years when goiters
various iodine atoms of the naturally occur- and cretinism, particularly in the Alps moun-
ring hormones, T, and T,, occupy the posi- tain regions of Europe, were linked to this
tions indicated by R,, R,, R,' and R,'. The gland. In the early 1800s, it was discovered
aromatic ring containing iodine substitution that iodine could reduce or prevent goiters,
2 Molecular Mechanism and Physiology of Thyroid Hormone Action 565

linker Gross and Pitt-Rivers (4) and, simultaneously,


Roche et al. (5) found and synthesized thyro-
nine (T,) in 1952. T, was found to be more
biologically active than T, and is presently
thought to be the predominant activator of the
thyroid hormone receptors. T, is the primary
hormone synthesized by the thyroid, and
while it is active in tissue, it is intracellularly
converted to T,.
Thyroid hormones exert a wide array of bi-
ologic activities and are important in growth,
development, and cellular metabolism. In
prime ring non-prime ring A

(outer ring) (inner ring) adults, thyroid hormones are critical for main-
taining basal metabolic rate, optimal cognitive
abilities, lipid metabolism, and cardiovascular
-
status. Because of the importance of these
myriad biologic activities, hypothyroidism and
hyperthyroidism are characterized by marked
physiologic effects that can have an enormous
impact on day-to-day function in patients. For
this reason, treatment of thyroid-related dis-
eases is of great clinical interest. Because of
the effects of thyroid hormones on metabolic
rate and lipid metabolism, the potential of thy-
romimetics as anti-obesity and lipid-lowering
although this was purely an empirical obser- agents also exists. However, controlling the
vation. In the late 1800s, the link between hy- side effects of such agents presents a daunting
pothyroidism and hyperthyroidism (or thyro- obstacle for drug development scientists.
toxicosis) and the thyroid gland was deduced.
A search for the active agent was undertaken, 2.2 Molecular Mechanism of Thyroid
and using iodine as a marker, Kendall isolated Hormone Action
the active substance in crystalline form in
1914 on Christmas day and named the com- 2.2.1 Background. Thyroid hormone re-
pound thyroxin (1).This name was derived ceptors are ligand-dependent transcription
from a contraction of thyroxyindole and was factors that belong to the nuclear receptor su-
based on his belief that the compound's molec- perfamily (6). Included in this family of pro-
ular structure had an indole nucleus and three teins are receptors for steroid hormones, reti-
iodine atoms. The correct structure, estab- noic acid, and vitamin D, as well as
lished by Harington in 1926 (2), showed the homologous proteins with unidentified li-
presence of four iodine atoms and an amino gands, i.e., orphan receptors (7). The first sug-
acid structure. It was accordingly renamed gestion that thyroid hormones act as regula-
thyroxine (terminal "ine" in common with the tors of transcription came from the early
names of the naturally occurring a-amino ac- observations that T, induces a rapid increase
ids, for example, alanine). Thyroxine, also in RNA synthesis that preceded de novo pro-
known now as T, (the subscript indicating the tein synthesis (8). Shortly thereafter, evidence
number of iodine atoms), was successfully that thyroid hormones acted through specific
synthesized in high yield in 1949, making it nuclear binding sites was shown using radio-
clinically and economically viable for thera- labeled T, (9,lO).Using photoaffinity-labeling
peutic use compared with treatment with des- procedures, thyroid hormone receptors were
iccated thyroid tissue (3). identified in both rat liver nuclei (11)and in-
The presence of a second thyroid hormone tact cells (12). Further in vitro studies using
was hypothesized, but not established until the rat growth hormone gene strongly sug-
Thyroid Hormones and Thyromimetics

AIB C D E F
Figure 11.1. Domain struc-
ture of nuclear hormone recep- AF1 DBD LBD
tors. activation DNA binding ligand binding

gested that the TRs recognized specific DNA been suggested (27). The TRP locus gives rise
elements (13). In 1986, two different subtypes to TRP,, TRP,, and the recently discovered
of high affinity thyroid hormone receptors TRP, (14,15,28). The TRP isoforms are tran-
that were homologs to the viral erbA oncogene scribed from separate promoters and alterna-
were independently cloned in two different tive splicing of the mRNA, which give rise to
laboratories (14,151.This started an extensive N-terminal variants. TRP,, like TRa,, is found
and detailed elucidation of the molecular in most tissues, however, at varying levels of
mechanisms for thyroid hormone acting as a expression. In the adult liver, TRP, predomi-
regulator of transcription. There are several nates in concentration over TRa,, whereas in
excellent reviews on this topic (16-18). the heart, the opposite is true (29). A more
restricted expression pattern is found for
2.2.2 Domain Structure of the Thyroid Hor- TRP, that is preferentially expressed in the
mone Receptors. The TRs are nuclear recep- pituitary and in distinct cells in the central
tors that share a common structure compris- nervous system (30).
ing three different functional domains: the
N-terminal transactivation domain (AFl),the 2.2.4 Transcriptional Regulation
DNA-binding domain (DBD),and the carboxy- 2.2.4.1 Thyroid Hormone Response Ele-
terminal ligand-binding domain (LBD; Fig. ments. TRs act by binding to specific DNA se-
11.1). The N-terminal All3 domain is the less quences, thyroid response elements (TREs),
well conserved region of the nuclear receptors, in the promoters of their target genes, thereby
and its exact role in transactivation is still un- stimulating or repressing transcription. Most
clear. The nuclear receptors interact with of these TREs are located upstream of the pro-
DNA through the central DNA-binding do- moter, but location in the 3' flanking region
main with a distinct "zinc fingers" structure. downstream from the coding region also has
The integrity of these zinc fingers are essen- been described (17,31).
tial for DNA binding and transcriptional activ- Mutagenesis studies of the rat growth hor-
ity (19-22). The hinge region (D) located be- mone promoter showed the consensus half site
tween the DNA-binding and ligand-binding to be GIAGGTCIGA. Considerable variation of
domains contains the nuclear recognition sig- this consensus sequence regarding primary
nal. The carboxyl terminal LBD also plays an nucleotides, numbers, spacing, and orienta-
important role in dimerization, transactiva- tion are found in TREs. The most prominent
tion, and transrepression (23, 24). The trans- half-site orientations with optimal spacing in-
activation function has been mapped to a clude a palindrome TRE (pal) with no spacing
highly conserved motif in the LBD termed nucleotides, direct repeats with four interven-
AF-2 (25). ing nucleotides (DR4), and inverted palin-
dromes with six spacing nucleotides (32).
2.2.3 TR Receptor Isoforms. TRs are en- 2.2.4.2 RXR Heterodimers. TRs bind to
coded by two distinct genes (TRa and TRP), these TREs usually as homodimers or het-
which give rise to several isoforms of TRs. The erodimers with auxiliary proteins. The major
TRa gene encodes for the ligand-binding re- heterodimer partner is RXR (retinoid X recep-
ceptor TRa, but also encodes for the TRa, tor) that belongs to the nuclear receptor su-
protein that does not bind T,, although it perfamily and has high homology with the
binds to DNA with reduced affinity (26). TRa, retinoic acid receptor (RAR). RXR binds 9-cis
and TRa, are co-expressed in most tissues. retinoic acid with high affinity and also het-
The biological function of TRa, is not clear, erodimerizes with vitamin D receptor (VDR),
but interference with normal TR function has RAR, peroxisome proliferator activator recep-
2 Molecular Mechanism and Physiology of Thyroid Hormone Action

TRE TRE

+ Transcription
*
Figure 11.2. Role of corepressor
and coactivators in transcriptional
regulation of NHRs.

tor (PPAR),liver X receptor (LXR), and farne- In a recent study, the critical surface and con-
syl X receptor (FXR). The RXRs (three differ- formational modes for NCoR interaction with
ent subtypes: a,P, and y ) are widely expressed TRP were characterized, suggesting the possi-
and therefore allow heterodimer formation bility of selective modulation of corepressor
with TR in most tissues (33,34). TR/RXR het- function by induction of specific receptor con-
erodimer formation increases the number of formations (44).
target genes that can be induced because heb Unliganded thyroid hormone receptors can
erodimers bind to a larger variety of TREs. In also stimulate the basal activity of certain neg-
the DR4 response element, a specific orienta- atively regulated promoters such as thyroid
tion of dimer is formed where TR is always stimulating hormone (TSH). The mechanism
oriented on the 3' side of RXR (35). Unlike by which TR exerts this function is less well
steroid receptors, the TR/RXR heterodimer understood, but it has been proposed to in-
binds to DNA in the absence of ligand and volve the same interaction with corepressors
thereby represses transcription. and coactivators, although with reversed func-
2.2.4.3 Role of Coactivators and Corepres- tional consequences (45).
sors. Nuclear hormone receptor (NHR) regu- Ligand binding to TR will increase the af-
lation of transcription involves important finity of the receptor to coactivators that inter-
roles played by numerous coregulator proteins act with the AF-2 domain of the LBD. These
that bind the receptorIDNA complexes. Those coactivators (such as SRC-1) do not bind DNA
coregulators that promote transcriptional ac- by themselves but have the capacity to in-
tivation by binding the ligand-bound form of crease transcription in vitro when either co-
the receptor are termed coactivators and those transfected with DNA-binding proteins or
involved in repression of transcription by fused with DNA-binding domains. When coac-
binding the ligand-free (apo) receptor are the tivators bind to TR they create an optimized
corepressors (Fig. 11.2) (36,37). environment for effective transcriptional acti-
In the absence of ligand, the TRs can re- vation supposedly by recruiting transcription
press basal transcription of many positively factors and histone acetyltransferase (HAT)
regulated genes that contain TR-binding sites activity. Many of the coactivators interact
in their promoters (14,38,39). Recruitment of with the receptors through a helical peptide
corepressor proteins like nuclear receptor motif with a core LXXLL sequence of amino
corepressor (NCoR) or RIP3 (40, 41), and si- acids (46-48). An X-ray structure determina-
lencing mediator of retinoic and thyroid recep- tion of TRP bound to a partial sequence of the
tor (SMRT) or TRAC (42, 43) that interact coactivator protein, SRC-1 (steroid receptor
with the unliganded TR, are involved in the coactivator-1), which contains two LXXLL
mechanisms of basal repression. Unliganded motifs, suggests specific interactions between
TRs recruit corepressor proteins that inhibit the hydrophobic region of the LXXLL helix
transactivation by either interfering with the and a hydrophobic cleft formed from portions
basal transcription machinery and/or recruit- of helices 3,4,5, and 12 of the receptor. Inter-
ing enzymes that inhibit transcription, e.g., estingly, the interaction domains on NCoR
histone deacetyltransferase (HDAC) activity. and SMRT exhibit a similar, but extended con-
Thyroid Hormones and Thyromimetics

sensus, sequence, LXXI/HWMI/L. This ex- scription of thyroglobulin, which is the start-
tended helix interacts with specific residues ing substrate for T, and T, production.
located in the same hydrophobic pocket that Given the pivotal role for TSH in regulat-
interacts with coactivators (49,501. This sug- ing biosynthesis and release of T, and T,, its
gests that ligand-induced conformational production in the pituitary is ideally suited as
changes involving the AF2 region of the LBD a site for feedback control. Increased plasma
influence the recruitment of coactivators and levels of T, or T, can reduce TSH release from
their interaction with the receptors. the pituitary within hours of administration
(56). This rapid reduction in TSH release is
followed by a continuing, but slower rate, of
2.3 Physiology of Thyroid Hormone Action secretion as long as T, is still present. Sup-
pression of TSH release is thought to be re-
2.3.1 Regulation of Thyroid Hormones. Be- lated to nuclear TR binding and therefore
cause thyroid hormones exert such diverse secondary to gene regulation. Reciprocal reg-
and profound biologic effects, precise regula- ulation of TSH release by TRH and T, nor-
tion of these hormones is required. Production mally results in a steady-state balance, al-
of T, and T, by the thyroid gland is under though some daily pulsatility can occur. T, not
negative feedback control, exerted at several only reduces production of TSH, but also acts
levels. TSH, also known as thyrotropin, is re- on the hypothalamus to reduce TRH release.
sponsible for normal thyroid gland function Because of its central regulatory function,
and thyroid hormone secretion. It is synthe- TSH is a good indicator of thyroid function.
sized in the anterior pituitary gland, and its Hypothyroidism caused by reduced T,/T, out-
secretion is controlled by thyroid releasing put from the thyroid gland will cause in-
hormone (TRH) that is synthesized in the hy- creased TSH output because of unrestricted
pothalamus. TRH, a G-protein-coupled recep- stimulation by TRH, as well as a reduced in-
tor (GPCR) agonist, causes a dose-dependent hibitory effect. Proper hormone replacement
and rapid release (in minutes) of TSH, but can therapy can be monitored by the return of
also up-regulate transcription and translation TSH to more normal values.
of TSH subunit genes through a mechanism TSH is released during the day in a some-
involving calcium influx and protein kinase C what pulsatile manner, with the highest levels
signaling pathways (51, 52). In addition to occurring during the night, although the
TRH, somatostatin, dopamine, and other cat- mechanism is not clear (57). Central regula-
echolamines can modulate TSH secretion and tion is likely and seems to be an interaction
release (53). between several modulatory mechanisms.
TSH is a glycoprotein with a- and P-sub- There are also several important extrinsic fac-
units, both of which are necessary for biologic tors controlling TSH secretion. First, fasting
activity. The major site of expression of the will reduce the TRH responsiveness of TSH,
TSH receptor is the thyroid, although it is also causing reduced tissue T, levels and a de-
expressed in other tissues, particularly adipo- creased metabolic rate (58). This would seem
cytes (54). TSH causes increased thyrocyte to be an adaptive response to energy restric-
CAMPlevels and associated protein kinase A tion. Another factor affecting TSH release is
(PKA) activity (55). The gene regulation oc- cold, which increases production of TSH (59).
curring secondary to PKA activation can occur The adaptive effects of increased thermogene-
within minutes to hours. TSH also stimulates sis during cold exposure seems to be a logical
PKC, causing release of inositol phosphates, response to this stress.
and therefore can cause release of intracellu-
lar calcium. Activation of TSH receptors 2.3.2 Thyroid Hormone Biosynthesis. There
causes increased thyrocyte iodide uptake. are several steps in thyroid hormone synthe-
TSH causes increased expression of thyroper- sis: (1) transport of iodide into the thyroid
oxidase (TPO), a central enzyme in thyroid gland; (2) mono- and di-iodination of the ty-
hormone biosynthesis necessary for organifi- rosine residues of the protein thyroglobulin
cation of iodide. TSH also increases gene tran- (Tg)in the thyroid follicular cells; (3)coupling
2 Molecular Mechanism and Physiology of Thyroid Hormone Action

of the iodotyrosines by TPO to form nascent 2.3.3 Thyroid Hormone Transport, Cellular
T, and T, as amino acid residues contained in Uptake, and Metabolism
Tg; and (4)proteolysis of Tg followed by secre- 2.3.3.1 Thyroid Hormone Transport in
tion of the formed free thyroid hormones. The Blood. Delivery of T, and T, from the thyroid
majority of the released thyroid hormone from gland through blood is primarily accomplished
the thyroid gland is in the form of T,. The through serum carrier proteins that have
primary source of T, is from enzymatic deio- varying affinities for the thyroid hormones. A
dination of T, to T, in extrathyroidal tissue. high percentage (99%)of T, is protein bound,
Iodide is actively transported into thyroid yet it can be readily released from these pro-
cells and is therefore ouabain sensitive (60). teins for entry into cells. Approximately 70%
Transport of iodide across the membrane of circulating T, and T, are bound by thyrox-
seems to be linked to sodium, and the relevant ine-binding globulin (TBG) and this is because
transport protein is thought to be a co-trans- of its high affinity for these hormones. TBG is
porter (61). TSH seems to be the most impor- a single-chain polypeptide that is a member of
tant factor in regulating iodide transport into the serine protease inhibitor (serpin) family.
follicular cells. Transthyretin [TTR or thyroxine-binding
Much of the iodine in the thyroid is bound prealbumin (TBPA)] accounts for approxi-
to Tg, and only a small fraction is present as mately 15% of hormone binding, but because
inorganic iodide. Iodide must be oxidized be- of its lower affinity, it is responsible for much
fore it can effectively iodinate the tyrosine res- of the immediate delivery of thyroid hormones
idues of Tg, and this is carried out by TPO to cells. TTR is also the main thyroid hor-
using hydrogen peroxide as the oxidant. Thy- mone-binding protein in cerebrospinal fluid.
roperoxidase also catalyzes both the iodina- Each TTR molecule has two binding sites, and
tion of tyrosyl residues and the coupling of numerous other compounds will also interact
iodinated tyrosyl residues to form T, and T, in with TTR.
Tg. TPO is a membrane-bound hemoprotein Albumin binds 15-20% of thyroid hor-
whose gene is up-regulated by TSH. TPO ac- mones, and they are even more readily disso-
tivity requires H20, and is generated on the ciated then they are from TTR. Lipoproteins
apical surface through a calcium-sensitive also transport small amounts of thyroid hor-
mechanism (TSH also increases H20, produc- mones, and their specific uptake by certain
tion). The iodination of Tg takes place at cells may be critical for some of the functions
the cell-colloid surface close to the apical of these cells.
membrane. These thyroid-binding proteins serve sev-
Tg structure is crucial for the coupling re- eral important functions. The proteins are
action responsible for synthesis of T,. The thought to primarily serve as buffers so that
coupling involves a reaction between either di- the hormone is released when needed. The
iodotyrosine (DIT) or monoiodotyrosine (MIT) binding proteins also seem to serve as an ex-
with DIT to form T, or T,, respectively. Both tra-thyroidal storage site for thyroid hor-
iodination and coupling occur on Tg. The mones. This is probably necessary for con-
mechanism of coupling most likely involves stant and equal distribution among the
radical formation (TPO-dependent) of DIT or tissues.
MIT. The primary hormone produced by the 2.3.3.2 Uptake and Metabolism of T,.
coupling reaction in the thyroid gland is T,, Whereas T, has intrinsic biologic activity, T,
although smaller amounts of T, are synthe- is thought to be more biologically important.
sized through coupling between MIT and DIT. Most T, production occurs through deiodina-
The iodinated Tg is stored as an extracellular tion of T, in target tissues and may represent
polypeptide in the luminal colloid. The colloid a noncirculating pool. A host of other deiodi-
is resorbed through pinocytosis, and Tg un- nation products are also generated by deiodi-
dergoes proteolysis in lysosomes. T, is then nase activity. The type I 5'-deiodinase cata-
released through the basal surface into the lyzes 5'-deiodination of T,, and it is felt this
bloodstream where it is bound by carrier pro- enzyme plays an important role in the produc-
teins. tion of T,. Propylthiouracil (PTU) completely
Thyroid Hormones and Thyrornirnetics

blocks this enzyme. This deiodinase shows ab- Increased metabolic rate has been reported
solute preference for L-enantiomers such as to be caused bv both increased ATP turnover
L-T, and is found in most tissues of the body. and enhancedproton leak and is associated
A second isoenzyme, type I1 5'-deiodinase, with increased mitochondrial size, density,
seems to be important in generating intracel- and surface area (80-82). The precise mecha-
lular T, for local use in pituitary, brain, and nism for increased respiratory rate may vary
brown adipose tissue and is not greatly sensi- by tissue. Early hypotheses placed a great deal
tive to PTU. Both enzymes are integral pro- of emphasis on the up-regulation of Nat/K+
teins of cellular membranes. 5'-Deiodinases ATPase as a primary mechanism for increased
also remove iodines from the 5- or 3-positions oxygen consumption. A component of the met-
of the tyrosyl ring (inner or non-prime ring) of abolic rate response to thyroid hormones can
iodothyronines. These processes represent a indeed be blocked by ouabain. Thyroid hor-
major component of T, and T, degradation. mones increase Na+/K+ ATPase activity as
Little is known about transport of thyroid well as density (and mRNA), and its time
hormones through plasma membranes. It has course of up-regulation is similar to the rate of
been suggested that transport proteins can increase of oxygen consumption. Neverthe-
.
have s~ecific interactions with cell surface re- less, the relative importance of up-regulation
ceptors that may facilitate transport. Alterna- of this ion exchanger is now thought to be less
tively, thyroid hormones may be transported then previously hypothesized. Respiratory en-
by diffusion (62-64). Thyroid hormones may zymes encoded in both the nuclear and mito-
also be transported into cells through trans- chondrial genomes are up-regulated by thy-
port proteins that seem to be linked to sodium roid hormones. Thyroid hormones increase
transport, and some transporters may be ATP cytochrome C and P-F,-ATPase expression,
dependent (63). Work by Barlow et al. (65) and putative TREs exist for these genes (75,
suggests that certain thyromimetics such as 83, 84). Glycero-3-phosphate dehydrogenase
SKF-94901 may exert tissue selectivity is rapidly induced by T,. These changes in re-
through differences in tissue transport. Rela- spiratory gene expression can contribute to in-
tively poorer uptake by the heart versus the creased ATP production and turnover.
liver was invoked to explain robust choles- Thyroid hormones can cause respiratory
terol-lowering activity with minimal cardiac uncoupling, particularly in thyrotoxic tissues
effects for SKF-94901. Some degree of selec- (67). The importance of increased uncoupling
tive liver uptake of GC-1 has also been shown to thermogenesis with lower doses of thyroid
and may partially explain some of the liver- hormone is, however, debatable (85). Thyroid
specific effects of this agent (66). hormones are known to increase expression of
mitochondrial uncoupling proteins (UCPs),
2.4 Biologic Actions of Thyroid Hormones which promote proton leak across mitochon-
drial membranes and therefore uncouple mi-
2.4.1 Metabolic Rate. TR activation will tochondria causing a thermogenic effect (67,
increase metabolic rate in a dose- and time- 81). UCP-3 is regulated by thyroid hormone,
dependent manner (67-70). Whole body oxy- particularly in skeletal muscle, and recent
gen consumption can be profoundly increased studies in UCP-3 overexpressing or knockout
by thyroid hormones, causing weight loss as mice suggest a thermogenic role for this pro-
well as cardiac acceleration (71-74). Most tis- tein, although evidence for a lack of a thermo-
sues show increased metabolic rates in re- genic function for UCP-3 must also be consid-
sponse to TR stimulation with the notable ex- ered (86, 87). UCP-1 in brown adipose tissue
ception of the brain (75, 76). Increased (BAT) is directly up-regulated by thyroid hor-
metabolic rate by TR activation is largely mones and indirectly through sympathetic
caused by changes in gene expression, partic- stimulation (81). Whereas a thermogenic role
ularly with regard to mitochondrial respira- for UCP-1 may be important in rodents, its
tion (75, 761, although changes in non-mito- importance in primates in not presently clear.
chondrial proteins such as Nat/K+ ATPase Recently published work suggests that
can also be involved (77-79). adaptive thermogenesis may be mediated pri-
2 Molecular Mechanism and Physiology of Thyroid Hormone Action 571

marily through activation of the TRa, recep- and biliary excretion are also increased. The
tor isoform, whereas TRP, may not be as ef- balance of these activities is such that LDL
fective (88).These results indicate that UCP-1 cholesterol in plasma is reduced. Apo A1 may
levels are directly regulated by TRP,, but that be increased by thyroid hormones that may
the action of thyroid hormone on sympathetic act to increase high density lipoprotein (HDL)
stimulation (which is required for adaptive cholesterol and enhance "reverse" cholesterol
thermogenesis) is regulated by TRa,. Other transport (95). There is evidence that thyroid
studies have further suggested the importance hormones can reduce the lipoprotein Lp(a),
of TRa, using TRa-I- mice, showing that in which is thought to be an important cardiac
the absence of TRa,, body temperature is re- risk factor (98-100).
duced (89). TRP,-'- mice have normal body
temperatures or metabolic rates, suggesting a 2.4.3 Carbohydrate Metabolism. Glucose
critical role for TRa in maintenance of meta- and carbohydrate use by non-hepatic tissue is
bolic rate (90). It is presently unclear whether markedly increased by thyroid hormones
stimulation of TRP, can cause increases in (101).The increased use occurs concomitantly
metabolic rate upon pharmacologic stimula- with increased glucose production through
tion, but studies suggest not only a potential liver gluconeogenesis (102). Thyroid hor-
role for TRP, in increasing metabolic rate, but mones increase peripheral glucose uptake pri-
a profile that may be different from that of marily through increased expression of glu-
TRa, stimulation (66, 91, 92). cose transporters such as GLUT-4 (103).
The ability of thyroid hormones to increase Whereas thyroid hormones can increase insu-
metabolic rate is critical for their capacity to lin release under certain circumstances, the
cause weight loss in hyperthyroid patients. increased uptake and use of glucose by tissue
Whereas uncoupling of mitochondria may such as skeletal muscle is relatively indepen-
contribute to increased metabolic rate, in- dent of insulin levels.
creased ATP turnover and use are most likely
more important. 2.4.4 Protein Metabolism. Excess thyroid
hormone causes a catabolic state character-
2.4.2 Lipid Metabolism. Thyroid hormones ized by increased metabolic rate as well as ac-
induce lipid synthesis, mobilization, and deg- celerated protein loss or wasting. Thyroid hor-
radation in a variety of tissues (93-95). Tri- mones cause both protein synthesis and
glycerides are turned over more rapidly and degradation, but under thyrotoxic conditions,
lipoprotein lipase and chylomicron triglycer- protein loss predominates. Variable degrees of
ide clearance are enhanced. Fatty acid synthe- muscle atrophy can occur depending on the
sis is increased in adipose tissue and liver, but thyroid state, as well as sympathetic input
degradation seems to be stimulated to a (68). Any thyromimetic design as a lipid-low-
greater extent (96). Therefore, adipose tissue ering or anti-obesity agent must not cause sig-
releases fatty acids at an accelerated rate after nificant loss of lean body mass or loss of skel-
thyroid hormone treatment. Thyroid hor- etal muscle mass and strength.
mones also increase sensitivity of lipolysis to
catecholamines (97). The increased triglycer- 2.5 Thyroid Hormone Effects
idelfatty acid cycling may also cause an in- on Specific Tissues
crease in oxygen consumption. Lipogenic en-
zymes in the liver are rapidly up-regulated, 2.5.1 Heart and Cardiovascular System.
including spot 14 and malic enzyme (90). Some of the most profound effects of thyroid
Plasma low density lipoprotein (LDL) cho- hormones are seen in heart. Thyroid hor-
lesterol levels are reduced by thyroid hor- mones cause dose-dependent increases in
mones. Hepatic LDL receptors are up-regu- heart rate (positive chronotropic effect) as
lated by thyroid hormones, causing increased well as effects on cardiac function (positive
LDL cholesterol uptake by liver (95). While inotropic and lusitropic effects) (72, 73). Car-
cholesterol synthesis rates are increased by diac output is increased by thyroid hormones,
thyroid hormones, cholesterol degradation and increases in ventricular muscle mass can
Thyroid Hormones and Thyromimetics

be observed (cardiac hypertrophy) (71, 104). creased expression of p-adrenergic receptors


Thyroid hormones typically lower peripheral (73,109). Thyroid hormone may also increase
vascular resistance and increase total blood the CAMP response to catecholamines, as well
volume. Studies in TRa-I- and TRP-I- mice as enhance the effects of CAMP. Increased cat-
suggest that much of the cardiac effects of thy- echolamine release is also reported for thyroid
roid hormone are caused by stimulation of hormones. Increased action or sensitivity to
TRa, (89), although some role for TRP may catecholamines may contribute to tachycar-
also be important (66,91). dia, ion channel disturbances, and arrhyth-
Total protein synthesis in the heart is in- mias in diseases such as hyperthyroidism.
creased by thyroid hormones, much of which
is consistent with observed changes in con- 2.5.2 Liver. An important site of action for
tractility and cardiac relaxation. The tran- thyroid hormones is liver, where numerous
scription of several critical genes is affected by metabolic processes are altered. Thyroid hor-
thyroid hormones, with the best characterized mones stimulate both lipogenesis and lipoly-
being myosin heavy chain (MHC), sarcoplas- sis. Lipogenic enzymes such as malic enzyme,
mic reticulum Ca2+ ATPase (SERCA), and fatty acid synthase, and the protein Spot 14
Na+/Kt ATPase genes (16). There are two are up-regulated rapidly (110 -112). Choles-
MHC genes (a and P ) with the a-gene predom- terol metabolism is also markedly affected,
inant in rodents and p-MHC predominant in and thyroid hormone can reduce serum cho-
man. a-MHC (myosin V1) has a higher veloc- lesterol, an effect caused by increased hepatic
ity of contraction and higher ATPase activity LDL receptor expression as well as increased
compared with p-MHC. Thyroid hormone in- cholesterol degradation. Thyroid hormones
creases the relative expression of a-MHC ver- may also regulate expression of other enzymes
sus P-MHC; this is caused by increases in involved with cholesterol metabolism (16).
a-MHC production as well as reduced P-MHC Cholesterol metabolism mav " also be affected
gene expression (105).The shift to the a-MHC by increased expression of apoA-1, which may
isoform is associated with increased contrac- explain the effects of thyroid hormones on
tility and energy use. Putative TREs have HDL cholesterol and the potential for "re-
been identified for both genes. verse" cholesterol trans~ort.
A

Several ion channels or transporters have Hepatic metabolic rate is increased by thy-
been shown to be affected by thyroid hor- roid hormones, and this is associated with pro-
mones. SERCA2 mRNA is markedly increased found up-regulation of Na+/K+ATPase levels.
by thyroid hormones (106).SERCA are critical Hepatic mitochondria1 respiration is also in-
for sequestration of calcium during relax- creased by thyroid hormones, and this may be
ation, and therefore, increased expression or caused by changes in mitochondrial density
activity of this pump can increase the rate of and respiratory enzymes, UCP expression,
ventricular relaxation. Other ion channels or and fatty acid "cycling."
transporters thought to be affected by thyroid
hormones are Kv1.5, KV 4.2, HCN2, HCN4, 2.5.3 Bone and Skeletal Muscle. Thyroid
and Na/Ca2+exchangers (16,107). Changes in hormones can increase both bone formation
these ion channels may be critical for the chro- and degradation and are essential for normal
notropic, action potential shortening, and ino- bone development (113, 114). Thyroid hor-
tropic effects of thyroid hormones. Thyroid mones increase both osteoclast and osteoblast
hormones may also exert acute effects on some activity, and indeed, cause enhanced calcifica-
critical cardiac ion channels such as sodium, tion accompanied by increased bone resorp-
and some of these changes can occur rapidly tion. Thyroid hormone may also indirectly af-
(108). Some of these rapid changes may result fect bone growth through effects on growth
from alternative, transcription-independent, hormone and consequently IGF-1. Whereas
T, signaling pathways (non-genomic),but this both bone formation and resorption are in-
is still under debate. creased by thyroid hormone, thyrotoxic pa-
Thyroid hormones also increase sensitivity tients often seem to be subject to net bone loss
to catecholamines, partially through in- and are at risk for osteoporosis (115).
2 Molecular Mechanism and Physiology of Thyroid Hormone Action 573

Table 11.1 Phenotypes of TR Receptor Knockouts


Name of
Targeted Remaining
Mutant Mice ReceptorM Phenotype References
TRal-'- TRa,, all Low heart rate and body temperature; aberrant
TRP repolarization by I,,; aberrant expression of
cardiac myosin; cerebellar development resistant
to hypothyroidism; deficient in cued response
test; aberrancies in brain cortex, decreased
lordosis behavior
all TRa Hyperthyroid; deaf; HR low-responsive to T3;
audiogenic seizures; deficiency in cholesterol
homeostasis; dysregulation of liver genes;
increased lardosis
TRa, Elevation of T,, T,, TSH; hypothyroid phenotype;
resistant to loss of weight by hypothyroidism;
pituitary and hypothalamic dysfunction; small
bone abnormalities; reduced body fat; low heart
rate; low body temperature; low basal metabolic
rate; cold intolerant; deaf; cochlear
misdevelopment; defective cholesterol
homeostasis, immense goiter
TRa,, all TRP Overexpress TRa, 6 X; mixed hypo- and
hyperthyroidism; low fertility
TRa, Rescue by elevated TRa, of deafness and pituitary
function, minor rescue of liver function
All TRa; No retinal M cones; color blind; mild elevation in
TRP, and TH, TSH
P3

Thyroid hormones cause changes in skele- been crossed to generate mice with combined
tal muscle that are consistent with other cell TR gene mutations: TRaIp1- TRp-I- (121),
types. Metabolic rate is increased concomi- TRa2-l- TRP-I- (122, 123),and the total TR
tantly with changes in mitochondrial density deficient mice TRa-I- TRP-I- (124).This sec-
and respiratory enzymes. Glucose uptake is tion reviews the phenotypic characteristics of
increased primarily because of up-regulation the various TR knockout mice (29).
of GLUT-4 (116). Thvrotoxicosis is associated TRP plays a key role in regulating the hy-
with net protein'turn&er, and therefore, mus- pothalamic-pituitary axis. This is manifested
cle wasting and weakness can be observed by the resistance to thyroid hormone and ele-
(16). Thyroid hormones have been shown to vated levels of TSH seen in the TRp-I- mice
up-regulate gene expression of ubiquitin-de- and even more pronounced in the TRa,-'-
pendent proteasomes, which account for the TRP-I- mice. Studies of these TR-deficient
major part of myofibrillar proteolysis and mice suggest that TRa, is important in con-
breakdown (117). trolling basal heart rate as well as body tem-
perature (89, 125). TRP is the major TR iso-
2.5.4 Receptor lsoform Knockouts. The form expressed in the liver, and it is therefore
role of the different subtypes of TR in vivo was not surprising that the cholesterol-lowering
partially clarified from the phenotypic charac- effect of thyroid hormone requires this TR in
terization of different TR knockout mice. A mice, demonstrated in the TRP-I- mice (126).
panel of mutated mice have been generated The phenotypic characteristics of most TR
and include mice that lack TRP-I- (118), knockout mice are summarized in Table 11.1.
TRp,-'- (301, TRa,-'- (891, TRa2-I- (1191, They reveal distinct and non-overlapping in
and TRa-I- (120). These mouse strains have vivo functions for the different TR subtypes.
Thyroid Hormones and Thyromimetics

Although distribution of the receptor isoforms Peripheral glucose uptake from gut and in pe-
may partially explain the differential tissue se- ripheral tissues is diminished, except in the
lective effects, differential receptor function brain.
cannot be ruled out. These results support the
proposal that isoform or subtype selective thy- 2.6.2 Hyperthyroidism and Thyrotoxicosis.
romimetics may prove to be of therapeutic Hyperthyroidism is a condition caused by in-
benefit (127). creases in thyroid hormone production or
secretion, whereas thyrotoxicosis is the meta-
2.6 Thyroid Diseases bolic response to increased T, or T, levels.
Therefore, the aforementioned terms are not
2.6.1 Hypothyroidism. Hypothyroidism is always synonymous. Examples of thyrotoxico-
a common thyroid-related disorder. This oc- sis associated with hyperthyroidism are as fol-
curs either through reduced thyroid produc- lows: Grave's disease (stimulatory TSH recep-
tion of thyroid hormones (primary or thyroi- tor antibodies), thyroid neoplasm or tumors,
dal hypothyroidism) or through diminished and less commonly drug-induced hyperthy-
pituitary TSH release by TRH (central hypo- roidism. The most common form of thyrotoxi-
thyroidism). Primary hypothyroidism is char- cosis without hyperthyroidism is excessive ex-
acterized by an elevated TSH in blood and of- ogenous thyroid hormone treatment.
ten results from destruction of thyroid tissue The clinical signs of thyrotoxicosis are var-
or altered hormone biosynthesis. This is ied but consistent with the known effects of
caused by autoimmune thyroiditis (e.g., thyroid hormones on tissues. Metabolic rate
Hashimoto's thyroiditis), irradiation, drug-in- increases are followed by increased appetite,
duced hypothyroidism, or infiltrative destruc- weight loss, hyperactivity, tremor, and cardiac
tion of thyroid tissue (e.g., sarcoidosis, palpitations. Despite the hyperactivity, a feel-
amyloidosis). ing of fatigue and weakness often accompanies
The effects of hypothyroidism are variable, thyrotoxicosis. The skin can be warm and
moist with abundant perspiration. Tachycar-
with symptoms ranging from overt to subclin-
dia is a common feature of thyrotoxicosis.
ical. Subclinical hypothyroidism is defined as
Ophthalmologic pathology is also noted and
increased TSH with little change in free characterized by a gritty sensation in the eyes,
plasma thyroid hormone concentrations. increased retroocular pressure, blurred vi-
While there is great variability in clinical sion, and photophobia. Ocular muscle pathol-
symptoms or signs, there are many common ogy can cause blurring as well as the classical
features. Gross symptoms include fatigue, Grave's ophthalmopathy characterized by a
lethargy, mental depression, weight gain, cold staring appearance of the eyes. Thyrotoxicosis
intolerance, dry skin, bradycardia, nonpitting can also be associated with osteoporosis
edema (myxedema), and constipation. caused by the preponderance of bone resorp-
The cardiovascular system is often affected tive activity that can be seen under this
by hypothyroidism with increased peripheral condition.
resistance concomitant with reduced meta- These signs and symptoms of thyrotoxico-
bolic demand. Cardiac performance is reduced sis are consistent with what is known about
both during systole and diastole. This is be- the action of thyroid hormones on various tis-
cause of modulation of numerous genes in- sues. Increased metabolic rate will cause
cluding MHC subtypes, SERCA, and respira- weight loss, and indirectly, tachycardia. Of
tory enzymes. Mean arterial blood pressure is course, thyroid hormones will also directly in-
usually unaltered, but cardiac output, blood crease heart rate and cardiac output. Muscle
volume, and cardiac mass are typically reduced. loss caused by increased protein turnover is
Lipid synthesis will typically predominate evident and may explain some of the muscle
over lipolysis, and therefore, hypothyroid pa- weakness. Sympathetic stimulation will exac-
tients have increased adiposity. Also, triglyc- erbate tachycardia, sweating, and increased
erides and LDL cholesterol are often elevated. metabolic rate.
3 Therapeutic Potential of Thyroid Hormone Analogs

3 THERAPEUTIC POTENTIAL 3.3 Obesity


OF THYROID HORMONE ANALOGS
Hyperthyroidism is associated with weight
loss, and this is most likely secondary to in-
3.1 Introduction
creased metabolic rate and reduced adiposity.
An obvious example of therapeutic use for thy- Thyrotoxic patients can also lose lean body
roid hormones is replacement therapy for hy- mass, which is not desirable, and therefore
pothyroidism. The purpose of this treatment metabolic rate can only be slightly increased
is to bring patients back to a "normal" meta- for there to be a selective loss of adipose tissue
bolic state. However in addition to replace- without undesirable cardiac and catabolic ef-
ment therapy, there is great potential for thy- fects. The dose-response curve for hyperme-
roid hormone agonists or antagonists for tabolism is so steep for T, that no therapeutic
treating a variety of disorders, although window exists. The requirement for a broad
achieving selectivity of action will be critical
dose range in which therapeutic 5-10% in-
for the development of such compounds. Se-
creases in metabolic rate can be observed is a
lective thyromimetics have long been sought.
Mechanistic explanations for some early selec- difficult hurdle to cross for thyromimetics.
tive agents focused on how affinity and trans- However, novel thyromimetics have been re-
port between cellular compartments (mem- ported that show promise in this regard, and
brane, cytoplasm, and nucleus) might be these will be discussed below. Obese patients
dissimilar among different cell types (65,138). are often at risk for diabetes. Selective thyro-
With modern understanding of the molecular mimetics may additionally provide an anti-di-
biology of thyroid receptors, it can now be en- abetic effect through glucose uptake by in-
visioned that selective agents might also re- creased skeletal muscle GLUT-4 activity.
sult from TR isoform selectivity or possibly
tissue specific transactivation arising from the 3.4 Cardiac Indications
selective recruitment of coactivator proteins
(139,140). Such newer approaches to design- Several studies have shown beneficial inotro-
ing safe and effective thyromimetics are start- pic effects for thyroid hormones (141). Thy-
ing to be described in the literature, and this roid hormones have been used for inotropic
chapter will discuss some key examples. support after surgical procedures such as cor-
The following is a short list of potential in- onary bypass and graft. Of course, these
dications for drug development of selective agents are non-selective and need to be used
thyromimetic drugs. acutely. The mechanism for the acute inotro-
pic effects are not clear, but several ion chan-
3.2 Lipid Lowering nels have been shown to be acutely affected by
Thyroid hormone analogs will reduce choles- thyroid hormones, perhaps through non-
terol levels by increased uptake and metabo- genomic mechanisms.
lism of LDL by the liver. HDL cholesterol may Interesting work from several investiga-
be maintained, or in some animal models, may tors suggested the possibility of efficacy for
be increased. Apo A1 is up-regulated by thy- congestive heart failure (73). This is based in
roid hormones and may exert a beneficial ef- part on the up-regulation of SERCA in myo-
fect through "reverse" cholesterol transport. cardium. This calcium ATPase is responsible
Lp(a) has been shown to be reduced by thyroid for re-uptake of calcium during diastole and
hormones, although this needs to be further would therefore be expected to improve dia-
studied. Thyroid hormone will reduce choles- stolic function. For such agonists to be useful,
terol in animal models but is associated with however, selectivity for SERCA would be crit-
numerous unwanted side effects at efficacious ical because cardiac acceleration and a hyper-
doses. Several agents have been reported that metabolic state would be contraindicated.
can lower plasma cholesterol without signifi- Some evidence has emerged suggesting that
cant cardiac effects, and these will be dis- TRP stimulation may up-regulate SERCA
cussed later in this review (66, 138). while exerting minimal heart rate effects (66).
576 Thyroid Hormones and Thyromimetics

This suggests the exciting possibility of selec- ment with a thyroid hormone analog in com-
tive improvement of diastolic function in the bination with a bone resorption inhibitor
failing heart. would result in net bone formation, which
TR antagonists may be useful for treating would be useful in treating osteoporosis (18).
cardiac arrhythmias. Amiodarone, which has
been characterized as possessing thyroid hor-
mone antagonist activity (however it also has 4 STRUCTURE-ACTIVITY RELATIONSHIPS
a myriad of ion channel effects), is a highly AND BIOLOGIC ACTIVITIES OF THYROID
efficacious anti-arrhythmic agent (142). By HORMONES AND ANALOGS
down-regulating the expression of cardiac po-
tassium channels, TRa blockade would be ex- 4.1 Introduction
pected to prolong action potential duration, The early foundations of structure-activity re-
increase refractory period, and therefore in- lationships (SAR) for thyroid hormone ana-
hibit reentrant arrhythmias. It is possible that logs were built largely through the use of in
a selective TRa antagonist would have class vitro receptor-binding assays and in vivo tests
I11 anti-arrhythmic activity without the re- for thyroid hormone activity, although inter-
verse use-dependency seen for many of the pretation of the latter may be complicated by
-
agents in this class that block repolarizing ion metabolic processes (activation or deactiva-
currents. tion) or pharmacokinetic factors. This discus-
sion will primarily focus on the in vitro SAR of
3.5 Hypothyroidism compounds. Although receptor isoforms and
Hypothyroidism was initially treated using subtypes for TR were uncovered in the late
thyroid gland extracts to replace the deficient 1980s,relatively little in vitro binding SAR for
hormone. Purification of thyroid hormones al- thyromimetics has been reported using the
lowed for effective and safe replacement ther- TRa and TRP receptor isoforms. In addition,
apy. Synthetic T, (Levothroid, Synthroid) is modern assays of nuclear hormone receptor
the most commonly used preparation, and it is activity now include transactivation assays in
well absorbed and has a long plasma half-life, genetically engineered reporter cell lines that
allowing for small daily fluctuation of plasma determine functional agonism and antago-
T,. A wide variety of pill strengths (25-300 pg) nism. Here again, there is relatively little data
allows titration that is readily followed using available for thyromimetics. In addition to de-
TSH and T, measurements. Accurate titra- lineating basic SAR, this section will also dis-
tion is critical to prevent excessive T, and as- cuss the in vivo activity of a selection of key
sociated cardiac, metabolic, and musculo-skel- examples from the published thyromimetic lit-
etal complications. T3 (Cytomel) is less erature. Excellent reviews of thyromimetic
advantageous because its rapid absorption SAR developed before 1996 are available
and short half-life can result in wide fluctua- (148-150).
tions in plasma levels.
4.2 Active Hormone Conformation
3.6 Other Disorders and Receptor-Ligand Structures
Thyroid receptors have been identified in skin It has long been established by a variety of
(143-145), a classic target tissue for thyroid experimental measurements and theoretical
hormone action. A topical T3-containing calculations that substitution at the R, and R,
cream has been shown to stimulate epidermal positions on the diphenylether scaffold of thy-
proliferation and dermal thickening (146). roid hormones and their analogs enforces a
The use of thyroid hormone analogs to treat perpendicular orthogonal relationship be-
skin atrophy has been proposed (147). tween the two aromatic rings as depicted in
As mentioned above, thyroid hormone in- Fig. 11.3 (i.e., where one ring is in the plane of
creases the activity of both osteoblasts and os- the page and the second is projecting out of the
teoclasts, and as a result, both bone formation plane of the page (151, 152). Furthermore,
and resorption are increased. However, treat- SAR studies, including the testing of confor-
Zure Relationships and Biologic Activities of Thyroid Hormones and Analogs 577

[Proximal] and imidazole ring of His-381 (TRa number-


ing) in the receptor seems to be important for
[Distal] binding as well as functional (agonist)activity.
Groups at R,' and R,' on the prime ring and
R, and R, of the non-prime ring bind into rel-
atively hydrophobic regions of the receptor.
The R, substituent, which generally contains
polar acidic functions such as the a amino acid
00 moiety in T, and T,, binds into a hydrophilic
Figure 11.3. Active hormone conformation. region of the receptor that features several ar-
ginine residues common to both receptor iso-
mationally constrained analogs, indicated forms. Interestingly, the amino group of T,
that for TH analogs in which R,' = H (e.g., seems to be deprotonated when bound to the
T,), the active conformation is one in which receptor because it is within hydrogen bond-
the R,' group is pointed away from the non- ing distance of the backbone amide nitrogen of
prime ring (153, 154). This orientation has Ser-271 (TRa numbering). This region of the
been termed the "distal" orientation, as op- ligand-binding cavity is also the location of the
posed to "proximal," where the group points only amino acid difference between the a and
back toward the non-prime ring (Fig. 11.3). p receptor isoforms (Ser-271in TRa and Asn-
X-ray crystallographic studies of the ligand- 331 in TRP) that exists in the immediate vicin-
binding domains of TRa and TRP with various ity of the bound ligands. This amino acid dif-
natural and synthetic ligands have fully cor- ference could theoretically be used in the
roborated the favored distal orientation of T, design of isoform selective thyroid hormone
and its analogs (155-158). analogs, although their similarity (both pos-
With the ability to clone and express the sessing neutral polar side-chains) presents
isolated ligand-binding domains of the thyroid significant challenges to selective thyromi-
hormone receptor isoforms, there are pres- metic drug design.
ently several X-ray crystallographic struc-
tures available of ligand-receptor complexes 4.3 Receptor Binding of Thyroid
(155-158). These structures reveal certain Hormone Analogs
common features of the various ligand-bind-
ing interactions of the natural hormone and 4.3.1 Background. Because of the rela-
its analogs. Merging this structural data with tively recent discovery of receptor isoforms for
established thyromimetic SAR, the nature of TR, the published in vitro SAR of thyromimet-
the ligand contacts to the receptor can be sum- ics contains little information about the bind-
marized as depicted in Fig. 11.4. The bound ing affinities of analogs to the TRa and TRP
ligand is fully engulfed by the LBD, suggesting receptors. Instead, early SAR work in the field
a role in protein folding and stabilization. A generated a considerable body of data using
hydrogen bond between the 4'-hydroxyl group competition binding assays with the readily

Figure 11.4. Hormone-receptor bind-


ing interactions.
578 Thyroid Hormones and Thyromimetics

Table 11.2 SAR of T, Analogs

R5'
In Vitroa In Vivob
Compound R4' R3' R5' X R3, R5 R1 (binding) (antigoiter)
(1)L-T3 OH I H L-Ala
(2) L-T4 OH I I L-Ala
(3)D-T3 OH I H D-Ala
(4) H I H DL-Ala
(5) OCH, I H L-Ala
(6) OH i-Pr H L-Ala
(7) OH H H L-Ala
(8) rT3 OH I I DL-Ala
(9) OH I H C0,H
(10) Triac OH I H CH,CO,H
(11) OH I H (CHz)ZCOZH
(12) OH I H (CHz),COzH
(13) OH I H DL-Ala
(14) OH I H L-Ala
(15) OH i-Pr H DL-Ala
(16) OH i-Pr H L-Ala
(17) OH i-Pr H DL-Ala
"Activity expressed relative to T3 = 100%in binding to solubilized and intact nuclear receptors from rat liver cells.
bActivityrelative to T3 = 100%for preventing goiter formation in the rat.

available nuclear TRs from sources such as rat that the in vivo activity of T, also reflects its
liver cells. I n vivo SAR was also studied pri- conversion to T, in most tissues via intracel-
marily using the classical rat antigoiter assay. lular deiodinase activity. The D-alanine ana-
In this assay, thyroid hormone activity was log (3)retains significant binding affmity and
measured as the ability of the test compound moderate in vivo activity. In compound (41,
to suppress TSH release and goiter formation some in vivo activity is retained likely result-
that is induced by antithyroid drugs such as ing from metabolic hydroxylation at the 4' po-
thiouracil(148).Because TSH suppression is a sition. Consistent with the binding model in
TRP process, it can be now appreciated that Fig. 11.5, compound (5) shows weak binding
the antigoiter assay suffers from the limita- caused by the loss of a critical H-bond interac-
tion that it is insensitive to the test com- tion. However, in vivo potency is retained in
pound's binding to the TRa receptor isoform.

4.3.2 Summary of Basic Thyromimetic


SAR. Table 11.2 summarizes key SAR fea-
tures, in vivo (antigoiter assay) and i n vitro, of R ~ J ~ R
analogs of T, (L-3,5,3'-triiodothyronine) (148,
153,159). As mentioned earlier, T, (1)is more R4' R1
active than T, (2) both in vitro (receptor-bind- R5'
ing affinity) and in vivo. Although the relative
in vitro and in vivo activities between these Figure 11.5. Thyroid hormone pharmacophore
compounds is consistent, it must be reiterated SAR.
4 Structure-Activity Relationships and Biologic Activities of Thyroid Hormones and Analogs 579

Table 11.3 SAR of 3'-Position of Thyromimetics

*u:L2
HO /

In Vitro
/

COOH
In Vitro
Compound R3' Binding* Compound R3 ' Binding"
(6) H 0.08 (30) F 0.76
(18) Methyl 0.6 (31) C1 5.1
(19) Ethyl 56 (32) Br 13
(20) nPropyl 45 (1) L-T3 I 100
(21) n-Butyl 44 (33) OH 0.04
(22) n-Pentyl 32 (34) CH,OH 0.39
(23) n-Hexyl 60 (35) CH,CH,OH 0.45
(7) i-Propyl 87 (36) COOH 0.004
(24) t-Butyl 25 (37) CH,COOH 0.03
(25) Cyclohexyl 9.8 (38) CH,NH, 0.01
(26) CH,-Cyclohexyl 36 (39) CHO 0.18
(27) Phenyl 7.3 (40) CO-CH, 0.37
(28) CH,-phenyl 22 (41) CO-Ph 0.37
(29) CH,CH,-phenyl 1.8 (42) NO2 0.13
"Binding to intact rat liver nuclei, relative to T3 = 100%.

(5)through metabolic activation, in this case drop in binding affinity. However, substitu-
de-alkylation of the 4' methyl ether. An impor- tion with methyl (16) or isopropyl (17) is far
tant modification commonly used in thyromi- more deleterious to binding.
metic structures is the replacement of the 3' The successful replacement of the 3' iodo
iodo substituent by isopropyl as shown in com- group in T, by isopropyl has been followed by
pound (6). This change retains the full binding many thyromimetics featuring a variety of 3'
affmity of T, and significantly improves the in groups. Table 11.3 illustrates the SAR at the
vivo antigoiter activity because (6) is impervi- 3' position for binding to thyroid hormone re-
ous to deactivation by 3' deiodinase activity. If ceptors from intact rat liver nuclei (160).
the prime ring is missing both the R,' and R,' Among alkyl substituents (18-261, isopropyl
substituents (7) or if the non-prime ring has seems to be optimal. A 3' phenyl group (27)
only one group at R, or R, (B), an analog exhibits about 10% of the binding affinity of
known as reverse-T, or rT,, activity is signifi- T,, and the addition methylene spacers (28,
cantly reduced. Compared with the large loss 29) have little effect. Iodine is clearly the best
of binding affinity incurred by removing the 3' halogen group at 3' (30-32). Compounds (33-
iodo of T,, considerable variation at R, is tol- 42) show that polar groups at 3' bind very
erated (9-12), with improved binding result- poorly relative to T,.
ing from removing the amino group in com- The following is a summary of the essential
pound (10) and its homolog (11).Compound in vitro SAR of thyroid hormone analogs de-
(10)is also known by its trivial name, "Triac," veloped before 1990 (Fig. 11.5). At R,, the L-
which is derived from triiodo and acetic (refer- amino acid moiety found in T, is not critical
ring to its R, group). Further homologation for activity, and the amino group can be dis-
(12) results in a loss of binding affinity. Hydro- pensed with entirely. A recent report de-
phobic, one atom linker replacements for oxy- scribes the successful incorporation of a thia-
gen at "X,"such as methylene (13)and sulfur zolidinedione acid surrogate (161). Relatively
(14), are successful. At R, and R,, bromine little information is available on analogs with
(15)substitutes for iodine with only a modest substituents at R, and its companion position,
Thyroid Hormones and Thyromimetics

R,. Positions R, and R, are essentially always lectivity of functional (agonist and antagonist)
substituted symmetrically with halogens or activity determined using TR-mediated tran-
small alkyl groups. Polar substituents are not scription assays. Thyromimetic SAR for func-
well tolerated. The linker X is a single atom, tional activity will also be described in the con-
oxygen being most common, but sulfur and text of both TR receptor isoforms and TR
methylene are also acceptable. On the prime response elements.
ring, R,' and R,' have rarely been substituted,
although successful simultaneous substitu- 4.4 Selected Biologically Active Thyroid
Hormone Analogs
tion at R,'and R,' has been reported by replac-
ing the prime ring with a tetrahydronaphthyl
4.4.1 Background. Although thyroid hor-
or naphthyl ring (162). As discussed earlier,
mones are associated with a variety of biologic
R,' is optimal with iodine or isopropyl, and activities, medicinal chemistry has primarily
varying degrees of suitable affinity can be re- focused on their lipid-lowering properties, an
tained with non-polar alkyl, aralkyl, or aryl emphasis motivated by the large market po-
groups. However, polar groups at R,' mark- tential and significant cardiovascular health
edly reduce binding affinity. Simultaneous benefits of such drugs. A key to the success of a
substitution at R,' and R,' generally leads to thyromimetic lipid-lowering drug is the ab-
poorer binding (i.e., T, versus T,). The phe- sence of undesirable cardiac effects. The un-
nolic hydroxyl group at R,' is critical to both natural enantiomer of T, was once believed to
binding and agonist activity, and a structural be such a compound. However, in human clin-
role in hydrogen bonding to a histidine side- ical trials, D-T, caused greater mortality than
chain imidazole ring from the receptor has placebo (167). Although there have been sub-
been elucidated through X-ray studies (as de- sequent reports of selective thyroid hormone
scribed earlier). There are no known signifi- analogs with the potential to safely lower lipid
cant alternatives to a phenol moiety at R,'. levels in man (discussed below), research ac-
For some of the newer thyromimetics, it tivity in this area has been somewhat modest
has become clear that the classic SAR conclu- because other approaches, especially the
sions based on T, may not hold for compounds HMG-CoA reductase inhibitors, have found
with different groups at R,. For example, significant success as established and safe
when the R, group is an oxamic acid (- NH- therapy. Recently, more research is being re-
COCOOH), the analog with methyl substitu- ported on the weight-reducing potential of
ents at R, and R, binds to rat liver nuclear TRs thyromimetics and their applicability to obe-
better than T, (163). This result contrasts sity, a major health problem in essentially all
with the comparison of T, to compound (16)in developed nations. The data for selected
Table 11.2 in which methyl substitution at R, thryomimetics with therapeutic potential are
and R, causes about a 100-fold loss in binding discussed below. Two excellent recent reviews
affinity. Further examples of SAR divergence are available (18, 168).
can be expected with thyromimetics having R,
groups differing from the a amino acid found 4.4.2 1-T, (Liothyronine), 1-T, (Levothyrox-
in T,. he), and Triac. Of the naturally occurring
Very little SAR has been reported describ- thyroid hormones available for therapeutic
ing the receptor isoform-binding affinities of use, L-T, (2, levothyroxine) is more commonly
thyroid hormones and their analogs. Although used than L-T, (1, liothyronine). Levothyrox-
there is some variation perhaps reflecting dis- ine is indicated for TH replacement therapy or
similar assays, several reports (164-166) indi- supplemental therapy for hypothyroidism. Be-
cate that T, does not discriminate in binding cause T, and T, are not selective in their in
to the TR isoforms (less than a twofold differ- vivo activities, they are not used for therapeu-
ence in binding affinity between the TRa and tic weight reduction. In man, the sodium salt
TRP receptors ). Future discussions of thyro- of (2) is well absorbed from the GI tract, and
mimetic in vitro SAR can be expected to ad- the drug is principally metabolized through
dress not only binding selectivity but also se- deiodination processes and conjugation to
4 Structure-Activity Relationships and Biologic Activities of Thyroid Hormones and Analogs 581

patients (combined thyroidectomy and radio-


iodine ablation for treatment of thyroid carci-
noma) compared the effects of Triac with L-T,
at equally TSH suppressive doses (170). Triac
significantly reduced total and LDL choles-
terol and increased serum sex hormone bind-
ing globulin (SHBG), whereas L-T, showed
little effect, suggesting increased liver-specific
actions for Triac relative to L-T,. Triac also
increased serum osteocalcin and urinary cal-
cium excretion, whereas L-T, showed no sig-
glucuronides and sulfates (which are excreted nificant effects. Metabolic rate, pulse, and
into the bile and gut where they undergo en- mean arterial pressure showed little change
terohepatic recirculation) (169). relative to baseline for treatment with either
Triac (lo),which is a known metabolite of drug. Despite these promising organ-specific
T,, has been shown to be of potential benefit activities, the changes in serum parathyroid
hormone and urinary calcium excretion in-
duced by Triac elicited concern that acceler-
ated bone demineralization may limit the
compound's therapeutic potential.

4.4.3 SKF-94901. SKF-94901 (43) was the


first thyroid hormone analog designed to take

(10) Triac

for the treatment of resistance to thyroid hor-


mone (RTH). RTH is a genetic disorder char-
acterized by diminished responsiveness to
thyroid hormones in the pituitary and periph-
eral tissues caused by mutations in the LBD of
the TRP, receptor. In a study comparing Triac
to T,, both agents were reported to bind TRa,
equally, but Triac showed 2.7-fold greater af-
finity for TRP, (164). In the same study, Triac HO Br COOH
produced higher maximal transactivation
than T, and attained 50% induction at a lower
concentration than T, in a TR@,lTRE-chlor-
amphenicol acetyltransferase (CAT) reporter advantage of the less stringent SAR at the 3'
assay run in transiently transfected COS-1 position on the prime ring of the thyromimetic
cells. In contrast, Triac activated TRa, to the pharmacophore, resulting in a major struc-
same degree as T,. With these results, it was tural departure from the natural thyroid hor-
argued that administration of Triac in cases of mones (138,160,171).Although replacing the
RTH augments TRP, activity with less TRa- iodine normally present at 3' with a meth-
mediated hyperthyroid effects. A second re- ylpyridazinone group markedly reduces bind-
port describing TR isoform specificity of Triac ing affinity (one-tenth the binding of T, to iso-
also supported these findings and furthermore lated rat liver nuclei), in animal models,
went on to show that Triac regulation of tran- compound (43) is an effective cholesterol-low-
scription can vary between different TREs ering agent with cardiac sparing properties. In
(166). Triac has also been investigated for po- addition, (43) increases metabolic rate and de-
tential therapeutic applications to hyperlipid- creases plasma TSH, properties consistent
emia and osteoporosis. A study in athyreotic with thyroid agonist activity. The absorption,
582 Thyroid Hormones and Thyromimetics

Table 11.4 SAR of Oxamic Acid Series

Compound
(1)L-T3 1.1
(44) Isopropyl H, H H OH 48
(45) Isopropyl 1, I H OH 0.10
(46) Isopropyl Br, Br H OH 6.0
(47) CGS-23425 Isopropyl '333, CH3 H OH 0.19
(48) Isopropyl (333, CH3 CH3 OH 120
(49) Isopropyl CH3, (3% H NH2 0.95
(50) Isopropyl '333, CH3 H OEt 0.23
(51) -CH,-p-C1-phenyl C1, C1 H OH 0.27
(52) -CHOH-p-C1-phenyl C1, C1 H OH 1.5
(53) -CHOH-p-F-phenyl CH3, CH3 H OH 0.8
(54) CGS-26214 -CHOH-p-F-phenyl '333, CH3 H OEt 0.16
(55) Cyclohexyl '333, CH3 H OH 2.2
(56) Phenyl '333, CH3 H OH 1.9
"Binding to isolated rat liver nuclei.

distribution, metabolism, and excretion prop- myocardial damage, none of the SKI?-94901
erties of (43) were investigated in the rat, dog, treated rats died before termination of the
and cynomolgus monkey (172). Evidence from study and their hearts showed no damage at
these studies show that bromine substitution autopsy. However, SKF-94901 did give rise to
at R, and R, blocks deactivation by deiodi- fibroplasia on peritoneal surfaces, the appear-
nase-like mechanisms and that there is less ance of multinucleate cells in the distal tu-
conjugation (sulfation or glucuronidation) of bules of the kidneys, and formation of new
the phenolic hydroxyl at R,', presumably be- trabecular bone in the femur. These effects
cause of the steric hindrance presented by the were also observed in the T,-treated animals.
pyridazinone ring. Because (43) binds equally As a result of these effects, the development of
to TRa and TRP, receptor isoform specificity SKF-94901 was suspended (149,174).
cannot account for the compound's selective
actions (hepatoselective and cardiac sparing) 4.4.4 Oxamic Acids. As discussed earlier,
in vivo (173). Evidence has been presented when the R, amino acid moiety present in the
that the biological specificity of SKI?-94901 is naturally occurring thyroid hormones is re-
caused by selective tissue uptake. The com- placed by an oxamic acid group, there is a di-
pound shows reduced binding to proteins in vergence from the classical SAR at positions
the cytoplasm of cardiac cells (65) and en- R, and R,. More of the SAR of the oxamic acid
hanced concentration in hepatic nuclei (173). series is presented in the analogs shown in
The processes by which SKF-94901 preferen- Table 11.4 (163,175).Compounds (44-46) ex-
tially enters certain cells remain unclear. hibit the familiar trend of iodo better than
SKI?-94901 was evaluated for safety in a 30- bromo and much better than lack of substitu-
day study in rats at doses of 0.02-10 mg/kg/day tion at R, and R,. However, analog (47), also
(with a comparator study of T, given at 1 mg/ known as CGS-23425, shows that affinity for
kg/day). Although many of the T,-treated rats TRs nearly equivalent to that of T, is achieved
died with their hearts showing evidence of with methyl substitution. In compound (481,
4 Structure-Activity Relationships and Biologic Activities of Thyroid Hormones and Analogs

N-methylation on the oxamate group mark-


edly reduces binding affinity. However, in yet
another departure from earlier established
SAR, the neutral oxamic amide (49) and ester
(50) both exhibit potent receptor binding com-
parable with T,, showing that an acidic func-
tion at R, is not strictly required. Aryl substi-
tution at 3' similar to the strategy used to (55) R = cyclohexyl
develop SKF-94901 also yields potent oxamic (56) R = phenyl
acids. The best analogs include those in which
the one atom linker between the aryl group
and the 3' position is either methylene terol in a dose-dependent manner, with a min-
(--CH,-) as in (51) or hydroxymethylene imum effective dose of 1 pgkg, giving a 35%
( 4 H O H - ) as in compounds (52, 53, and reduction after 7 days of drug treatment. The
54). Analog (54), another example of a potent safety of CGS-26214 was tested in conscious
oxarnate ester, is also known as CGS-262214. dogs treated for 7 days at 100-fold the minimal
Oxamic acids with cyclohexyl(55) and phenyl effective lipid lowering dose and compared
(56) directly attached to the 3' position are with L-T, given at 10-fold above its lipid-low-
also good TR binders. The in vivo activities of ering dose. Although there were slight in-
(47,54,55), and (56) are discussed below. creases in heart rate and whole body oxygen
consumption, these effects were not statisti-
cally significant, and the authors concluded
that (54) was free of cardiac and thermogenic
effects. CGS-26214 exhibited a 100-fold pref-
erence for binding in vitro to nuclei of HepG2
cells over cardiac myocytes, thus implicating
selective tissue uptake to account for the
compound's cardiac selectivity. Although
CGS-26214 advanced to phase I clinical trials
for treatment of hyperlipidemia, development
of the compound was subsequently discontin-
ued (176).
CGS-23425 (47) was the second oxamate to
be discussed for its potential selective lipid-
lowering properties (177, 178). In hypercho-
lesterolemic rats, CGS-23425 gave a 44% de-
crease in LDL cholesterol with 10 mgkglday
HO

HO
H
a COOEt
doses administered over 7 days. Rats given up
to 10 mgkg doses of (47) were free of cardio-
toxicity, but adverse cardiac effects began to
appear at 40 mgkg. This margin of safety was
reported to be similar to that of CGS-26214
(and SKF-94901). Based on earlier reported
CGS-26214 (54) was the first oxamate to be correlations of L-T, cardiac effects with its
reported with extensive biologic studies in binding to plasma membranes (179),the bind-
vivo and in vitro. The compound has been pro- ing of (47) to hepatic plasma membranes was
posed to be a potent cholesterol-lowering shown to be relatively weak compared with T,
agent free of cardiovascular and thermogenic and SKF-94901, but similar to CGS-26214. In
activity with a reported 25-fold dose separa- transient transfection assays in human fetal
tion between minimal lipid lowering and the hepatoma cells using CAT reporters with rat
observation of cardiovascular effects (74). In apoAl promoters, compound (47) increased
hyperlipidemic rats, (54) reduced LDL choles- transcription with an EC,, = 0.002 nM for
Thyroid Hormones and Thyromimetics

TRP, versus an EC,, = 1 nM for TRa,. Citing binding selectivity for the TRP isoform. Tran-
the greater abundance of TRP, in the liver scriptional activation by GC-1 was also TRP
versus TRa, in the heart (180, 181), it was selective when measured in HeLa cells trans-
proposed that subtype selectivity contributes fected with TRE-luciferase reporter plasmids
to the hepatic selectivity and relative cardiac and either human TRa, or TRP, expression
safety of CGS-23425. plasmids. GC-1 was a full agonist under these
The cholesterol-loweringactivities of 3'-cy- conditions. The structure of GC-1 is halogen-
clohexyl (55) and 3'-phenyl (56) oxamic ana- free and features an oxyacetic acid group at R,
logs show an interesting divergence. Both along with a distinctive one carbon-linker
compounds show similar in vitro receptor- (-CH,-) joining the prime and non-prime
binding affinities (see Table 11.4) and give phenyl rings. An X-ray structure of (57) bound
equal cholesterol lowering in hypercholester- into the LBD of TRP, is available (158). It sug-
olemic rats (4050% reduction after 20 p&g gests that the observed P selectivity is because
oral doses administered over 7 days). How- of a greater opportunity for H-bonding inter-
ever, in rats given drug at 25 mg!kg for 7 days, actions with the R, group of GC-1 in the com-
the 3'-cyclohexyl analog (55)caused increases plex with TRP than with TRa.
in atrial heart rate, atrial tension, and heart KB-141 possesses a structure reminiscent
weight, whereas the 3'-phenyl analog (56) of Triac, with the difference being the pres-
showed no significant effects. No explanation ence of chloro at R, and R, rather than iodo.
was provided for this seemingly subtle struc- The in vitro activity of (58)also reflects a mod-
tural change on in vivo SAR (175). erate level of selectivity for the P receptor (91).
KB-141 has an IC,, = 24 nM for TRa, and an
4.4.5 CC-1 and KB-141. Studies in TRa IC,, = 1.1 nM for TRP, (IC,, is the concentra-
and TRp-I- mice suggest that selective TRP tion achieving 50% inhibition of the binding of
selective agonists may reduce cholesterol with radiolabeled T,). This is in contrast to Triac,
less heart rate effects. The role of TRP on met- which has been reported to show only a 2.7-
abolic rate has only recently been described fold greater affinity for TRP. In CHO cells ex-
(91, 92). Knowledge of the potential of this pressing human TRa, or TRP,, KB-141 is a
receptor isoform for drug development has full agonist and is TRP selective, being essen-
been increased by studies of the modestly se- tially equipotent to T, for the P receptor but
lective TRP agonists, GC-1 (57) and KB-141 about 10-fold less potent than T, for the a
receptor. T, is slightly TRa selective in this
assay.
Both GC-1 and KB-141 have been shown in
animal models to lower cholesterol at doses
free of undesired cardiac effects. In hypercho-
lesterolemic rats, GC-1 lowered cholesterol
levels at doses where there was no evidence of
increased heart rate (66, 92). In cholesterol-
fed rats given drug orally over 7 days, GC-1
(58). GC-1 binds human TRa, receptors with was 28-fold selective for cholesterol lowering
KD = 0.44 nM and TRP, receptors with KD = versus tachycardia relative to T, (which was
0.067 nM (165). Thus, GC-1 shows modest non-selective). Tissue distribution studies in-
dicated that GC-1 is localized to a greater ex-
tent in the liver versus the heart, thus impli-
cating selective tissue uptake as part of the
explanation for the compound's activity. KB-
141 was similarly 27-fold selective for choles-
COOH terol lowering versus tachycardia relative to
HO T, in cholesterol-fed rats treated with drug
orally over 7 days (91). In the rat, KB-141 also
shows a lower heartlplasma distribution ratio
and Things

than T,. This suggests that favorable tissue darone (501, have been shown to antagonize
uptake may also contribute to this com- TR activity in vitro (142, 183, 184). However,
pound's selective biologic activity. Because there are no reports of demonstrated TR an-
both compounds show in uitro selectivity for tagonist activity in cells or in uiuo for deseth-
TRP over TRa, receptor isoform selectivity ylamiodarone. Amiodarone itself has low bind-
has also been proposed to play a role in their ing affinity for TRs, and as a pharmacologic
activity profiles. agent, it is associated with numerous activi-
Both GC-1 and KB-141 give modest, but ties (183,185). The research group that devel-
significant, separations between increasing oped GC-1 also reported the synthesis and TR
metabolic rate and cardiac effects when given antagonist properties of the related analog
orally over 7 days to cholesterol-fed rats (91, (59) (186). Compound (59) was designed by
92). GC-1 showed a sevenfold dose window for
a 5-10% increase in metabolic rate versus a
15% increase in heart rate. For the same pa-
rameters, KB-141 exhibited a 10-fold dose
window. Under the same experimental proto-
col, T, was non-selective for the separation of
metabolic rate from tachycardia. In addition,
both GC-1 and KB-141 gave dose-response
curves for metabolic rate that were signifi-
cantly shallower than that of T,. It is presently
unclear whether a 10-fold dose range is large
enough to safely and effectively cause weight
reduction. It is also unclear whether TR ago-
nist-mediated increases in metabolic rate can
be achieved without increasing appetite,
thereby nullifying any anti-obesity effects.
Nevertheless, these agents support the sug- comparing the X-ray structures of the LBDs of
gestion that selective thyromimetics may find TR (155) and the estrogen receptor (1871, and
application in the treatment of obesity with by modeling the binding to ER of ICI-164,384
the anticipated benefit of cholesterol-lowering (60), a known ER antagonist (188-190). Com-
activity (182).

5 RECENT DEVELOPMENTS
AND THINGS TO COME

5.1 Antagonists
TR activation has been associated with ion
channel disturbances, action potential short-
ening, and arrhythmias, particularly in hyper-
thyroid patients (73). The idea that TR antag-
onists may represent a potential means of pound (59)binds TRa, with K,,= 112 nMand
inhibiting arrhythmia generation has been TRP, with KD = 148 nM. In a cell-based trans-
further suggested by the clinical efficacy of activation assay, (59) shows competitive an-
arniodarone, which among other activities, tagonism.
may be a TR antagonist. Although safe and Another approach recently used to identify
effective thyroid antagonists are potentially of antagonists of NHRs has been to devise com-
therapeutic benefit, progress in this area has pounds that block the receptor's ability to as-
been limited by a lack of proven TR antago- sume the conformation needed for coactivator
nists. The antiarrhythmic drug amiodarone binding. DIBRT (63) was designed to feature a
(61) and its major metabolite, desethylamio- 5'-isopropyl group that would disrupt the po-
Thyroid Hormones and Thyromimetics

(61) Amiodarone: R = Et
(62) Desethylamiodarone: R = H RTH therapy. HY1 (64) was designed using
GC-1 (44) as a starting point because of its
intrinsic p selectivity (193). The target was
the RTH-associated mutation, TRp(R320C),
for which T, shows reduced affmity (194,195).
The available X-ray structure of the T,ITRP
complex (155) was used to design compound
(64), in which a neutral alcohol group at R,
better accommodates the mutational replace-
ment of arginine by the neutral amino acid,
cysteine. In a transactivation assay, HY1 was
(63) DIBRT five times more potent than GC-1 as an ago-
nist for the mutant receptor, TRp(R320C),
thus demonstrating the potential for this ap-
sitioning of helix-12 of the TR LBD (191). He-
plication of structure-based design.
lix-12 has been shown to be a key component
It is anticipated that the future of drug de-
of the coactivator binding surface of many
sign for small molecule modulators (agonists
NHRs (187, 192). Although a relatively weak
competitive inhibitor (IC,, - 1 pA4 for both
and antagonists) of nuclear hormone recep-
tors will involve more than solely considering
TRa and TRP), DIBRT showed modest antag-
X-ray structures of receptor-ligand complexes,
onist potency blocking both a positive TRE re-
such as in the exercise described above. This
sponse and a negative T, response, measured
type of structure-based design will be supple-
with the TSHp promoter (cellular assays). In
mented by new technologies that take into
similar assays, full agonist efficacy was dis-
consideration more of the full complexity
played by the corresponding analog in which
known for the molecular mechanism of tran-
the 5'-isopropyl was replaced by hydrogen.
scriptional regulation by NHRs (as described
With the ever increasing knowledge of
earlier). The LBDs are only one part of the
structure-function in the nuclear hormone re-
multi-domain structure common to these re-
ceptor family, more examples of designed
ceptors. Furthermore, the surfaces of the re-
antagonists for TR can be anticipated. The re-
ceptors interact with multiple and diverse co-
sulting molecules should find useful applica-
regulatory proteins, with the resulting
tion to further elucidate the biology of thyroid
complexes further interacting with the impor-
hormones and may yet lead the way to devel-
tant histone modifying enzymes, HAT and
oping therapeutically useful TR antagonists.
HDAC. Recently introduced technology uses
phage display to detect the different receptor
5.2 Drug Design
conformation states induced by ligands associ-
As discussed earlier, RTH is a disorder with ated with distinct biologic activities (agonism,
genetic origins traced to various mutations in antagonism, etc.) (196). When applied to the
the ligand-binding domain of the TRp recep- estrogen receptor (ER), this method success-
tor isoform. Reasoning that compounds pos- fully classified known selective estrogen recep-
sessing affinity and selectivity for mutant tor modulators (SERMs) with distinct, ligand-
forms of TRP versus TRa might be useful for induced receptor surface shapes (139, 140).
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CHAPTER TWELVE

Fundamentals of Steroid
t Chemistry and Biochemistry
ROBERT W. BRUEGGEMEIER
PUI-KAILI
Division of Medicinal Chemistry and Pharmacognosy
College of Pharmacy
The Ohio State University
Columbus, Ohio

Contents
1 Introduction, 594
2 Steroid Chemistry, 595
2.1 Structure and Physical Properties
of Steroids, 595
2.2 Steroid Nomenclature, 597
2.3 Chemical Synthesis and Microbial
Transformations of Steroids, 599
2.3.1 Synthesis of Cortisone, 599
2.3.2 Microbial Steroid Biotransformations,
602
2.3.3 Selected Chemical Reactions of
Steroids, 604
3 Steroid Biochemistry, 606
3.1 Steroidogenesis, 606
3.1.1 Cholesterol Biosynthesis, 606
3.1.2 Formation of Pregnenolone, 609
3.1.3 Biosynthesis of Adrenal Steroids, 612
3.1.4 Biosynthesis of Progesterone, 616
3.1.5 Androgen Biosynthesis, 616
3.1.6 Estradiol Biosynthesis, 618
3.2 Steroid Metabolism, 620
3.2.1 Corticosteroids Metabolism, 620
3.2.2 Progesterone Metabolism, 620
3.2.3 Androgen Metabolism, 620
3.2.4 Estrogen Metabolism, 620
3.3 Steroid Hormone Action, 622

Burger's Medicinal Chemistry and Drug Discovery


Sixth Edition, Volume 3: Cardiovascular Agents and
Endocrines
Edited by Donald J. Abraham
ISBN 0-471-37029-0 0 2003 John Wiley & Sons, Inc.
593
Fundamentals of Steroid Chemistry and Biochemistry

1 INTRODUCTION unorthodox, this technique of preparing tissue


extracts and evaluating the effects of the ex-
Steroids are a unique class of chemical com- tracts was eventually adapted for the isolation
pounds that are found throughout the animal of biologically active constituents from the ex-
and plant kingdom, and this class includes ste- tracts.
rols such as cholesterol and ergosterol, bile ac- The modern era of steroid research began
ids, and steroid hormones. Modern scientific with steroid chemistry in the early 1900s. Dr.
research on steroid chemistry and biochemis- Adolf 0. Windaus, a Gottingen chemist,
try began in the early twentieth century, and worked for over 20 years on the isolation of
several major treatises on the subject have steroids, development of assays for detecting
been published (1-9). This chapter is intended steroids, and the use of classical chemistry to
to provide a general summary of the steroid elucidate steroid structures (12). Dr. Windaus
chemistry and biochemistry rather than a received the Nobel Prize in 1928 for his re-
comprehensive review. Subsequent chapters search on the "constitution of sterols." During
in this current volume of Burger's Medicinal the same period, Dr. Heinrich 0.Wieland of
Chemistry contain more detailed discussions Munich was engaged in natural products
on the individual classes of steroids. chemistry, including bile acids (13), and was
The biological and medical significances of awarded the Nobel prize in 1928 for his re-
steroids have been observed since ancient search on this subject. The original chemical
times, even though the exact chemical nature structures of cholesterol and other steroids
and properties of steroids began to be under- proposed in 1928 were subsequently found to
stood only in the late 1920s and early 1930s. In be incorrect, and correct structures were iden-
the literature of the ancient Greeks, Hip- tified in 1932 (14-16). Chemical studies on
pocrates referred to gallstones and the term compounds involved in reproduction began in
cholesterol is derived from the Greek terms for the 1920s,and in 1929 Adolf F. Butenandt and
bile (chole) and solid (steros). Aristotle identi- Edward A. Doisy independently reported the
fied the effects of castration in birds and in isolation of an active steroid sex hormone. es-
humans, and agricultural practices of castra- trone, from the urine of pregnant women (17,
tion to produce sterility, alter aggressiveness, 18). Throughout the 1930s, many steroid hor-
and affect body size in domestic animals has mones were isolated and structures deter-
been known since early times. Egyptians and mined, including progesterone by Butenandt
Romans used extracts of plants such as purple (19) and corticosteroids by Reichstein (20).
foxglove to treat dropsy. The physical effects The synthesis of steroids followed shortly
of castration were well recognized in eunuchs thereafter by research groups led by Bach-
and in medieval castrati choirs. mann, Woodward, Robinson, and Cornforth
Scientific observations in more modern (21-23).
times began examining the biological conse- Steroid biochemistry began with studies on
quences of hormones without the realization the biosynthesis and metabolism of steroids,
of nature of the chemicals involved. Berthold, and early studies in the 1930s and 1940s used
a Gottingen physiologist, reported the effects large amounts of unlabeled compounds (6).
of implanted testis in studies with cocks in With the production of radiolabeled mole-
1849 (10). In 1855, Addison discovered the re- cules, studies then used more physiological
lationship of the adrenal glands with a partic- levels of steroids and steroid precursors, with
ular disease characterized by bronze skin color an early significant demonstration that all the
( l l ) , and this disease of chronic adrenal insuf- carbon atoms of cholesterol are derived from
ficiency is now referred to as Addison's dis- the two carbons present in isotopically labeled
ease. In 1889 Brown-Sequard prepared a tes- acetate (24). Research on steroid biosynthesis
ticular extract and tested on himself, and metabolism began in the 1950s and 1960s
reporting enhanced rejuvenation. Although and continues to the present time. Studies on
2 Steroid Chemistry

4 6

cyclopentanoperhydrophenanthrene

Figure 12.1. Basic steroid structure. Figure 12.2. Side-chains on steroid scaffold.

the biochemical mechanism of action of ste- phenanthrene, describing the three rings of
roids began in the late 1950s with the use of phenanthrene (rings A, B, and C) and the cy-
tritiated estrogens of high specific activity. clopentane ring (ring Dl. In steroids, the
Jensen and Jacobson reported the accumula- phenanthrene ring system is completely satu-
tion of physiological levels of estrogen in tar- rated (hydrogenated) and is thus referred to as
get organs and postulated the presence of a a perhydrophenanthrene. This steroid scaf-
receptor (25). Research on the biochemistry fold contains 17 carbon atoms, and the num-
and molecular biology of steroid hormone ac- bering of the carbon atoms begins with the
tion has exploded in the past few decades and carbons of the phenanthrene and is then fol-
constitutes a major effort in the field today. lowed by numbering the remaining carbons of
Two important discoveries in the late 1940s the cyclopentane ring (Fig. 12.1). Additional
and early 1950s had a dramatic effect not only carbon atoms on steroids include angular
on steroid research but also on the pharmaco- methyl groups attached to C13 and C10 and
logical applications of steroids. The first was alkyl substituents on C17 (Fig. 12.2).
the clinical report by Hench et al. (26) from When the steroid nucleus is drawn in a two-
the Mayo Clinic on the significant improve- dimensional representation, the steroid scaf-
ment in patients with rheumatoid arthritis fold appears planar and substituents on car-
following cortisone treatment. The second bons of the steroid scaffold may be located
was the application of estrogen and progestin either above or below the "plane" of the ste-
preparations for contraception, demonstrated roid. Substitutents located above the plane are
by Pincus and colleagues (27). These two se- drawn with solid lines or with solid wedges,
ries of studies showed for the first time that and these moieties are referred to as being in
steroids could be considered as drugs. As a re- the p-configuration. Substitutents located be-
sult, extensive research on the medicinal low the plane are drawn with dashed lines and
chemistry, pharmacology, and clinical studies are referred to as having the a-configuration.
of steroid agonists and antagonists has The angular methyl groups numbered 18 and
evolved and continues to provide new insights 19 are attached in the p-configuration (above
and new medicinal agents for therapies in the steroid plane) to C13 and C10, respec-
many different diseases and chemoprevention tively. Side-chains at position 17 are always /3
strategies. unless indicated by dotted lines or in the no-
menclature of the steroid. The stereochemis-
try of the rings and the substituents on the
2 STEROID CHEMISTRY
steroid scaffold markedly affects the biologic
activity of a given class of steroids.
2.1 Structure and Physical Properties
The three-dimensional shapes of the rings
of Steroids
in the steroid scaffold are actually not planar.
Steroid molecules possess a common chemical The cyclohexane rings of steroids exist in the
skeleton of four fused rings, consisting of preferred chair conformation. As a result, sub-
three six-membered rings and a five-mem- stitutents on the cyclohexane rings can be
bered ring (Fig. 12.1). Chemically, this hydro- located in either the axial or the equatorial
carbon scaffold is a cyclopentanoperhydro- position. The cyclopentane ring exists in a
Fundamentals of Steroid Chemistry and Biochemistry

5p-steroid

Figure 12.3. Representations of


5wsteroid and 5P-steroid.

half-chair or open-envelope conformation. Al- remaining bonds of a steroid can be assigned


though the cyclohexane ring may undergo a easily. The orientation of the remaining bonds
flip in conformation, steroids are rigid struc- on a steroid may be determined if one recalls
tures because they generally have at least one that groups on a cyclohexane ring that are po-
trans fused-ring system, and these rings must sitioned on adjacent carbon atoms (vicinal,
be diequatorial to each other. Endogenous ste- -C,H-C,H-) of the ring (i.e., 1, 2 to each
roids contain two trans fused rings, one die- other) are trans if their relationship is 1,2-di-
quatorial trans fusion between rings B and C axial or 1,2-diequatorial and are cis if their
(carbons 8 and 9) and the other diequatorial relationship is 1,2-equatorial-axial.
trans fusion between rings C and D (carbons The backbone of the steroid molecule can
13 and 14). Two possible ring fusions are ob- be referred to by a series of carbon-carbon
served in endogenous steroids between rings A bonds and the cis or trans relationship of the
and B. The diequatorial trans fusion between four rings (Fig. 12.4). The 5a-steroid molecule
rings A and B results in the hydrogen atom at has a trans-anti-trans-anti-trans backbone. In
position 5 being on the opposite side of the this structure, all the fused rings have trans
rings from the angular methyl group at posi- (diequatorial) stereochemistry (i.e., the A/B
tion 19; the 5a notation is used for this hydro- fused ring, the B/C fused ring, and the C/D
gen. The overall three-dimensional shape of fused ring are trans). The term anti is used in
the 5a-steroid is a nearly flat, pleated shape. backbone notation to define the orientation of
The axial-equatorial cis fusion between rings rings that are connected to each other and
A and B results in the hydrogen atom at posi-
tion 5 being on the same side of the rings with
the angular methyl group at position 19; the
5P notation is used for this hydrogen. The two-
dimensional and three-dimensional represen-
tations for the 5a-steroid and the 5P-steroid
are illustrated in Fig. 12.3. These ring junc-
tions (Ail3 and B/C) and the chair conforma-
tion of the six-membered rings result in an
overall topography of the steroid scaffold that
is rather rigid. Some minor flexibility is ob-
served in the conformation of the D ring. -- H H
Because the angular methyl groups at posi- H
tions 18 and 19 are P and have an axial orien- trans-anti-trans-anti-trans
tation (i.e., perpendicular to the plane of the
rings), the conformational orientation of the Figure 12.4. Steroid backbone.
2 Steroid Chemistry

ing carbons 20 and 21). Because C20 is not a


ring carbon, the preferred designation for the
stereochemistry on C20 is determined based
on the Cahn-Ingold-Prelog sequence rules
(the R,S system). The R,S system is also used
to designate the stereochemistry of other po-
sitions on the steroid side-chains.
Figure 12.5. a and P substituents on carbon num-
Important physical properties of steroids
ber 20. include the physical state of the molecules and
the solubility of steroids. The overall class of
steroids is found almost entirely as solids. The
have a trans-type relationship. For example, melting points for steroids range from approx-
the bond equatorial to ring B, at position 9, imately 100 to 250°C. Molecules of one partic-
which forms part of ring C, is anti to the bond ular steroid compound may crystallize in ei-
equatorial to ring B, at position 10, which ther an anhydrous form or in a hydrated or
forms part of ring A. A 5P-steroid has a cis- solvated form, resulting in different melting
anti-trans-anti-trans backbone, in which the points observed for the two forms. Also, indi-
Ah3 rings are fused cis. The term syn is used to vidual molecules of one steroid compound may
define a cis-type relationship in a similar fash- pack in different arrangements in crystals
ion as anti. No naturally occurring steroids from different solvents, resulting in polymor-
exist with a syn-type geometry, although such phic forms (5). Regarding solubility, steroids
compounds can be chemically synthesized. are generally insoluble in water, whereas they
Thus, the conventional drawing of the steroid are reasonably soluble in organic solvents
nucleus is the natural configuration and does such as ethanol, acetone, chloroform, and di-
not show the hydrogens at the 8P,9a, or 14a oxane. Steroids with a phenolic hydroxyl
positions. If the carbon at position 5 is satu- group on the aromatic A ring (estrogens) are
rated, the hydrogen is always drawn, either as soluble in dilute sodium hydroxide.
5a or 5P. Also, the conventional drawing of a
steroid molecule has the C18 and C19 methyl
2.2 Steroid Nomenclature
groups shown only as solid lines.
Many of the biologically important steroids Many naturally occurring steroids are re-
contain a carbon-carbon double bond between ferred to by their common or trivial names,
positions 4 and 5 or 5 and 6, and consequently such as cholesterol, cortisol, progesterone, tes-
there is no cis or trans relationship between tosterone, and estradiol. As more steroid mol-
rings A and B. The symbol A is often used ecules were being discovered andlor synthe-
to designate a carbon-carbon double bond sized, it became clear that a more systematic
(C=C)in a steroid. If the carbon-carbon dou- method for naming steroids was needed. Be-
ble bond is between positions 4 and 5, the com- ginning in the 1950s, nomenclature rules for
pound is referred to as a A*-steroid. If the car- steroids were being developed, and the most
bon-carbon double bond is between positions 5 recent IUPAC-IUB Joint Commission rules
and 10, the compound is designated a A 5(10)- for systematic steroid nomenclature were
steroid. Addition of a double bond also in- published in 1989 (28, 29). The systematic
creases the flexibility of the ring; for example, names for steroids are based on the steroid
the A ring of a A4-steroid exists primarily in a hydrocarbon system, and the particular sys-
half-chair conformation. tematic name begins by selection of the stem
Aliphatic side-chains at position 17 are al- name based on the hydrocarbon system (Fig.
ways assumed to be p-configuration, which is 12.6). Cholestane is the term used for steroids
the configuration found in endogenous ste- with 27 carbon atoms (i.e., the C,, steroid
roids. By long-standing convention, the a and structure). Pregnanes are steroids with 21
p terms have been applied to substituents on carbon atoms, androstanes have 19 carbon at-
carbon number 20 (Fig. 12.5) on steroids con- oms, estranes have 18 carbon atoms, and go-
taining a two-carbon side-chain (i.e., contain- nanes have 17 carbon atoms.
Fundamentals of Steroid Chemistry and Biochemistry

androstane (C19) estrane (Cia) gonane (C17)

Figure 12.6. Structures of steroid stem names.

If there are any double bonds present in the Examples of the trivial names, systematic
steroid scaffold, the "ane" ending of the stem names, and chemical structures for common
name is replaced with "ene" if one double steroids are illustrated in Fig. 12.7. Choles-
bond is present, "diene" if two double bonds terol is the central steroid of the animal king-
are present, "triene" if three double bonds are dom and functions as an essential component
present, and so forth. The position(s) of the of cell membranes and as a biosynthetic pre-
double bond is indicated by placing the lowest cursor to other steroids in the body. Choles-
number of the carbon atom of the double bond terol has 27 carbon atoms, a hydroxyl group in
in front of the "ene" ending. If the number of the P-configuration at carbon 3, and contains a
the first carbon atom indicates ambiguous po- carbon-carbon double bond between carbons 5
sitions, then this first number is followed by
and 6. Cholesterol is referred to as a A5-steroid
the number of the other carbon atom, placed
or, more specifically, a A5-sterol because it is
in parentheses. When saturation is present at
an unsaturated alcohol. The systematic name
the 5 position, a designation of either 5a- or
5p- is required and is placed before the stem for cholesterol is cholest-5-en-3p-01.The adre-
name. A suffix for the stem name is selected nocorticoids (adrenal cortex hormones) are
based on the following priorities: pregnanes and are exemplified by cortisol,
which is an 11/3,17a,21-trihydroxypregn-4-
carboxylic acid (or derivative)>carbonyl> ene-3,20-dione. Progesterone (pregn-4-ene-
3,20-dione),a female sex hormone synthesized
by the corpus luteum, is also a pregnane ana-
log. The male sex hormones (androgens) are
In adding the suffix to the stem name, the final based on the structure of 5a-androstane. Tes-
"e" in the stem name is always dropped when tosterone, an important naturally occurring
the suffix begins with a vowel. The carbon androgen, is named 17p-hydroxyandrost-4-
number of the substituent (and stereochemis- en-3-one. Finally, the estrogens are female sex
try, if present) is placed in front of the suffix. hormones synthesized by the follicular cells of
Remaining substituents are denoted as pre- the ovaries and are estrane analogs containing
fixes, are preceded by the position number and an aromatic A ring. Although the A ring does
stereochemistry, and are placed in alphabeti- not contain isolated carbon-carbon double
cal order. bonds, these analogs are named as if the bonds
2 Steroid Chemistry

cholesterol cortisol

progesterone testosterone estradiol

Figure 12.7. Structures of common steroids.

were in the positions shown in estradiol. Es- cabeza de negro, to pregnonolone acetate in
tradiol, a typical member of this class of drugs, essentially two steps (37,381. His work had a
is named estra-1,3,5(10)-triene-3,17p-diol. significant impact on the industrial produc-
tion of steroids. One unique feature of re-
2.3 Chemical Synthesis and Microbial search on steroid chemistry is the equal con-
Transformations of Steroids tribution on the field from both academia and
A total of six Nobel prizes were awarded to the pharmaceutical industry. The historical
scientists working in the area of steroids, with perspective on steroid chemistry has been de-
four out of the six prizes in steroid chemistry. scribed in detail by a series of papers (7-9).
The pioneer work of Adolf Windaus and Hein- The development of corticosteroids and
rich Wieland, in the 19209, in structural eluci- oral contraceptives in the late 1940s and early
dation of a number of important steroids set 1950s reflected the significance of chemical
the stage for the many significant discoveries synthesis in steroid research. In this chapter
in the steroid area from the 1930s to the we use the chemical synthesis of cortisone as
1950s. The total synthesis of equilenin was an example to illustrate some of the synthetic
first reported by Bachmann et al. (21) and transformations in steroid chemistry. In addi-
later by Johnson and coworkers (30). The total tion, the high costs of the chemical synthesis
synthesis of estrone was reported by Anner of corticosteroids eventually led the research
and Mieschler (31), followed by the work of on microbial biotransformation of steroids de-
other well-known chemists on the synthesis of scribed in the next section.
cholesterol and sex hormones (32-35). In ad-
dition to the total synthesis of steroids, Russell 2.3.1 Synthesis of Cortisone. The synthesis
Marker reported the use of sapogenins as the of cortisone was first reported by Sarett and is
starting material for the synthesis of cortico- shown in Fig. 12.8a (39,40). Methyl bisnordes-
steroids and sex hormones (36). In a series of oxycholate (I) was used as the starting mate-
studies reported in numerous brief communi- rial for the synthesis. The synthetic scheme
cations in the early 19409, Marker reported a constitutes three basic operations: transposi-
chemical degradation process that converted tion of the 12a-hydroxygroup to the C11 posi-
diosgenin, a sapogenin from the Mexican yam, tion (from compound I to V), cleavage of the
VI

11. NaN3
2. Dilute HOAC

H3C
Y
NH2

HNOl
Pyridine

XI1 XI X

1 1.KOH
2. Succinic anhydride

- oe
CH20Ac

oe
I
HCOAc
1. AC20

HO'
@
- - - then
c r KoH
o3
-
H
-
:.a --
H
Xlll XIV xv

CH20H CH20Ac
1
Br2

CH20Ac
I I I

@
-
HCOH
-@ K O
HCOAc
Pyridlne
c -- @
-
HCOAc

/
0
/ 0 0 -
H
XVlll XVll Br
XVI
I Ac20

XIX Cortisone acatate

Figure 12.8. (a) Synthesis of cortisone acetate. (b) Improved synthesis of cortisone acetate.
2 Steroid Chemistry 601

-BzCI, Py
. Se02,KOH

HO'" H

8 & 8
.
...--
0
8
H
/
H3
NaHC03
C---

HO'" H
IH _
H3CHCl
HCI
13

HO'" H
IH H3 Hi
CH30H,

HO"' H
1 H H3

I 1.Z-HOAc
2. Meystre-Miescher
degradation

XVI xv XIV Xlll

-.
XVll XVlll XIX XX

0 CH20Ac

&
,,\OH
CH~COCOZH,HOAc
CHCI,, HBr (GO2)

*
O H (NOZ)~C~H~NH
cortisone acetate XXI

Figure 12.8. (Continued.)


Fundamentals of Steroid Chemistry and Biochemistry

Curvularia lunata
Progesterone

1 1 p-Hydroxyprogesterone

Figure 12.9. 11-Hydroxylationof progesterone.

bile acid side-chain and incorporation of the duction of a 3,4-double bond afforded corti-
dihydroxyacetonemoiety (from V to XIII), and sone acetate. The total synthesis of cortisone
generation of the 4-ene-3-one system (from was reported in 1951 by Woodward and col-
XI11 to cortisone). The yield for the conversion leagues (45).
of V to M was low. Eventually, an improved
synthesis of cortisone was reported by Sarett 2.3.2 Microbial Steroid Biotransformations.
(41, 42). The improved synthesis began with The research on microbial transformation of
deoxycholic acid I. Protection of the 3a-hy- steroids was stimulated after World War I1
droxy group in I followed by oxidation yielded when the anti-inflammatory properties of cor-
the ketone 111. The next step involved the for- tisone were reported (26). Efficient synthesis
mation of the unsaturated ketone IV with se- of corticosteroids was then required for
lenium dioxide (43). The next few steps re- scale-up synthesis as well as structure-activity
quired the transposition of the ketone at C12 relationship studies. As shown in Fig. 12.8, the
to C11. The unusual step is the conversion of synthesis of cortisone required a total of 31
VII to VIII, to form the 3a,9a epoxide (44).The steps (39, 40). One of the particularly chal-
epoxide ring was opened with HBr and subse- lenging conversions was the transposition of
quent transformations yielded the important the 12a-hydroxy group in the bile acid to the
intermediate XIII. The remaining steps are C11, which required 12 steps. In 1952, Peter-
the improved procedure for the introduction son and Murray of Upjohn reported the first
of the dihydroxyacetone side-chain as com- patented process of direct lla-hydroxylation
pared to the previous procedure (Fig. 12.8b). of progesterone through the use of Rhizopus
Reacting XI11 with HCN formed the cyanohy- arrhizus and Rhizopus nigricans (46,47) (Fig.
drin XIV.Dehydration followed by acetylation 12.9). In the same year, Fried and coworkers
obtained XVI. Reacting XVI with osmium tet- of Squibb Institute reported a similar micro-
roxide yielded the osmate ester XVII. The os- bial transformation with Aspergillus niger
mate ester served as a protecting group for the (48). In addition to progesterone, deoxycorti-
C,,-C,, double bond in addition to the intro- costemne, 11-deoxy-l7a-hydroxycorticostemne,
duction of the C17a alcohol group. Oxidation and 17a-hydroxy-progesteronecould also be
of the 3a-hydroxy group followed by the intro- the substrates for the transformation. Three
2 Steroid Chemistry

Side chain cleavage

Figure 12.10. Selected site of


microbial transformation of
steroids.

years later, Schull and Kita of Pfizer reported 7a- and 9a-Hydroxylations. 7a-Hydroxyan-
the stereoselective llp-hydroxylation with drostenedione is an important intermediate in
Curvularia lunata by use of the same proges- the production of diuretics. Its production
terone substrates reported by Fried (5) (Fig. with microbial transformation from 3a,7a-di-
12.9). As a result of the incorporation of the hydroxydp-holanic acid has been reported
microbial transformation in the synthesis, the (59). Microbial 9a-hydroxylation of steroids
cost of production of hydrocortisone was low- was first observed by Peterson and Murray
ered from $200 per gram in 1948 to $3.50 per (60). This type of transformation has practical
gram in 1955. significance, given that the 9a-hydroxy moiety
In this chapter, only selected microbial can easily be converted to the 9,ll-dehydro
transformations of steroids are described. The functionality. The 9,ll-dehydro system is an
reader is directed to refer to articles with more
extensive reviews on this topic (49-55). The
microbial transformations are described with
regard to the type of reaction and the carbon of
the steroid skeleton bearing the reaction. Fig-
ure 12.10 is a diagrammatic illustration of the
selected site of microbial transformations of
steroids by use of a cholestane steroid skele-
ton.
16a-Hydroxylation. Every carbon in a ste-
roid molecule is accessible for microbial trans- Progesterone
formation (56). Hydroxylations at the l l a , #

llp, and 16a positions of steroids are rou- Streptomyces


tinely used industrially through microbial argenteolus
transformation. Hydroxylations at the l l a
and l l p positions of progesterone were de-
scribed above. The first exam~leof microbial
A

16a-hydroxylationwas the conversion of pro-


gesterone to 16a-hydroxyprogesteronewith S.
argenteolus reported by Perlman and cowork-
ers (57) (Fig. 12.11). In addition to progester-
one, 16a-hydroxylation on synthetic cortico-
steroid was also accomplished with S.
roseochromogenus (58) and subsequently led
to the discovery of triamcinolone, a widely
used anti-inflammatory steroid. Figure 12.11. 16a-Hydroxylationof progesterone.
&
Fundamentals of Steroid Chemistry and Biochemistry

EcH

0
Norethindrone Finasteride

0vCH20H
OH

Betamethasone ICI 182,780

Figure 12.12. Steroids with different pharmacological and therapeutic indications.

important intermediate in the production of conversion of the steroids to androst-1,4-


9a-fluoro andlor 11-hydroxysteroids. diene-3,17-dione by Mycobacterium has been
14a-Hydroxylation. 14a-Hydroxysteroids reported (65).Two years later, Liu reported an
are of practical significance. 14a-Hydroxyan- improved method for the conversion of choles-
drost-4-ene-3,6,17-trione was recently shown terol to testosterone in a 43% yield (66).
to have aromatase inhibitory activity in hu-
man placenta and uterine tumors (61-63). 2.3.3 Selected Chemical Reactions of Ste-
The derivative was obtained through micro- roids. Because it is not feasible to describe all
bial oxidation of androst-4-ene-3,17-dione the chemical transformations reported in ste-
with Acremonium strictum. roid literature in this chapter, we decided to
Many fungi, such as Mucor griseocyanus choose several chemical reactions used in the
and Actinomucor elegans, have been shown to synthesis of steroids with significant pharma-
introduce a 14a-hydroxy group to progester- cological and therapeutic indications (Fig.
one and other steroids (60, 64). The l4a-hy- 12.12). (For a complete review of steroid chem-
droxy steroids can serve as important inter- istry and reactions, refer to Refs. 1-3,7-9,67-
mediates in the production of steroids with 75.) The steroids in Fig. 12.12 have distinct
14P-hydroxy-5ppregnane nuclei, a common chemical structures and functionalities in the
structural framework that exists in many car- steroid skeleton. They serve as good examples
dioactive steroids. Chemical transformation of to illustrate the different chemical transfor-
steroids from the 14a-hydroxy configuration mations in the steroid backbone.
to 14P-hydroxy substituents can be accom- Norethindrone. Norethindrone (l7a-ethi-
plished through 14P,15p-epoxide intermedi- nyl-17P-hydroxyestr-4-en-3-one) is a widely
ates. used orally active progestin in oral contracep-
Side-Chain Cleavage. Many of the cheap tives and possesses a 19-norandrostane ste-
and readily available natural products such as roid skeleton. This particular steroid, first
sitosterol, campesterol, and cholesterol have synthesized in the Syntex laboratories, is ob-
been considered waste products because of the tained by applying Birch reduction on estrone
lack of efficient methods for the cleavage of 3-methyl ether to form the 1,4-dihydroben-
their saturated side-chains. A process for the zene en01ether moiety (Fig. 12.13). Oxidation
2 Steroid Chemistry

1-J Li I NH3, ethanol 1 L

Estrone &methyl ether


I H+

Norethindrone
Figure 12.13. Synthesis of norethindrone.

and ethynylation of the en01 ether affords the double bond yields the 4-aza steroid backbone
l7a-ethynyl derivative. Norethindrone can in finasteride (78, 79) (Fig. 12.14).
then be obtained by hydrolysis of the en01 Betamethasone. Betarnethasone is a widely
ether moiety (76). The double isomer, 17a- used anti-inflammatory glucocorticoid. The
ethinyl-l7p-hydroxyestr-5(10)-en-3-one (nor- 9a-fluoro group greatly enhanced the glu-
ethynodrel), was first prepared in the Searle cocorticoid activity of the compound. The in-
laboratories through use of the Birch reduc- corporation of the 9a-fluoro moiety can begin
tion method (77) and is also used in oral con- with the dehydration of the 11-hydroxylgroup
traceptives. Norethynodrel is converted by to form 9,ll-olefin. The olefin is first treated
gastric juices in the stomach to norethindrone. with hypobromus acid (formed from N-bro-
Finasteride. Finasteride is a 5a-reductase moacetamide in water), which can be cyclized
inhibitor used for the treatment of benign in the presence of base to form the 9,ll-epox-
prostatic hyperplasia (BPH)and alopecia (78). ide. Ring opening of the epoxide affords the
It contains a distinct 4-aza androstane nu- 9a-fluoro and llp-hydroxy moieties in beta-
cleus. The synthesis of the 4-aza moiety begins methasone (80) (Fig. 12.15).
with steroid with a 4-ene-&onesystem. Oxida- Faslodex (ICI 7 82,780). Faslodex (ICI
tion of the enone yields the seco-acid, which 182,780) is a pure steroidal antiestrogen and
can be cyclized with ammonia at high temper- was recently approved by the U.S. Food and
ature to form the unsaturated lactam. NaBH, Drug Administration for the treatment of es-
reduction of the double bond between C5 and trogen-receptor positive metastatic breast
C6 followed by dehydrogenation of the C1-C2 cancer (81).The long hydrophobic alkyl group
Fundamentals of Steroid Chemistry and Biochemistry

180 "C
1
Ethylene glycol
Liq. NH3

H
Figure 12.14. Synthesis of 4-aza steroid 4-aza steroid
backbone in finasteride. skeleton

at the 7a position converts the estradiol from the 7a and 7P epimers. The advantage of this
an estrogenic molecule to a pure antiestrogen. scheme is that the 70 epimer can be quantita-
The key reactions to incorporate the 7a-sub- tively converted to the 7aepimer with NaOMe
stituent begin with the 1,6-conjugated addi- in methanol. Deoxygenation and deprotection
tion of the dimethyl t-butyl-silyloxyundecyl yielded the final product.
bromide to the dienone followed by the aroma-
tization of the A ring (82) (Fig. 12.16). An im-
proved synthetic scheme for the synthesis of 3 STEROID BIOCHEMISTRY
an analog of faslodex (ICI 164,384) was re-
ported (83). The synthesis begins with the 3.1 Steroidogenesis
treatment of the 6-keto estradiol derivative
with potassium t-butoxide in dry 1,2-dime- 3.1 .I Cholesterol Biosynthesis. Cholesterol
thoxyethane followed by quenching of the eno- is the central steroid of the animal kingdom
late with alkyl iodide (RI) to form a mixture of and functions as an essential component of

1. N-bromoacetamide,
2. Base

9a-F, 11 &OH steroid

Figure 12.15. Synthesis of 9a-fluoro-llp-hydroxy steroids.


3 Steroid Biochemistry

1. Separation of 7a and 78
isomers
2. Ac20/H20
3. Ac20/Pyridine
v

Faslodex ---
OTHP
HO & ' { ( C H ~IOAC
)~
cuBL
r/B
iH
r..

OTHP
o& '/(CH~)~~OAC

OH

0 R = (CH2)10CON(CH3)C4H9
6-keto estradiol ether ICI 164,384

Figure 12.16. Synthesis o f 7a-substituted estradiol derivative.

cell membranes and as a biosynthetic precur- The first steps in cholesterol biosynthesis
sor to other steroids in the body. The two ma- use three molecules of acetyl-CoA and involve
jor sources of cholesterol are (1) dietary reversible enzymatic reactions. Two acetyl-
sources and (2) biosynthesis within the cell. CoA molecules are condensed together by a
All animal cells have the capacity to biosyn- thiolase enzyme to form acetoacetyl-CoA. The
thesize cholesterol; the principal sites of syn- third acetyl-CoA is combined with acetoacetyl-
thesis in humans are liver, skin, and intestinal CoA by the enzyme 3-hydroxy-3-methylglu-
mucosa. The biosynthesis of cholesterol con- taryl-CoA synthase to form S-3-hydroxy-3-
sists of a complex pathway involving the par- methylglutaryl-CoA (HMG-CoA). The next
ticipation of both cytosolic and membrane- step in cholesterol biosynthesis is the conver-
bound enzymes (4,6). The 27 carbon atoms of sion of HMG-CoA to mevalonate (or mevalonic
cholesterol are all derived from the acetate acid) by the enzyme HMG-CoA reductase (Fig.
two-carbon unit, and the biosynthesis uses 18 12.17). The enzymatic step uses two molecules
acetyl-CoA molecules for the construction of of NADPH, obtaining the reduction of the
cholesterol. The biosynthesis of cholesterol thioester to an alcohol, resulting in R-meval-
can be divided into two major stages: (1)the onate. This enzymatic step is irreversible and
conversion of acetyl-CoA substrates to the C,, is the rate-limiting step in cholesterol biosyn-
hydrocarbon polyene squalene and (2)the con- thesis. HMG-CoA reductase is an integral
version of squalene to cholesterol. membrane protein of the smooth endoplasmic
Fundamentals of Steroid Chemistry and Biochemistry

- -

II thiolase II II
2 H3C-C-S-COA H3C- C -CH2- C -S-COA
7
J
CoA
' -
II
(-H3C-C-S-CoA
S-HMG-CoA acetyl-CoA
synthase
,L
booH
COA

fio0
HO 3 S-HMG-COA
r e
.

HO CoA NADP' NADPH


+ H+ COA-S 0
R-mevalonate S-hydroxymethylglutaryl CoA
(S-HMG-COA)

Figure 12.17. Formation o f mevalonate f r o m acetyl CoA.

reticulum and is under complex regulation. group forming isopentenyl pyrophosphate,


HMG-CoA reductase can exist as an inactive the basic "isoprene unit" (a C, molecule).
phosphorylated form and as an active dephos- Isomerization of isopentenyl pyrophosphate
phorylated form, thus enabling rapid regula- produces dimethylallyl pyrophosphate. Iso-
tion of its enzymatic activity by hormones pentenyl pyrophosphate and dimethylallyl py-
such as insulin and glucagons. At the tran- rophosphate then condense in a head-to-tail
scriptional level, increased intracellular levels fashion by the enzyme prenyl transferase to
of cholesterol can inhibit the gene regulating form geranyl pyrophosphate (a C,, molecule).
HMG-CoA reductase. This enzyme is an im- Prenyl transferase then catalyzes the head-to-
portant target for pharmacological regulation tail condensation of another isopentenyl pyro-
of cholesterol biosynthesis and is inhibited by phosphate to form farnesyl pyrophosphate (a
the important class of hypercholesterolemic C,, molecule). Finally, two farnesyl pyrophos-
agents referred to as the statins (84). Lova- phates combine in a head-to-head fashion to
statin (Mevacor),pravastatin (Pravacol), sim- form squalene (a C,, molecule). This last step
vastatin (Zocor), and atorvastatin (Lipitor) is catalyzed by the membrane-bound enzyme
are examples of drugs from this therapeutic squalene synthase, uses a molecule of
class (Fig. 12.18). The lactone ring present in NADPH, and eliminates both pyrophosphates
some of these medicinal agents is hydrolyzed in the reaction.
in vivo to the active form, and the open-chain The second major stage of cholesterol bio-
derivative structurally resembles R-meval- synthesis is the conversion of squalene to cho-
onate. lesterol (Fig. 12.21). Squalene is cyclized to
The next series of enzymatic steps convert form the basic steroid scaffold through a two-
six molecules of mevalonate into the C,, poly- step process. The first step is catalyzed by
ene squalene and involve a series of phosphor- the enzyme squalene epoxidase, or squalene
ylation events involving ATP (Figs. 12.19 and monooxygenase, and uses molecular oxygen
12.20). Mevalonate is first phosphorylated by and NADPH. Squalene epoxidase is a flavin
the enzyme mevalonate kinase by use of ATP monooxygenase present in the smooth endo-
to produce 5-phospho-mevalonate. A second plasmic reticulum and adds one oxygen atom
molecule of ATP is used to form 5-pyrophos- from molecular oxygen to the end of squalene,
pho-mevalonate. The third molecule of ATP is forming the epoxide 2,3-oxidosqualene. The
used in a concerted reaction that involves de- second step involves cyclization to lanosterol
carboxylation and loss of the tertiary hydroxyl and is catalyzed by the enzyme 2,3-oxidosqua-
3 Steroid Biochemistry

R=CH3 X=H lovastatin (~evacor?


R = OH X=H pravastatin (~ravachol?
R = CH3 R = CH3 sirnvastatin (~ocor@)

Figure 12.18. Inhibitors of HMG-CoA


reductase.

lene cyclase. The enzyme mediates a protona- complex that consists of three proteins, cyto-
tion of the oqgen atom of the epoxide, and the chrome P45OSo (also referred to as cyto-
opening of the epoxide drives the series of cy- chrome P450 llAl), ferrodoxin, and ferro-
clizations to produce a protosterol cation in- doxin reductase (85). Three oxidation steps
termediate. The protosterol cation intermedi- are involved in the enzymatic conversion, and
ate undergoes a series of rearrangements that three molecules of NADPH and molecular ox-
include 1,Phydride and methyl shifts, result- ygen are consumed for each molecule of cho-
ing in the C,, sterol lanosterol. Conversion of lesterol converted to pregnenolone (Fig.
lanosterol to cholesterol is a complex, 19-step 12.22). The first oxidation results in the for-
process that involves the removal of three mation of cholest-5-ene-3P,22R-diol, followed
methyl groups (at C4 and C14), reduction of by the second oxidation, yielding cholest-5-
the side-chain double bond, and rearrange- ene-3P,20R,22R-triol. The third oxidation
ment of the 8,9-double bond to the 5,6-double step catalyzes the cleavage of the C2,-C,, bond
bond in the B-ring. These transformations of to release pregnenolone and isocaproic acid.
lanosterol involve multiple enzymes found in This conversion of cholesterol to preg-
the smooth endoplasmic reticulum, and di- nenolone is the initial step in steroidogenesis
verging and converging pathways exist to and is often considered the rate-limiting enzy-
many of the intermediates. matic step. However, several steps must pre-
cede the oxidation of the side-chain of choles-
3.1.2 Formation of Pregnenolone. Choles- terol. These early steps are regulated by
terol is converted by enzymatic cleavage of its pituitary trophic hormones, which bind to G-
side-chain to pregnenolone (3p-hydroxypregn- protein-coupled receptors to elevate intracel-
5-en-20-one), which serves as the biosynthetic lular CAMPlevels and initiate steroidogenesis
precursor of the steroid hormones. This side- within minutes (Fig. 12.23). First, cholesterol
chain cleavage biotransformation is catalyzed is stored in steroidogenic tissues (such as the
by a mitochondrial cytochrome P450 enzyme adrenal cortex, the ovary, the testis) in lipid
Fundamentals of Steroid Chemistry and Biochemistry

HOOC mevalonate
OH kinase

mevalonate

phosphomevalonate
kinase

H6
ADP
CO2 +Pi ATP

-,I/ ! O
0- P- 0- P- 0-
II -i2k-J-
pyrophosphomevalonate HOOC
decarboxylase
0
0- P- 0- P- 0-
11
I I I I
isopentenyl pyrophosphate

0- 0-
dimethylallyl pyrophosphate
Figure 12.19. Formation of isopentenyl pyrophophate from mevalonate.

droplets as cholesterol oleate esters. Choles- gen. The cytochrome P45OsCc protein re-
terol esterase is activated by CAMP mecha- ceives reducing equivalents from NADPH by
nisms and cleaves the esters to liberate choles- an electron-transport process that occurs in
terol. The free cholesterol is transported in the the mitochrondrial matrix. The FAD-con-
cytoplasm by sterol carrier proteins to the mi- taining flavoprotein, ferrodoxin reductase,
tochondria. The cytochrome P45OsCc is lo- is reduced by NADPH and transfers elec-
cated on the inner membrane of the mitochon- trons to ferrodoxin, a two-iron, two-sulfur
dria. For cholesterol metabolism to occur, the protein. Ferrodoxin then transfers the elec-
cholesterol must be transferred to the inner trons to the cytochrome P45OsCc to activate
mitochondrial membrane for side-chain cleav- the oxygen atom. In the adrenal gland, these
age. This transfer of cholesterol across the proteins are referred to as adrenodoxin re-
mitochondrial membrane is accomplished by ductase and adrenodoxin. As noted above,
a protein referred to as steroidogenic acute three oxidation steps are needed to convert
regulatory protein (or StAR protein), and cholesterol to pregnenolone by cytochrome
this transfer of cholesterol has been sug- P45OsCc. The cytochrome P45OsCc enzyme
gested to be the real rate-limiting step (86, is encoded by a single human gene
87). Cholesterol binds to the cytochrome (CYPllAl), and the gene for cytochrome
P45OsCc protein, followed by molecular oxy- P45OsCc lies on chromosome 15 (88,891.
3 Steroid Biochemistry

L ;r II
0- P- 0- P- 0- +
0
s
0-p-0-P-0-
0
I1
I
I I I
0- 0- 0- 0-
dimethylallyl pyrophosphate isopentenyl pyrophosphate

PPi
1 prenyl transferase

geranyl pyrophosphate

isopentenyl
pyrophosphate
prenyl transferase

0 0
II I1
0- P- 0- P- 0-
I I
0- 0-

farnesyl pyrophosphate

farnesyl
pyrophosphate

NADPH

NADP+
r
-\
1 squalene synthase

2 PPi

Figure 12.20. Formation of squalene.


Fundamentals of Steroid Chemistry and Biochemistry

squalene
epoxidase

7-i-Y
0 -
2 NADPH NADPi
squalene + Hi

1squalene
cyclase

lanosterol \ protosterol cation

I8 steps \

cholesterol
Figure 12.21. Conversion of squalene to cholesterol.

3.1.3 Biosynthesis of Adrenal Steroids. Preg- The peptide hormone in the anterior pituitary
nenolone serves as the common precursor in that influences glucocorticoid biosynthesis is
the formation of the adrenocorticoids and adrenocorticotropic hormone (ACTH; cortico-
other steroid hormones. This C,, steroid is tropin), whereas the peptide hormone in the
converted through enzymatic oxidations and hypothalamus is corticotropin-releasing fac-
isomerization of the double bond to a number tor (CRF). CRF is released by the hypothala-
of physiologically active C,, steroids, includ- mus and is transported to the anterior pitu-
ing the adrenocorticoids cortisol (hydrocorti- itary, where it stimulates the release of ACTH
sone), corticosterone, and aldosterone (90, into the bloodstream. ACTH is transported to
91). The glucocorticoids and mineralocorti- the adrenal glands, where it stimulates the
coids are secreted under the influence of pep- biosynthesis and secretion of the glucocorti-
tide hormones secreted by the hypothalamus coids. The circulating levels of glucocorticoids
and anterior pituitary (adenohypophysis). act on the hypothalamus and anterior pitu-
3 Steroid Biochemistry

*
HO 02, NADPH
cholesterol cholest-5-ene-3j?,22R-diol

HOOC
02, NADPH

'7
isocaproic acid
J

02, NADPH

HO HO
pregnenolone cholest-5-ene-3j?,20R, 22R-triol
Figure 12.22. Conversion of cholesterol to pregnenolone.

itary in a negative feedback mechanism to reg- 17a-hydroxyprogesterone.Another hydroxyla-


ulate the release of both CRF and ACTH. A tion occurs by the action of 21-hydroxylase
variety of stimuli, including pain, noise, and (cytochrome P450,, or C!YP21) to give rise to
emotional reactions, increase the secretion of 11-deoxycortisol,which contains the physiologi-
CRF, ACTH, and, consequently, the glucocor- cally important ketol (-COCH,OH) side-chainat
ticoids. Aldosterone secretion rates are influ- the 17Pposition. The final step in the biosynthe-
enced to a greater extent by the octapeptide sis is catalyzed by the enzyme llp-hydroxylase
angiotensin 11. (cytochrome P45OIlp), a rnitochondrial cyto-
The biosynthesis of adrenal steroids occurs chrome P450 enzyme complex. This results in
in adrenal cortex cells and involves enzymes the formation of cortisol (hydrocortisone), the
found in both the mitochondria and the most potent endogenous glucocorticoid secreted
smooth endoplasmic reticulum (Fig. 12.24). by the adrenal cortex. More detailed discussions
Once pregnenolone is formed in the mitochon- about the enzymology and regulation of adrenal
dria, this steroid is then transported to the steroidogenesis are provided in several reviews
endoplasmic reticulum for further metabolism. (6,88,89,92).
I
i
Hydroxylation of pregnenolone at position 17 The pathway for the formation of the po-
I by the enzyme l7a-hydroxylase (cytochrome tent mineralocorticoid molecule aldosterone is
'
!
P45Ol,,) produces 17a-hydroxypregnenolone. similar to that for cortisol and uses several of
In one step, 17a-hydroxypregnenolone is oxi- the same enzymes (Fig. 12.24). The preferred
dized and isomerized by the dual action of pathway involves the conversion of preg-
the enzyme 5-ene-3P-hydroxysteroid dehydro- nenolone to progesterone by 5-ene-3P-hy-
genasel3-oxosteroid-4,5-isomeraseto produce droxysteroid dehydrogenasel3-oxosteroid-4,5-
Fundamentals of Steroid Chemistry and Biochemistry

Trophic
hormone2
Plasma membrane

GTP GDP
7
Mitochrondia CAMP + PP,

Lipid droplet

Cholesterol Cholesterol esterase Cholesterol


esters

Other steroids
Endoplasmic reticulum
Figure 12.23. Trophic hormones and steroidogenesis.

isomerase. Hydroxylation at position 21 of lase is CYPllBl and is found on chromosome


progesterone by 21-hydroxylase results in 21- 8 in humans. Chromosome 8 is also the loca-
hydroxyprogesterone (deoxycorticosterone). tion of CYPllB2, the gene for 18-hydroxylase.
llp-Hydroxylase catalyzes the conversion of 17a-Hydroxylaseand 21-hydroxylaseare cyto-
deoxycorticosterone to corticosterone, which chrome P450 enzymes of the smooth endo-
exhibits mineralocorticoid activity. The final plasmic reticulum. Both of these enzymes use
two oxidations involve hydroxylations at the the flavoprotein NADPH-cytochromeP450 re-
C18 methyl group and are catalyzed by 18- ductase to receive reducing equivalents from
hydroxylase (cytochrome P45Ol, or 18-oxi- NADPH. The cytochrome P45017, enzyme is
dase). These reactions produce, first, 18-hy- encoded by a single human gene (CYP17), and
droxycorticosterone (not shown) and then the gene for cytochrome P45Ol7, lies on chro-
aldosterone, the most powerful endogenous mosome 10 (88,93).Two genes for cytochrome
mineralocorticoid secretion of the adrenal cor- P450,, (gene CYP21B and pseudogene
tex. The aldehyde at C18 of aldosterone exists CYP21A) have been identified in the human
in equilibrium with its hemiacetal form. genome on chromosome 6, but only CYP21B is
Thus, the formation of the adrenal cortico- active and encodes for the enzyme (94).
steroids from pregnenolone involves several The other key enzyme in adrenal steroido-
cytochrome P450 enzymes, present in either genesis is the enzyme 5-ene-3P-hydroxysteroid
the mitochondria or the smooth endoplasmic dehydrogenase/3-oxosteroid-4,5-isomerase (also
reticulum. The mitochondrial enzymes, l l p - referred to as 3P-HSD). As noted above, this
hydroxylase and 18-hydroxylase, are cyto- membrane-bound enzyme exhibits dual enzy-
chrome P450 proteins that receive reducing matic activity in a single protein molecule.
equivalents from NADPH through an elec- Two human genes, HSD3B1 and HSD3B2, en-
tron-transport process in the mitochrondrial code for 3P-HSD Type I and 3P-HSD Type 11,
matrix involving adrenodoxin reductase and respectively. These two genes and closely re-
adrenodoxin, analogous to the side-chain lated pseudogenes are all located on chromo-
cleavage enzyme. The gene for 1lp-hydroxy- some 1 in humans. Finally, genetic mutations
cholesterol pregnenolone

HO
progesterone

corticosterone

cortisol aldosterone

Figure 12.24. Adrenal steroidogenesis. Enzyme activities: (a) side-chain cleavages, (b)3P-hydroxy-
steroid dehydrogenase/4,5-isomerase,(c) l7a-hydroxylase, (d) 21-hydroxylase, (e) llp-hydroxylase,
(f) 18-hydroxylase.
61 6 Fundamentals of Steroid Chemistry and Biochemistry

in these key enzymes of adrenal steroidogene- increase in intracellular cAMP levels through
sis result in congenital adrenal hyperplasia activation of a G-protein and adenyl cyclase.
(CAH), characterized by ambiguous genitalia Cholesterol is liberated from lipid stores and is
and adrenal insufficiency in newborns (95). then converted in mitochondria to preg-
The most common form of CAH arises from nenolone through the side-chain cleavage re-
defective 21-hydroxylase, but rare forms of action, as described earlier.
CAH can also occur from defects in llp-hy- Two major pathways are involved in the
droxylase, l7a-hydroxylase, or 3P-HSD. conversion of pregnenolone to testosterone,
referred to as the "4-ene" and "5-ene" path-
3.1.4 Biosynthesis of Progesterone. Proges- ways. The "5-ene" pathway involves the con-
terone is biosynthesized and secreted by the version of pregnenolone to dehydroepiandros-
corpus luteum of the ovary during the luted terone (DHEA) and is quantitatively more
phase of the reproductive cycle (96, 97). Lu- important in humans (99). Pregnenolone is
teinizing hormone (LH),the anterior pituitary converted by l7a-hydroxylase to 17a-hy-
glycoprotein hormone, binds to the LH recep- droxypregnenolone and then to DHEA
tor on the surface of the cells to initiate pro- through 17-20 lyase. DHEA is the primary an-
gesterone biosynthesis. As in other endocrine drogen secreted by the adrenal cortex in both
cells such as adrenal cortical cells, the binding men and women. In the male testis, DHEA is
of LH results in an increase in intracellular converted by 5-ene-3P-hydroxysteroid dehy-
cAMP levels through activation of a G-protein drogenasel3-oxosteroid-4,5-isomeraseto the
and adenylyl cyclase. One of the processes in- 17-ketosteroidal androgen, androstenedione
fluenced by elevated CAMPlevels is the activa- (androst-4-ene-3,17-dione).Testosterone is
tion of cholesterol esterase, which cleaves cho- formed by reduction of the 17-ketone of andro-
lesterol esters and liberates free cholesterol. stenedione by 17P-hydroxysteroid dehydroge-
The free cholesterol is then converted in mito- nases, and testosterone and androstenedione
chondria to pregnenolone through the side- are metabolically interconvertible. Several
chain cleavage reaction described earlier, and isozymes of 17Shydroxysteroid dehydroge-
progesterone is formed from pregnenolone by nase have been reported (100-103), with the
the action of 5-ene-3P-hydroxysteroid dehydro- specific types catalyzing either a reductive re-
genasel3-oxosteroid-4,5-isomerase(Fig. 12.25). action or an oxidative reaction. Testosterone
is secreted by the Leydig cells and can act in a
3.1.5 Androgen Biosynthesis. Androgens negative feedback fashion in the hypothala-
(male sex hormones) are synthesized from mus and pituitary to decrease the release of
cholesterol in the testes and adrenal cortex gonadotropins.
and formed in the liver from circulating C2, The most potent endogenous androgen is
steroids (90, 98). The major pathway for the the 5a-reduced steroid, 5a-dihydrotestoster-
biosynthesis of testosterone, the major circu- one (5a-DHT,17P-hydroxy-5a-androstan-3-
lating androgen in males, is shown in Fig. one). This molecule is formed in androgen tar-
12.20. In males, the hypothalamus regulates get tissues by the enzyme 5a-reductase, which
the anterior pituitary gland through luteiniz- has been located in both the microsomal frac-
ing hormone releasing factor (LHRH), which tion and the nuclear membrane of homoge-
stimulates the release of follicle-stimulating nized target tissues. The 5a-reductase enzyme
hormone (FSH) and luteinizing hormone catalyzes an irreversible reaction and requires
(LH). The two gonadotropins have separate NADPH as a cofactor, which provides the hy-
functions in males, with FSH promoting sper- drogen at C5. Two different isozymes of the
matogenesis and LH stimulating the biosyn- enzyme are present in humans (104-108).
thesis and secretion of androgens. The pri- The first cDNA isolated and cloned that en-
mary source of testosterone is the Leydig cells coded 5a-reductase was designated Type 1,
of the testes. LH binds to its receptor on the and the second was designated Type 2. The
surface of the Leydig cells to initiate testoster- gene 5AR1 encoding Type 1 is located on chro-
one biosynthesis. As in other endocrine cells, mosome 5, whereas the gene 5AR2 encoding
the binding of the gonadotropin results in an Type 2 is located on chromosome 2. The two
cholesterol pregnenolone

0
progesterone

&
0
,,,,OH

0 17~-hydroxyprogesterone
dehydroepiandrosterone

HO
androstenedione androstenediol

0
estrone testosterone

estradiol

Figure 12.25. Biosynthesis of sex steroid hormones. Enzyme activities: (a) side-chain cleavage, (b)
3P-hydroxysteroid dehydrogenasd4,5-isomerase,(c) l7a-hydroxylase, (d) 17,20-lyase, (e) l7P-hy-
droxysteroid dehydrogenase, (f) aromatase.
Fundamentals of Steroid Chemistry and Biochemistry

-NADPH NADPH

0
androstenedione

estrone peroxy enzyme


- intermediate -

Figure 12.26. Biosynthesis of estrogens by aromatase.

human 5a-reductases have approximately amounts of these hormones are also synthe-
60% sequence homology. These two isozymes sized by the testes in the male and by the ad-
differ in their biochemical properties, tissue renal cortex, the hypothalamus, and the ante-
location, and function (106, 107). Type 1 5a- rior pituitary in both sexes. The major source
reductase exhibits an alkaline pH optimum of estrogens in both postmenopausal women
(6-8.5) and has micromolar affinities for ste- and men occurs in extraglandular sites, partic-
roid substrates. On the other hand, Type 2 ularly in adipose tissue.
5a-reductase has a sharp pH optimum at 4.7- In endocrine tissues, cholesterol is the ste-
5.5, has higher affinity (lower apparent K,) roid that is stored and is converted to estro-
for testosterone, and is more sensitive to in- gen, progesterone, or androgen when the tis-
hibitors than the Type 1 isozyme. The Type 2 sue is stimulated by a gonadotropic hormone.
isozyme is expressed primarily in androgen The major pathways for the biosynthesis of
target tissues, the liver expresses both types, sex steroid hormones are summarized in Fig.
and the Type 1 form is expressed in various 12.25. In the ovary, FSH acts on the preovula-
peripheral tissues. Type 2 5a-reductase ap- tory follicle to stimulate the biosynthesis of
pears to be essential for masculine develop- estrogens. The thecal cells of the preovulatory
ment of the fetal urogenital tract and the ex- follicle convert cholesterol into androgens,
ternal male phenotype, whereas the Type 1 whereas the granulosa cells convert andro-
isozyme is primarily a catabolic enzyme. gens to estrogens. After ovulation, LH acts on
the corpus luteum to stimulate both estrogen
3.1.6 Estradiol Biosynthesis. Estrogens are and progesterone biosynthesis and secretion.
biosynthesized from cholesterol, primarily in Cholesterol is converted by side-chain
the ovary in mature, premenopausal women cleavage into pregnenolone, which can be con-
(96, 97). During pregnancy, the placenta is verted to estrogens through several enzymatic
the main source of estrogen biosynthesis steps. Pregnenolone is converted to the andro-
and pathways for production change. Small gens androstenedione (androst-4-ene-3,17-di-
3 Steroid Biochemistry

Reduction of
20 keto group
/
Reversible oxidation OH Oxidation of
of 11 hydroxyl group 17~-hydroxy
group
HO

Reduction of
3-keto g r o u p ~ o
A/'4
Reduction of
4,5 double bond
Metabolism of hydrocortisone

R = 1 1 ,%OH,Tetrahydrocortisol 11-hydroxyetiocholanolone

#
R = 11 keto, Tetrahydrocortisone

& O H

HO\'" HO\"' HO\"' -


H H H
R = 1 1 p-OH, 20p-Cortol R = 1 1 p-OH, 20~-Cortol 1 1-hydroxyandrosterone
R = 11 keto, 20p-Cortolone R = 11 keto, 20~-Cortolone

Figure 12.27. Metabolites of corticosteroids.

one) and testosterone, as described earlier. microsomal cytochrome P450 enzyme com-
Loss of the C19 angular methyl group and aro- plex termed aromatase. Androstenedione is
matization of the A-ring of testosterone or an- the preferred substrate for aromatization and
drostenedione results in 17p-estradiol or es- three molecules of NADPH and three molecules
trone, respectively. l7p-Estradiol and estrone of oxygen are necessary for conversion of one
are metabolically interconvertible, catalyzed molecule of androgen to estrogen (109, 110).
by 17&hydroxysteroid dehydrogenases. Aromatization proceeds through three succes-
The final step in the biosynthesis is the con- sive steps catalyzed by a single enzyme com-
versions of the C,, androgens to the C,, estro- plex, consisting of the cytochrome P450,,,
gens. This enzyme reaction is catalyzed by the protein and NADPH-cytochrome P450 reduc-
620 Fundamentals of Steroid Chemistry and Biochemistry

tase, present in the endoplasmic reticulum of


the cell. The gene CYP19 expresses cyto-
chrome P450,,,, and the gene is located on
chromosome 15 in humans. The mechanism of
aromatization is illustrated in Figure 12.26. The
first step involves oxidation of the angular C19
methyl group to provide 19-hydroxyandro-
stenedione (19-hydroxyandrost-4-ene-3,17-di-
one). 19,19-Dihydroxyandrostenedione, iso-
lated as 19-oxoandrostenedione,is formed by "
Progesterone
the second oxidation step. The exact mecha-
nism of the last oxidation remains to be fully
determined, with the likely intermediate be-
ing an enzyme-peroxyspecies (111-113). After
the last oxidation, the Cl0-C,, bond is cleaved
and aromatization of the A-ring occurs. In this
last step, the C19 carbon atom is lost as formic
acid and oxygens from the first and last step
are incorporated into the formic acid. The lp-
hydrogen and 2P-hydrogenatoms are also lost
during aromatization.
3.2 Steroid Metabolism
Figure 12.28. Structure of progesterone and
Because of their high potencies, steroid hor- 5P-pregnane-3a,ZO~ol.
mones are secreted in minute amounts and
their secretions are tightly regulated. In addi- forward, and reduction can occur on the 4,5
tion, the secretion and the metabolism of en- double bond and at the 3 and 20 keto groups
dogenous steroids also play a role in control- (115). The major urinary metabolite of proges-
ling their concentrations. Both phase I and terone is the glucuronide conjugate of 5P-
phase I1 pathways are involved in the metab- pregnane-3a720P-dio1(Fig. 12.28).
olism of steroids in vivo.
3.2.3 Androgen Metabolism. As described
3.2.1 Corticosteroids Metabolism. Under in Section 3.1, both androstenedione and testos-
normal conditions, there is an interconversion terone are the substrates of he enzyme aro-
between hydrocortisone and cortisone. Both matase to form estrone and estradiol, respec-
hormones are metabolized extensively before tively. In addition, the true active endogenous
excretion. Reduction can occur both in the 4,5 androgen 5a-dihydrotestosterone is the reduc-
double bond and at the 3 and 20 keto groups of tive metabolite of testosterone by the enzyme
hydrocortisone to form various metabolites such 5a-redudase. Metabolism of androstenedione
as tetrahydrocortisols, tetrahydrocortisones, and testosterone are carried out primarily in the
cortols, and cortolones (114) (Fig. 12.27). A mi- liver. Androstenedione and testosterone can be
nor metabolic pathway also occurs through interconverted by the enzyme 17P-hydrosteroid
cleavage of the C,,-C,, bond to form 11-hy- dehydrogenase. They can be converted to vari-
droxyetiocholanolone and 11-hydroxyandros- ous metabolites by 3a- and 3P-hydroxysteroid
terone. All the metabolites containing the 3-hy- dehydrogenase and by 5a- and 5P-redudases
droxy group go through phase I1 metabolism in (1151, as shown in Fig. 12.29. Androsterone and
liver and to a lesser extent in kidney to form etiocholanolone are the major urinary metabo-
predominantly water-soluble glucuronide con- lites excreted primarily in the form of gluc-
jugates. uronides and to a lesser extent as sulfates (116).

3.2.2 Progesterone Metabolism. The me- 3.2.4 Estrogen Metabolism. The metabo-
tabolism of progesterone is relatively straight- lism of estrogens is carried out primarily in
3 Steroid Biochemistry

&-@ 0 - -

& &-I_-ii-;
H H
5a-Dihydrotestosterone 5a-Androstane9a, 17P-diol

/
0
Testosterone
0 HO
H H

Androstenedione 5p-Androstan-3,17-dione R = 3a-OH, Eticholanolone


R = 3p-OH, 3pHydroxy-5pandrostan-
17-one
0

5a-Androstan-3,17-dione R = 3a-OH, Androsterone


R = 3p-OH, 3p-Hydroxy-5a-androstan-17-one

Figure 12.29. Metabolites of testosterone.

the liver. Estrone and estradiol can be inter- Estrogens also go through phase I1 metab-
converted by 17p-hydroxysteroid dehydroge- olism to form to sulfate and glucuronide con-
nases. One of the major metabolic pathways jugates. Estrone sulfate is the most abundant
for estrone is l6a-hydroxylation to form 16a- estrogen conjugate in females (1-2 nM) (120,
hydroxyestrone, which is then converted toes- 121). There is strong evidence that the high
trio1 (117). Another major pathway is C2 hy- concentration of estrone sulfate may repre-
droxylation to form 2-hydroxyestrone, which sent an important reservoir of active estro-
can then be converted to 2-methoxyestrone by gens for estrogen-dependent illnesses. Es-
the enzyme catechol-0-methyl transferase trone sulfatase [sterylsulfatase EC (3.1.6.2)l
(118). Other minor metabolic pathways of es- has been consistently found in human breast
trogens include the formation of l7a-estra- cancer cells and the conversion of estrone sul-
diol, 6-hydroxylated estrogens, and 16-epiest- fate to estrone has been demonstrated (122-
riol (119),as outlined in Fig. 12.30. 125).
Fundamentals of Steroid Chemistry and Biochemistry

Estradiol
0

1 6p-Hydroxyestrone Estrone 1 6a-Hydroxyestrone

16-Epiestriol 2-Hydroxyestrone Estriol

Figure 12.30. Metabolism of endogenous estrogens.

3.3 Steroid Hormone Action with high affinity to intracellular receptors,


The steroid hormones such as the adrenocor- resulting in the formation of steroid-receptor
ticoids, progestins, estrogens, and androgens complexes that regulate gene expression and
are present in the body only in very low con- protein biosynthesis (Fig. 12.31). The li-
centrations (e.g., 0.1-1.0 nM). Yet, these hor- pophilic steroid hormones are transported in
mones exert potent physiologic effects on sen- the bloodstream reversibly bound to serum
sitive tissues at those low concentrations. carrier proteins. The unbound steroids can
Over the past several decades, the general diffuse through the plasma membrane and en-
mechanism of steroid hormone action has ter the cells. Cells that are sensitive to the
been extensively studied (126-134). The ste- particular steroid hormone (referred to as tar-
roid hormones act on target cells by binding get cells) contain steroid receptors capable of
3 Steroid Biochemistry

Steroid (S)

Autocrine and paracrine effects

Figure 12.31. Mechanism of steroid hormone action.

high affmity binding with the steroid. These ferentiation, and playing central roles in nor-
receptors are soluble intracellular proteins mal physiological processes, as well as in many
that can both bind steroid ligands with high important diseases.
affinity and act as ligand-dependent transcrip- The steroid receptors proteins are mem-
tional factors through interaction with spe- bers of the nuclear receptor superfamily based
cific deoxyribonucleic acid (DNA) sites and on their general protein structure and func-
other proteins associated with the chromatin. tion. The nuclear receptor superfamily in-
Initially, the unoccupied steroid receptors cludes receptors for steroids, vitamin D, thy-
were thought to be located solely in the cyto- roid hormones, and retinoids. The steroid
plasm of target cells; however, investigations receptors include glucocorticoid receptor
on estrogens, progestins, and androgens indi- (GR), mineralocorticoid receptor (MR), pro-
cate that active, unoccupied receptors are also gesterone receptor (PR), estrogen receptors
present in the nucleus of the cell. The interac-
alpha and beta (ERa and ERP), and the andro-
tion of the steroid with its receptor results in a
gen receptor (AR); these receptors function
conformational change of the receptor, trans-
location to the nucleus, and steroid-receptor through the formation of homodimers. The
complex homodimerization. The steroid-re- thyroid receptor (TR), vitamin D receptor
ceptor homodimers interact with particular (VDR), retinoid receptors ( M a , RARP,
regions of the cellular DNA, referred to as hor- RARy), and peroxisomal proliferator-acti-
mone-responsive elements (HREs) present in vated receptors (PPARa, PPARP, PPARy,
the promoter region of responsive genes. Bind- PPARG) interact with retinoid X receptor
ing of the nuclear steroid-receptor complexes (RXR) to form heterodimers. A third group of
to DNA and interaction with various nuclear nuclear receptors are involved in regulation of
transcriptional factors initiate the transcrip- steroid and lipid metabolism, such as constitu-
tion of the gene to produce messenger ribonu- tive androstane receptor (CAR), pregnane X
cleic acid (mRNA).The elevated mRNA levels receptor or steroid xenobiotic receptor (PXR
result in increased protein synthesis in the en- or SXR), and farnesyl X receptor (FXR). Fi-
doplasmic reticulum. These proteins include nally, orphan receptors whose ligands are yet
enzymes, receptors, and secreted factors that to be discovered are also members of this su-
subsequently result in the steroid hormonal perfamily, such as steroidogenic factor 1
response regulating cell function, growth, dif- (SF-1) and X-linked orphan receptor (DAX-1).
Fundamentals of Steroid Chemistry and Biochemistry

Transcription DNA Ligand


activation binding binding
domain domain domain

--(
H ~ N AJB

- Nuclear localization
- Dimerization
Figure 12.32. General struc- -
ture of the steroid receptors. Transactivation

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CHAPTER THIRTEEN

/ Female Sex Hormones,


E
i
Contraceptives, and
Fertility Drugs
PETER C. RUENITZ
College of Pharmacy
University of Georgia
Athens, Georgia

Contents
1 Introduction, 630
2 Clinical Applications, 630
2.1 Current Drugs, 630
2.2 Adverse Effects and Precautions, 631
3 Drug Metabolism, 633
3.1 Oxidation and Reduction
of Steroidal Estrogens, 633
3.2 Fate of 17a-Ethynyl Analogs
of Estradiol (I), 634
3.3 Fate of Conjugated Equine Estrogens, 635
3.4 in Vivo Reversible Monoesters of (11,636
3.5 Olddative Metabolism of Triarylethylenes,636
3.6 Biotransformation of Diethylstilbestrol (241,
637
3.7 Electrophilic (Carcinogenic ?) Metabolites of
ER Ligands, 638
3.8 Aspects of Glucuronide Conjugation, 639
3.8.1 Termination of Effects of ER Ligands,
639
3.8.2 Net Glucuronidation, 640
3.8.3 Enterohepatic Recycling, 640
3.9 Biotransformation of Progesterone (30) and
Its Analogs, 640
4 Aspects of Biosynthesis, 642
4.1 Steroid Aromatization, 642
4.2 Aromatase Inhibitors, 643
4.3 Genesis of Equine Estrogens, 644
5 Physiology and Pharmacology, 645
5.1 Topography of Estrogen Receptors, 645
Burger's Medicinal Chemistry and Drug Discovery 5.2 Ubiquitous Distribution of ER Isoforms, 648
Sixth Edition, Volume 3: Cardiovascular Agents and 5.3 Molecular Endocrinology of ER, 648
Endocrines 5.4 Assessment of Estrogenic Activity, 648
Edited by Donald J. Abraham 5.5 Progesterone Receptors, 649
ISBN 0-471-37029-0 O 2003John Wiley & Sons, Inc. 5.6 Assessment of Activity of PR Ligands, 650
629
630 Female Sex Hormones, Contraceptives, and Fertility Drugs

5.7 Progestin and Antiprogestin Effects, 650 7.2 Two Caveats Concerning the Meaning
5.8 Environmental and Dietary Estrogens of ER Affinity, 658
and Progestins, 651 7.3 Nonsteroidal ER Ligands, 659
5.9 Tissue-Selective Estrogens and Progestins, 7.3.1 Vicinal Diarylethylenes, 659
652 7.3.2 Diarylethylene Estrogen Antagonists,
5.10 Modulation of Ovulation, 654 660
6 History, 655 7.3.3 Triarylethylenes, 661
6.1 Discovery of SERMS, Including Tamoxifen 7.3.3.1 Conformational Analysis, 665
(16a),655 7.3.3.2 Affinity Labels for ER, 665
6.2 Other Discoveries, 656 7.3.4 Triarylheterocycles, 665
7 Structure-Activity Relationships, 656
7.4 Progesterone and Steroidal PR Ligands, 666
7.1 Steroidal Analogs of (I),656
7.5 Nonsteroidal PR Ligands, 667
7.1.1 7&ubstitution, 657
7.1.2 llp-Substitution, 657 8 Recent Developments and Things to Come, 668
7.1.3 Comparison of Estrone (2) with Equilin 9 Retrieval of Related Information, 669
(52c),658 10 Abbreviations, 669
7.1.4 Nonbenzenoid Steroid (-Like) ER
Ligands, 658

1 INTRODUCTION structure-activity studies of female sex hor-


mone agonists and antagonists, (2) estrogen
The roles women take in our society have un- biosynthesis, and (3) biotransformation of es-
dergone a dramatic redefinition over the past trogens, progestins, and their nonsteroidal an-
half-century, and medical use of estrogen and alogs.
progestin mimetics and antagonists has con- Structures of endogenous estrogens, pro-
tributed substantially to this change. It fol- gestins, and certain of their biosynthetic pre-
lows that these substances are among the cursors and metabolites, as well as those of
most intensively prescribed therapeutic analogs of these hormones, appear at points in
agents in use today. Steroidal estrogens and the text where they are most needed for com-
their conjugates are used in estrogen-replace- prehension of the narrative.
ment therapy in postmenopausal women, and
estrogen-progestin combinations are used in
2 CLINICAL APPLICATIONS
birth control. Nonsteroidal estrogen antago-
nists, especially tamoxifen, are used in the
prevention and control of postmenopausal 2.1 Current Drugs
breast cancer. Exciting prospects for tissue- Estrogens, antiestrogens, estrogen biosynthe-
selective estrogen therapy have emerged from sis (aromatase) inhibitors, progestins, and an
clinical and experimental endocrinologic stud- antiprogestin in current clinical use in the
ies. Molecular mechanistic studies involving United States are summarized in Table 13.1.
these substances, using materials and proce- The steroidal estrogens, l7p-estradiol(1) and
dures made available by advances in biotech- its 17-valerate and &sulfate esters (estropi-
nology, have begun to clarify our understand- pate) as well as estrogen conjugates (&sulfate
ing of the structural basis for the effects of esters of (2), (52), (53),and other steroids),
estrogen and progesterone throughout the hu- and the nonsteroidal estrogen dienestrol(71)
man body. are used in estrogen-replacement therapy
Besides describing the medicinal chemistry (ERT) in postmenopausal women (1).Besides
of therapeutically established and experimen- the serious discomfort that can accompany es-
tally promising female sex hormones and ana- trogen deficiency, progressive irreversible de-
logs, this chapter focuses on important devel- terioration of the cardiovascular and skeletal
opments over the last 10-15 years relating to systems can occur. Products containing mix-
(1) biochemical mechanisms of action and tures of estrogen conjugates isolated from the
2 Clinical *pplications

urine of pregnant mares have enjoyed wider women (5). Also, progestins have been used to
clinical acceptance than single-component treat gynecological disorders, such as endome-
products. Also used for ERT is the triaryleth- triosis, caused by hormone deficiency or im-
ylene chlorotrianisene (121, which has a pro- balance.
longed duration of oral estrogenic activity and
has estrogen antagonist effects in some tissues
2.2 Adverse Effects and Precautions
(2).
More recently, raloxifene (29) has been in- Endometriosis and an increased risk of endo-
troduced as a selective estrogen receptor mod- metrial cancer have been associated with use
ulator (SERM), which has the advantage over of steroidal estrogens in ERT, including (9),
earlier estrogen preparations of having a when administered alone (6). As indicated
greatly reduced incidence of reproductive above, ERT using estrogen-progestin combi-
tract side effects (3). Thus, (29) is indicated in nations resulted in a reduced incidence of en-
postmenopausal patients with an elevated risk dometrial cancer (and endometriosis), but also
for breast cancer andlor an elevated risk of resulted in a 20% increase in the risk of breast
endometrial cancer associated with prospec- cancer compared to women on estrogen-only
tive use of conventional estrogens. therapy (7).
The antiestrogens toremifene (22) and, in Because (16a) and (22) both have signifi-
particular, tamoxifen (16a),are used for sup- cant residual partial estrogen agonist activity
pression and prevention of postmenopausal in humans. endometriosis has been associated
breast cancer. Indeed, (16a) has now been ad- with long-term use of these triarylethylenes
ministered to over 1,000,000 women world- (8, 9). Linked to (16a)'s partial estrogenicity,
wide. Breast cancer death rates in the United and possibly to the way it undergoes biotrans-
States and Britain have declined by 25% over formation (see Section 3.71, is an elevated risk
the past 10 years, in large part because of the of endometrial cancer. This aspect of (l6a) has
use of (16a). In addition, (16a)was the first raised concern about its use in breast cancer
substance shown to have tissue-selective es- prevention (10).
trogenicity, by virtue of its ability to suppress On the other hand, use of (291, which has
bone loss and reduce serum cholesterol levels less residual reproductive tract estrogenicity
in patients undergoing therapy for breast can- than that of (16a),for these therapeutic appli-
cer (4). cations, has not been associated with develop-
Clomiphene (23) and, less commonly, (16a) ment of endometriosis or endometrial cancer
are used to stimulate fertility (ovulation) in (9).
patients who wish to become pregnant. Ami- However, neither (16a)nor (29) prevents a
noglutethimide (45),an estrogen biosynthesis common overt symptom of estrogen defi-
inhibitor, has been used alone or in combina- ciency, the one most likely to prompt patients
tion with (16a), in breast cancer treatment. to seek treatment. Lack of adequate estrogen
Ethynylestradiol(9) and its 3-methyl ether levels in certain regions of the central nervous
(mestranol, 10) and 3-cyclopentyl ether system can result in intermittent marked fluc-
(quinestrol, l l ) , and the 17-(3-cyclopentyl) tuations in body temperature known as hot
propionyl ester (cypionate)of (I),are used pri- flushes or hot flashes (11). Although deterio-
marily in combination with progestins in mod- ration of skeletal and cardiovascular systems
ulation of fertility. This is the major applica- is a more serious long-term consequence of es-
tion of the progestins [analogs of progesterone trogen deficiency, such deterioration is not ex-
(30)] listed in Table 13.1, some of which [levo- perienced early on by the patient. However,
norgestrel(361, medroxyprogesterone acetate hot flush episodes eventually (6-12 months)
(34)] are formulated without added estrogen. abate because of bodily adjustment to lower
Besides their applications in birth control, endogenous estrogen levels. Thus, their im-
progestins have been used in combination pact on compliance might be diminished in
with estrogens in hormone-replacement ther- patients that are aware of this and of the con-
apy: the addition of progestin reduces the risk sequences eventually arising from discontinu-
of endometrial cancer in postmenopausal ance of therapy.
632 Female Sex Hormones, Contraceptives, and Fertility Drugs

Table 13.1 Estrogen, Antiestrogen, and Progestin Pharmaceuticals


Generic Name Dose
(Structure) Trade Name U.S. Manufacturer Chemical Class (mglda~)~
Estrogens
Chlorotrianisene (12) Tace Hoechst, Marion, Triarylethylene 12
Roussel
Dienestrol R.W. Johnson Diarylethylene 0.01%
cream
Climara and Berlex and others Estrane 0.05b
others
Estradiol valerate Delestrogen BristolMyers Squibb Estrane 1040"
Estrogens, conjugated Premarind Wyeth-Ayerst Estrane-3-sulfates 0.3-2.5
equine (2,52,53, etc.)
Estropipate (2, 3-sulfate) Ogen Abbott Estrane-3-sulfate 0.75-6
Ethinyl estradiol(9) Estinyl Schering Estrane 0.02-0.05
e e
Mestranol(10) Estrane 0.1'

Aromatase inhibitors
Arninoglutethimide (45) Cytadrend Novartis Glutarimide 250
Anastrozole (50) Arimidexd AstraZeneca Triazole 1
Exemestane (51) Aromasind Pharmacia Androstane 25
Letrozole (48) Femarad Novartis Triazole 2.5

Antiestrogens
Clomiphene citrate (23) Clomid Hoechst, Marion, Triarylethylene 50
(Serophene) Roussel (Serono)
Raloxifene HCl(29) Evistad Lilly Diarylethylene 60
Tamoxifen citrate (16a) NolvadeP AstraZeneca Triarylethylene 20 (base)
Toremifene citrate (22) Fareston Orion Triarylethylene 60 (base)

Progestins
Ortho-Ceptf R.W. Johnson, Estrane 0.15
Desogen, Mircettg Organon
Ethynodiol diacetate Demulend f
Pharmacia Estrane 1
(97)
Levonorgestrel(36) Norplant (Population Council) Estrane 3tig
Medroxyprogesterone Depo-Provera Pharmacia Pregnane 100"
acetate (34) Lunelleh 25'
Megestrol acetate (93) Megace BristolMyers Squibb Pregnane 20
Norethindrone = Norinyli Pharmacia, R.W. Estrane 1
norethisterone (35) Ortho-Novumf Johnson
Norethynodrel(96) Enovidi Pharmacia Estrane 5
Norgestimate (98a) Ortho Cyclen-21f R.W. Johnson Estrane 0.25
Norgestrel(36j) Ovraldaf Wyeth-Ayerst Estrane 0.3-0.5

Antiprogestin
Mifepristone (55) Mifeprex Danco Estrane 600
"Administered orally unless otherwise noted. fcombined with 0.025-0.05 mg of e t h y l estradiol.
bAdministeredas a 24-h transdermal patch. gAdministered as a 30-day implant.
'Administered as a depot intramuscular injectable. hCombined with 5 mg of estradiol cypionate.
dHomepage is accessible on the internet. 'Combined with 0.1 mg of mestranol.
'Only administered in combination with a progestin. 'Mixture of (36)and its enantiomer.
F
I
i
i
3 Drug Metabolism
i
E
L
Table 13.2 Interaction of Progesterone (30) and Its Analogs with Steroid Receptors a
1
c RBAb for
1i
Compound PR GC MC AR Reference
' (30) (progesterone) 40 (23) 10 [I71 100 <0.1 [lo] 224, (651, [2251
(34) (medroxyprogesterone acetate) 115 (55) 29 160 5 224, (226)
(93) (megestrol acetate) (48) 226
(94) (ORG 5020) 100 17 53 <0.1 224
(95) (ORG 2058) 350 (100) 3 27 <0.1 224, (65)
(35)(norethindrone) (32,551 - - 13", 15 65,226
(96) (norethynodrel) (6) - <0.5 226
(37) (4,5a-dihydro(35)) (7) - - 1OC 65
(36) (I-norgestrel) 120 (113) 0.6 70 45,16 224, (226)
(98a) (norgestimate) (50) - - 0.3 227
(98b) (38) - - 1.3 227
(38) (desogestrel) (2) <0.5 226
3-keto (38) (120)
(55) (mifepristone) (99) [2411 300d [7641 - 25 [lo] 131 [2251
(104) [81 LO.061 - [0.031 123

"Affinity for human (rabbit) uterine PR, rat liver glucocorticoid (GC)receptors, rat kidney mineralocorticoid receptors
(MC), and rat prostate androgen receptors (AR) was determined. Relative binding affinities (RBA)for PR are expressed as
percent relative to promegestone (94). Those for GC, MC, and AR are as percentages of those of dexamethasone, aldosterone,
and methyltrienolone, respectively. Affinities of ligands for recombinant hPRA, hGC and hAR are in brackets.
b ~ RBAs,
R compared to (1)in human myometrial cytosol, were <0.2 for (35),(361, (38),3-keto (38); that of (96) was 1.5
(226).
"Determined using mouse kidney cytosol.
d~eterminedusing rat thymus cytosol.

The most common side effect associated


with use of progestins is residual androgenic
(masculinizing) activity. All currently pre-
scribed progestins have some degree of affinity
for androgen receptors (see Table 13.21, al-
though this varies over a wide range.
The low dose of steroidal estrogen (9) (or
10) used in oral contraceptives (Table 13.1: HO
progestins, footnotes f and i)is enabled by bac-
(1)
terial hydrolysis (see section 3.8.3) of estrogen
conjugates accumulated in the gut, allowing
facile reabsorption to proceed. Coadministra-
tion of antibiotics, or other substances that
have an adverse effect on normal gut bacterial
populations, can interrupt this mechanism of
estrogen conservation, resulting in a failure of
the administered oral contraceptive to pre-
vent ovulation. HO

(2)
3 DRUG METABOLISM
rings, and are illustrated in Fig. 13.1. Plasma
3.1 Oxidation and Reduction
and tissue levels of (I),estrone (2), and their
of Steroidal Estrogens
metabolites are modulated primarily by he-
Major biotransformation pathways of 17P-es- patic enzymes. Thus, specific 17ghydroxyste-
tradiol (1)involve oxidations of its A- and D- roid dehydrogenases catalyze, in turn, the re-
Female Sex Hormones, Contraceptives, and Fertility Drugs

3.2 Fate of l7a-Ethynyl Analogs


of Estradiol (1)
Potent, orally effective steroidal mimics of (1)
feature l7a-ethynyl substituents, as exempli-
fied by ethynyl estradiol (9) and its 3-methyl
and 3-cyclopentyl ethers mestranol (10) and
quinestrol(11). The ethynyl group in (9) does
not impede estrogen receptor (ER)affinity
(Table 13.3) (16) or estrogenic potency or effi-
cacy. The extent of D-ring biotransformation,
which otherwise leads to less active or inactive
metabolites, is greatly reduced (17). After oral
absorption, methyl ether (10)is readily O-de-
methylated by liver oxidase(s) to (9) (18).

Figure 13.1. Oxidative metabolism of 17P-estra-


diol(1) and estrone (2).These enzymatically inter-
convertible estrogens undergo D-ring hydroxylation
to (3) and (4), or alternatively, A-ring hydroxyla-
tion, ultimately generating guiacol metabolites (7)
and (8).

Studies with human liver microsomes have


duction of 17P-hydroxyestrogens and the implicated cytochrome P450 2C9 in this con-
oxidation of 17-ketoestrogens (12). Cyto- version (19). Lipophilic ether (11)gives pro-
chromes P450 1A2 and 3A4 are responsible longed estrogenic activity after oral dosing, at-
for 2- and 16a-hydroxylations (13). tributed to deposition in adipose tissues, from
Estrogenic activities of (1)and (2) are sim- which it is released slowly and converted to (9)
ilar as a result of interconversion, and hy- in a manner similar to that of (10) (20).
droxylated metabolites (3) and (4) retain es- However, the l7a-ethynyl group does not
trogenic and other effects (see Section 3.7) always impede D-ring hydroxylation of steroid
(13a).Indeed, (4) was as effective as (1)in pre- hormones. Thus, for moxestrol(67), the llp-
venting elevation of both serum cholesterol methoxy analog of (9), the total extent of uri-
and bone turnover, but its uterotrophic effect nary elimination of 15a-, 16a-, and 16P-hy-
was significantly less than that of in the ovari- droxylated metabolites was about three times
ectomized rat (14). However, methoxyestradi- greater than that of (1)and accounted for over
01s (7) and (8)are only weakly estrogenic if at 40% of the administered dose (17). Incidental
all (15).Also, it is unlikely that estrogenic or to these studies was the finding that the ethy-
other effects are manifested by catecholic es- nyl group of (67) also underwent significant
tradiol metabolites (5) and (6) because these oxidation, ultimately leading to a D-ring-en-
are very rapidly 0-methylated. larged keto metabolite.
3 Drug Metabolism 635

Table 13.3 ER Affinities of 17B-Estradiol(1)and Selected Analogs"


Compound RBA~ ER from Reference
(1)(17p-estradiol)
(2) (estrone) rat (mouse) [hERal
(3)(estriol) rat (mouse) [hERa]
3-deoxy (1) human
17-deoxy (1) human
3, 17-dideoxy (1) human
17-deoxy-17P-amino(1) calf
17-epi-(1) human (mouse) [hERal
2-hydroxy-3-deoxy (1) human
4-hydroxy-3-dwxy (1) lamb, human
( 5 2 ~(equilin)
) human
(52b) (17p-dihydroequilin) human
(9) (17a-ethynylestradiol) human (calf)
17-epi (9) rabbit
(10) (mestranol) rabbit
(64) (ICI 164,384) rat (calf) [hERal
(65) [ici (ZN) 182, 7801 rat (calf)
(66) rat
(67) (moxestrol) human (mouse) [hERal
(68) (RU 51625) human
(69) (RU 53637) human
(70) mouse (rat)
"Values were obtained using uterine cytosolic ER from the indicated sources, or using recombinant human ERa.
*RBA values were calculated using the formula: 100 x IC,, of l/IC,, of the test compound. All values are relative to (1).

3.3 Fate of Conjugated Equine Estrogens dro El-S (52c sulfate) were twice as effective
in the ovariectomized (OVX)rat uterotrophic
Conjugated equine estrogens are composed of
more than 10 aromatic A-ring &sulfate esters assay (section 5.41, as were the respective un-
(see Section 4.3). The major component of this conjugated estrogens (21). Clearly, sulfa-
mixture is the sulfate ester of (21, estrone sul- tase(s) play a critical role in generating decon-
fate or El-S (Fig. 13.2). After oral administra- jugated steroids subsequent to or during entry
tion, El-S and its counterparts are subject to of these into estrogen-responsive tissues.
efficient absorption. Indeed, El-S and 7-dehy- Clinical pharmacokinetics and biotransfor-
mation of one of the components of conjugated
equine estrogens have been studied systemat-
Gut ically. Thus, oral administration of 17P-dihy-
El -S droequilin (52b) &sulfate to postmenopausal
I women resulted in the urinary identification
a l of (S2b-c) and (53b-c) (structures, Section
t 4.3), and a high percentage (5141%)of more
El El polar metabolites suggested to be polyhy-
Blood droxylated derivatives of the four identified
metabolites (22). These results suggest that
Figure 13.2. Fate of orally administered conju- (52b) 3-sulfate ester is subject to absorption/
gated estrogens, exemplified by estrone sulfate (El- deconjugation as summarized in Fig. 13.2, fol-
S). After absorption, El-S is subject to deconjuga- lowed by oxidative metabolism. They extend
tion by sulfatase (a) in the liver, or possibly in the
findings of an earlier study in which levels of
blood or during transport to affected tissues. Decon-
jugation could occur to some degree in the gut as (I),(2), and ( 5 2 4 were measured in the serum
well. (b) This reaction is reversible, mainly in the of patients on conjugated equine estrogenic
liver, by sulfotransferase(s). therapy (23).
636 Female Sex Hormones, Contraceptives, and Fertility Drugs

3.4 In Vivo Reversible Monoesters of (1) ether, by parenteral dosing (2a). Thus, meta-
The estrogenic potency of (1)is much greater bolic 0-demethylation appears to play a major
parenterally than orally. Furthermore, esteri- role in mediating estrogenic potency of (121,as
fication of the 17P-hydroxyl group of (1) it does in the case of the steroidal estrogen
results in very prolonged (up to 14 days) mestranol(10) (18).
parenteral estrogenic effects, ostensibly at- Information about the metabolic fate of ta-
tributable to the prolonged dissolution rate of moxifen (16a) has emerged in an eclectic way
the poorly water soluble esters from the site of over the past 30 years, as implied by the letter-
administration (24). Examples are (1)-17-val- code designations used to denote its metabo-
erate, and (1)-17-(3-cyclopenty1)propionate lites (Table 13.4). Efforts to identify metabo-
(cypionate)(Table 13.1, footnote h) (25). Once lites were stimulated by the early finding that
absorbed into the blood from the injection site, 4-hydroxytamoxifen (la),a major metabolite
nonspecific esterases release (1)from these es- of (16a) in certain animal species, was a high
ters (26). affinity ER ligand and potent antiestrogen
(30). Comparative ER relative binding afini-
3.5 Oxidative Metabolism of Triarylethylenes ties (RBAs), and serum levels in patients on
Chlorotrianisene (12) was more effective long-term therapy, of (16a) and its metabo-
orally than parenterally and did not interact lites (17-21) are summarized in Table 13.4.
Taken together, these data suggest that (16a)
expresses its effects in large part through its
metabolites.
Studies with human liver microsomes
showed that conversion of (16a) to (17) and
(18) was catalyzed, in turn, by cytochromes
P450 2D6 (2C9,3A4 minor contributors) and
cytochrome P450 3A4 (34). Interpatient vari-
ability
" in hepatic levels of these enzymes
A " could
account for the wide range of serum levels of
(17) and (18)(Table 13.4).
Systematic biotransformation studies of
toremifene (22) revealed a pattern of metabo-
lism reminiscent of (16a), in human subjects
and in the female rat (35).
Despite its structural similarity to (16a)
and (221, clomiphene (23) was found to be less
susceptible to oxidative biotransformation.
with uterine estrogen receptors (27). Thus, it Thus, in the female rat, (23) was accompanied
was thought to undergo metabolic activation by only low levels of metabolites (36). Clinical
by drug-metabolizing enzymes of the intesti- studies indicated differential pharrnacokinet-
nal mucosa or liver. In vitro studies with oxi- ics of (23a) and (23b), but levels of metabo-
dase enzymes from rat liver suggested that lites were not reported (37). Unlike (16a) and
(12) was converted to a chemically reactive (22), (23) is suitable only for short-term ther-
metabolite that interacted irreversibly with apy because of unacceptable side effects. Per-
added estrogen receptors (27). Related studies haps this is attributable to its lack of conver-
demonstrated the conversion of (12) to its sion to multiple active metabolites expressing
mono- and bisphenolic metabolites, (13) and together a more favorable "therapeutic ratio"
(14) (28). Bisphenol (151, a close structural than that of the parent drug. This speculation
analog of (141, had estrogen receptor affinity aside, studies with (16a), (22),and (23) clearly
comparable to that of (1)(29). The estrogenic indicate that minor structural variation in
potency of (15) in the immature mouse was these triarylethylenes can greatly influence
200 times greater than that of its bis-methyl the extent of oxidative metabolism.
3 Drug Metabolism

3.6 Biotransformation of ical carcinogens are known or thought to re-


Diethylstilbestrol (24) quire activation by drug-metabolizing - en-

Years ago, (24) was widely used therapeuti- zymes (see below), considerable attention has
cally before its carcinogenic and teratogenic been focused on the ways in which (24) under-
potential became known. Because most chem- goes biotransformation. Because it is a li-
pophilic phenol, (24) is subject to O-glucu-
ronidation and, like steroidal phenolic
glucuronides (see Fig. 13.31, the resulting con-
jugate is capable of enterohepatic recycling
(38). In addition, (24) is subject to extensive
metabolic oxidation. Catechol and diene me-
/
HO#OH CH3 tabolites
other have (40).
products beenInidentified (39), as have
animals, dehydrogena-
tion of (24) to its diene metabolite is now
(24) thought to proceed spontaneously from a qui-
Table 13.4 ER RBAs and Clinical Steady-State Blood Levels of Tamoxifen (16a)
and Its Metabolitesa
Compound Metabolite Designation RBA Serum Level (ng/mL)

- -

"The RBAs were determined using ER from rat uterus (RBA of 1 = 100) and are from Ref. 31 unless otherwise
noted. Serum levels are ranges of values reported by different laboratories (32).
bDeterminedusing calf uterine cytosol(33).
"RBAis for a 1:lmixture of cis and trans isomers.
638 Female Sex Hormones, Contraceptives, and Fertility Drugs

Liver Blood types to (1)(46). Alternatively, reactivity of


glucuronyl catechol metabolite (6) was demonstrated by
E-G 4
transferase (t its ability to undergo electroreductive cou-
I
I
I pling at C-1 with the C-8 of adenine (47).
bile duct portal vein A quinone-type intermediate (251, pro-
duced by interaction of (24) with peroxidases,
1 bacterial hydmlase \
E-G rn E
Small intestine

Figure 13.3. The enterohepatic recycling system.


Effectiveness of estrogens (E)such as (11, (21, and
(9) is amplified by this process because each of the
respective glucuronide conjugates (E-G),after intes-
tinal accumulation, is a substrate for deconjugating
enzymes of normal gut flora. This results in reab-
sorption of the free estrogens.
was proposed to interact covalently with tubu-
none-type intermediate metabolite (below) lin and possibly with other macromolecules
generated by enzymatic oxidation of (24) (41). (48).Another nonsteroidal estrogen, hexestrol
(26),exhibited tumorigenicity in the male Syr-
3.7 Electrophilic (Carcinogenic 1)
Metabolites of ER Ligands
Long-term administration of steroidal estro-
gens such as (1)and (2) has been associated
with a slight increase in the incidence of repro-
ductive tract cancer. Similarily, a small but
significant incidence of adverse reproductive
tract effects, including cancers, in female off-
spring of women receiving diethylstilbestrol
(24) during pregnancy, has been reported
(42).This resulted from the unfortunate use of ian Golden hamster similar to that of (24)
(24) to prevent miscarriage, before its charac- (49), but ostensibly through a mechanism of
terization as a teratogen and carcinogen (43). metabolic activation more like that of (1)and
Moreoever, despite results indicating (16a) to (2). The major metabolite of (26) is 3'-hydroxy
be less estrogenic than steroidal estrogens in (26), which in turn was subject to further ox-
reproductive tissues, an increased risk of en- idation to a 3',4'-ortho-quinone by certain cy-
dometrial cancer has been associated with its tochrome P450s and/or peroxidases. This elec-
use @a). trophilic quinone was reactive toward purine
Metabolic "activation" to electrophilic me- moieties: it arylated N-7 of 2'-deoxyguanosine
tabolites is generally thought to be a pre- units in DNA (50).
requisite to chemical carcinogenicity (44). In the rat, long-term administration of
Electrophilic metabolites of steroidal and non- high doses of (16a)resulted in development of
steroidal ER ligands have been characterized. hepatic tumors. Several specific routes of oxi-
Thus, a hydroxylated metabolite of (2),specif- dative biotransformation have been impli-
ically (4), forms a stable adduct with albumin, cated in conversion of (16a) to electrophilic
suggested to arise from Heyns rearrangement metabolites proposed to account for tumorige-
of an a-hydroxyimine intermediate, itself pro- nicity (51). A stabilized quinoid intermediate
ducted by reaction of (4) with a lysine residue (27), generated by spontaneous cycloelimina-
of albumin (45). DNA damage was associated tion of sulfate from proximate metabolite (281,
with exposure of mouse mammary cells to (4), is an example of an electrophilic species (52)
but was not observed on exposure of these cell suggested to originate from (16a).
3 Drug Metabolism

In summary, the connection between for-


mation of chemically reactive metabolites of
ER ligands and their carcinogenicity risk in
humans has become clearer. However, other
factors besides conversion to electrophilic me-
tabolites might contribute to carcinogenicity
(57). Chronic exposure of cells containing ER
to full or partial estrogen agonists such as (1)
and (16), respectively, might be a major caus-
ative factor, given that carcinogenicity of
these substances in humans is generally re-
stricted to ER-containing organs.

3.8 Aspects of Glucuronide Conjugation


Large lipophilic molecules containing phe-
nolic moieties are often subject to glucuronide
conjugation. Many ER ligands fit this descrip-
tion, although the impact of this metabolic
pathway on the observed potency of a particu-
lar ER ligand depends on several factors.

3.8.1 Termination of Effects of ER Ligands.


Both oral bioavailability and duration of ac-
tion of raloxifene (29) are affected by its sus-

Electrophile (27) was suggested to N-alky-


late deoxyguanosine residues in DNA in the
rat (53, 54), and it might do so in humans.
Thus, DNA analysis of endometrial tissue
samples from patients receiving (16a) re-
vealed, in eight of the 16 patients studied, the
presence of a-(N-2-deoxyguanosiny1)-(16a)
and -(16b); levels ranged between 0.2 and 12
and 1.6 and 8.3 adducts/lOs nucleotides, re-
spectively (55).
Although long-term high dose administra- ceptibility to glucuronide conjugation, which
tion of (16a) has been associated with hepato- occurs predominantly at its 4'-hydroxyl group
carcinogenicity in the rat, this has not been in humans (58). Thus, despite its 40-fold
the case in humans. However, the possible higher affinity for ER compared to that of
link between use of (16a) in breast cancer (16a)and 1000-fold greater in vitro estrogen
treatment and human hepatocarcinogenicity antagonist potency (59), the oral potency of
has not been investigated thoroughly because (29) is one-half that of (16a), based on daily
of the assumption that any liver tumors in doses (Table 13.1) normalized for differences
(16a)-treated patients would arise secondarily in molecular weights. In female patients,
to breast cancer metastasis (56). Thus, the in- (16a) is accompanied by its active, oxidized
cidence of human hepatocarcinogenicity asso- metabolites (Table 13.4) and is evidently not
ciated with administration of (16a) might be subject to glucuronide conjugation; (291, in
significant. contrast, is present in serum primarily
640 Female Sex Hormones, Contraceptives, and Fertility Drugs

(>go%) as its 4'-0-glucuronide conjugate. Be- 3.9 Biotransformation of Progesterone (30)


cause ER affinity of (29)-4'-0-glucuronide is and Its Analogs
less than 1/10 that of (29) (60), it is unlikely Because it is subject to rapid reductive metab-
that this metabolite contributes to estrogenic/ olism, (30)is not orally effective as a proges-
antiestrogenic effects.
Because SERMs with high ER affinity al-
ways contain at least one phenolic hydroxyl
group, the possibility of bioavailability-limit-
ing glucuronide conjugation is always a drug
design consideration.

3.8.2 Net Glucuronidation. Susceptibility


to glucuronide conjugation, after absorption,
is governed not just by the rate of O-glucu-
ronidation of the phenolic SERM by uridine
diphosphate (UDP) glucuronyl transferases,
but also by the degree of exposure of the re- tin. The major urinary metabolite of endoge-
sulting conjugate to glucuronidase (61). Both nous or parenterally administered (30) is
enzymes are located to some degree in gastric pregnanediol (31),which is eliminated as a
mucosa, but mainly in hepatocyte endoplas- glucuronide conjugate. Alternatively, 3-keto-
mic reticulum. Thus, the high net gluc- and 4,5a-reduction of (30) affords (321, a me-
uronidation of (29) in serum is presumably a
consequence of minimal exposure of (29)-4'-
0-glucuronide to enteric and hepatic gluc-
uronidase. Net glucuronidation of a SERM
might depend on its structure: a SERM gluc-
uronide with maximal access to glucuronidase
would exhibit reduced net glucuronidation, re-
gardless of the affinity of its parent for UDP
glucuronyl transferase. Alternatively, im-
provement in SERM bioavailability and po-
tency by incorporating structural changes
that reduce susceptibility to UDP glucuronyl
transferase has been carried out successfully
(see Section 7.3.2) (62).

3.8.3 Enterohepatic Recycling. Besides be-


ing capable of enzymatic deglucuronidation in
vivo, the effectiveness/potency of estrogens
such as (I),(2), and analogs such as (9)is pri-
marily conserved by a second mechanism.
Glucuronide conjugates of these estrogens are
accumulated in the bile duct and thus trans-
ported into the small intestine. There, these
particular glucuronide conjugates are sub-
strates for bacterial hydrolases. The regener-
ated steroids are efficiently absorbed from the
intestine (Fig. 13.3). This process of enterohe- tabolite that potentiates the y-aminobutyric
patic recycling accounts for the prolonged du- acid-A receptor and appears to be responsible
ration of effect of endogenous and adminis- for the anticonvulsant effect of (30) in labora-
tered estrogens (63). tory mice (64). Formation of (32) was shown to
3 Drug Metabolism

proceed by initial dihydrotestosterone reduc-


tase-catalyzed saturation of the 4,5-double
bond, followed by oxidoreductase-catalyzed
3-keto reduction.
Several close structural analogs of (30)are
noteworthy with regard to their prolonged
progestational effects when given by injection.
Thus, 17a-hydroxyprogesterone~ p r o a t (33)
e

(35). In animal studies, (37) exhibited proges-


tin antagonist activity (65). Carbonyl reduc-
tion of (37) affords the 3P-hydroxy-5a-dihydro
metabolite of (35), which had a very low PR
affinity. Thus, this metabolite is unlikely to
express effects through these receptors. Alter-
natively, A-ring aromatization of estrane pro-
gestins has also been observed. Thus, (35)un-
dergoes conversion to (9) to the extent of 2.3%
in perimenopausal women (66), an extent that
appears to be pharmacodynamically signifi-
cant.
Desogestrel (38) undergoes A-ring allylic
is a long-acting progestin. Also, medroxypro- oxidation to its 3-keto metabolite in patients
gesterone acetate (34) has prolonged paren-
teral effects and is moderately active orally. It
is much more effective by these routes than its
6-normethyl counterpart.
Estrane progestins can undergo 4,5-ethyl-
enic bond reduction with and without keto re-
duction. Thus, norethindrone (35) and i-nor-

(67). This metabolite, 3-0x0 (38), has PR affin-


ity 60 times greater than that of (38) and thus
is probably responsible for the activity of this
progestin.
The reductive metabolites of gestodene
(39), especially (40b), exhibited affinity for
human ER and estrogenic activity in vitro, un-
like (39)itself (68), despite the absence of the
gestrel (36) are biotransformed to several aromatic A-ring seen in most steroidal estro-
A-ring-reduced (dihydro and tetrahydro) de- gens (Section 7.1). Thus, the estrogenic effects
rivatives. The progesterone receptor (PR) and of (39)seen in viuo might be the result of re-
androgen receptor affinities of (37) (Table ductive conversion to its structurally novel ER
13.21, the 5a-dihydro metabolite of (36), sug- ligand, (40b), rather than of A-ring aromati-
gest that it contributes to the overall effects of zation, as was the case with (35).
Female Sex Hormones, Contraceptives, and Fertility Drugs

The ovary is the major source of these estro-


gens except during pregnancy, in which pla-
cental production dominates. Adrenal cortex
and adipose tissues produce smaller amounts
of these estrogens. After menopause, these
tissues become predominant sources.
A novel and unifying structural feature of
endogenous estrogens is aromaticity. [The
biosynthetic sequence by which progesterone
(30) and testosterone arise has been clearly
established (70; see also previous editions).]
Consequently, attention is focused on the
mechanistic basis by which steroid aromatiza-
tion, the rate-determining step in estrogen
4 ASPECTS OF BIOSYNTHESIS
biosynthesis, takes place. Androgens were
shown to be precursors: thus, androstenedi-
Plasma and tissue levels of female sex hor- one (41) was a precursor to (2) in endocrine
mones are controlled by follicle-stimulating tissue (71). Androgen aromatization is cata-
hormone (FSH) and luteinizing hormone lyzed by aromatase (estrogen synthetase), an
(LH). These polypeptide gonadotrophins are enzyme found at high levels in placental and
secreted by the anterior pituitary, and control ovarian tissue. Aromatase is a member of the
the activity of enzymes, located predomi- cytochrome P450 family of intracellular mem-
nantly in the ovary, which sequentially oxidize brane-bound, mixed-function oxidases (72).
cholesterol to progesterone (30), and 17P-es- Mechanistic studies indicated that it catalyzed
tradiol (1)plus related estrogens. Together, androgen aromatization through three oxida-
(1)and (30) are important in the development tion steps. Thus, (41) initially undergoes se-
of female sexual characteristics, in regulating quential conversion to a 19-0x0 intermediate,
fertility, and in maintenance of normal func- (43) (73) (Fig. 13.4). This is followed by an
tion (homeostasis) in tissues and organs en- addition-elimination sequence believed to be
dowed with ER and PR. Although (1)and (30) initiated by a novel peroxyanion stabilized by
are the most important female sex hormones, heme iron at the active site of aromatase (74).
it is noteworthy that smaller but significant This mechanism was supported by the finding
amounts of androstenedione (41) and its 17p- that 0, was the main (90%)source of the "sec-
hydroxy counterpart (testosterone) are also ond" oxygen in HCOOH released during aro-
released from the ovary. matization of (43). In addition, the A2-3-deoxy
analog of (43) underwent aromatization. This
4.1 Steroid Aromatization
suggested that positioning of the lp-hydrogen
Estrogens (1-3)are the three most abundant is critical for rearrangement to occur, and that
endogenous estrogens. These were first iden- 2,3-enolizationof (44) and (43) might thus fa-
tified in the urine of pregnant women (69). cilitate aromatization (75).
4 Aspects of Biosynthesis

Figure 13.4. Aromatase-catalyzed conversion of androstenedione (41) to estrone (2). Seauential


hy&oxylation of (41) at (2-19gives (421, which undergoes dehydration to (43). This aldehyde reacts
with nucleophilic ferric peroxyanion, generated at the catalytic site of aromatase, to give (44),which
undergoes spontaneous rearrangement to produce (2) and formic acid.

4.2 Aromatase Inhibitors


Considerable attention has been directed to-
ward identification of drugs capable of selec-
tive, potent aromatase inhibition. Such drugs
were envisioned to facilitate reduction in lev-
els of circulating estrogens, thus offering a
means of suppression of estrogen-dependent
tumors.
Clinically relevant aromatase inhibition has
been found in nonsteroidal as well as steroidal
structures.Some examples of the former include
aminoglutethimide (G), initially evaluated for
its effects on the central nervous system. Other
enzymes involved in steroid hormone biosynthe-
sis besides aromatase are inhibited by (45).

hibitors with maximal aromatase selectivity are


vorozole (49) and anastrozole (50) (77).
The androstane derivative exemestane (51)
However, imidazoles such as fadrozole (46)and is a potent, selective, irreversible inhibitor of
triazoles such as (47) and letrozole (48) (76) in- aromatase (78). Like (4860), (51) is advocated
terfere with estrogen biosynthesis by more se- as a "second-line" treatment for patients whose
lective inhibiton of aromatase. Molecular mod- breast cancer is no longer suppressed by (16a)
eling studies with the active S-(-)-enantiomer (77, 79). Furthermore, because (51) did not
of (46)suggested that its N-heteroaromatic ring show cross-resistance with nonsteroidal aro-
interacts with the heme iron of aromatase at its matase inhibitors (79b), resistance was sug-
sixth ligand-bindingsite (76b).Examples of tria- gested to arise from changes in neoplastic cells
zole ring-containing nonsteroidal aromatase in- not directly linked to aromatase.
Female Sex Hormones, Contraceptives, and Fertility Drugs

4.3 Genesis of Equine Estrogens


Equine estrogen conjugate mixtures are com-
posed of a reasonably fixed proportion of phe-
nolic sulfate esters of estrone (2) accompanied
) equilenin (53~1,
by those of equilin ( 5 2 ~and
together with their respective 17a- and 17p-
hydroxy derivatives (80)(Table 13.5). Trace
amounts of a large number of related com-
pounds accompany these substances, but their
structural identities and biologic effects have
not been reported yet.
Unlike virtually all other natural product
extracts from which single purified compo-
nents have emerged as therapeutic agents,
mixtures of conjugated equine estrogen ex-
tracts are still preferred in estrogen-replace-
ment therapy.
Biosynthetically,the seven ring B-dehydro-
genated estrogens in Table 13.5 are produced
by pathways not involving squalene or choles-
terol, intermediates common to the biosynthe-
sis of all steroid hormones in humans. The
horse is an excellent source of (2), but only the
pregnant mare produces these dehydroge-
nated estrogens, which originate by pathways
that do not involve (I),(2), or their hormonal
precursors, (30)and (41) (83).
About 19% of equine estrogen conjugates
consists of 17a-hydroxy steroids (17-epi-(l),
(52a), and (53a),but the corresponding 17p-
hydroxy derivatives account for a much
smaller percentage. Although (2) undergoes
reversible reductive metabolism to (1)in hu-
mans (Fig. 1 3 1 , 17-epi-(1) is not produced
from (2). It is therefore unlikely that these
l7a-hydroxy steroids interconvert with their
17-0x0 counterparts in humans, and thus
their pharmacodynamic effects would be ex-
pressed independently of (1)and (2).
5 Physiology and Pharmacology 645

Table 13.5 Percentage Composition and Estrogenic Effects of Steroids in Coqjugated


Estrogen Tablets
Component Avg. wt %" RBA~ Uterotrophic E f f i c a e
17-epi (1)
(1)
(2)
A8~g-dehydro
(2)
(52a)
(52b)
(52~)
(53a)
(53b)
(53~)
Numerous other steroids
"Data are for 3-sulfate esters (80).
bRelativeto (52b)or (I),for human endometrial ER. Data in this column are from Ref. 81 and 82.
"The ability of each unconjugated steroid to stimulate uterine weight gain in the immature rat, at an i.p. dose of 2
pglanimdday for 3 days, with respect to vehicle-treated control: efficacy = 1.0, average uterine wet weight = 33.8 mg; P <
0.01 with respect to control for all steroids except 17-epi (1)(82).

5 PHYSIOLOGY A N D PHARMACOLOGY specific) binding of [3H]1, displacable by


coadministration of excess amounts of unla-
Endogenous estrogens (1-3)affect the cellular beled (I),was observed in uterine cytosol, and
roles of genes involved in homeostasis, cell di- in uterine and ovarian tissues of animals re-
vision, protein expression, and cell communi- ceiving [3Hl1. In the 30-plus years since these
cation. This control is mediated by interaction discoveries, our perception of the ER's struc-
with estrogen receptors. ture in the absence and presence of its ligands
The endogenous progestin progesterone has grown considerably.
(30) has major roles in preparation of the There are two isoforms of human ER, des-
uterus for pregnancy, maintenance of preg- ignated as hERa and hERP. These show a high
nancy, and in maintenance of the female re- degree of amino acid sequence homology in
productive organs. It is a central biochemical their hormone-binding domains (Fig. 13.51,
intermediate in the biosynthesis of estrogens, especially with regard to amino acid func-
androgens, and corticosteroids. It is produced tional groups making direct contact with li-
in the ovaries (corpus luteum), adrenal glands, gands, or lining the binding pocket (86); how-
and in the placenta during pregnancy. As sug- ever, there are subtle differences in their
gested from the receptor-binding data in Ta- respective character. So, although observable
ble 13.2, (30) interacts with other steroid aMinity of many ER ligands for each isoform is
receptors besides PR. Interaction with miner- similar, a few such ligands exhibit isoform
alocorticoid and glucocorticoid receptors ac- preferences. The homology of ERa and ERP
counts in part for its physiologic effects. Also, varies slightly or greatly, depending on do-
(30)antagonizes oxytocin at uterine oxytocin main, relative to respective isoforms found in
receptors (84), suggesting its pregnancy- experimental animals (87,88). ER is found in
maintaining effect is in part mediated non- monomeric, (mixed) dimeric, and oligomeric
genomically. states (89). Most of the ER within affected
cells is located in the nucleus, although ER has
5.1 Topography of Estrogen Receptors
been identified in cell membranes (see below).
The first direct evidence for the presence of Thus like PR, ER appears to mediate effects of
ER in estrogen-sensitive tissues was enabled ER ligands through nongenomic as well as
by the availability of radiolabeled (1)of suffi- well-established genomic mechanisms (90).
cient specific activity for use in tissue distribu- Each ER isoform is composed of six do-
tion studies (85). Thus, saturable (receptor- mains, as shown in Fig. 13.5 (91). The ligand-
Female Sex Hormones, Contraceptives, and Fertility Drugs

AIB C D E F

N1
-I DBD 1 LBD 1 C hERa

Figure 13.5. Diagram of hERa and hERP primary structures. Numbers starting from the N-termi-
nal end of the peptide indicate positions of amino acids at junctions of the six respective domains
(A-F). Each hormone (ligand) binding domain (E) contains the hormone-dependent transcription
activation function 2 (TAF-2).The hormone-independenttranscription activation function 1 (TAF-1)
is located in respective domains A and B. Domain C contains the DNA-binding domain (DBD).
Percentages indicate the degree of sequence homology of hERP domains to those in hERa.

binding domain (LBD)is embedded in Domain AG for all of these specific interactions (-14.2
E. Domain B, which contains the hormone- kcallmol) is in reasonable agreement with that
independent transcription activation factor 1 determined experimentally.
(TAF-1) and domain E, which in addition to Crystallization of chromatographically pu-
the LBD contains the hormone-dependent rified hERa LBD complexed with (1) was
transcription activation factor 2 (TAF-21, are achieved after initial carboxymethylation
of special significance. Studies with a series of with iodoacetate, which fortunately affected
truncated ERas indicated that only TAF-2 is only cysteine units not directly involved in li-
required for transcriptional activity of (I),but gand interactions. X-ray diffraction studies of
certain other ER ligands such as tamoxifen this complex showed that the ionized side-
( 1 6 4 required modified ERa with both TAF-1
chain functional groups of Glu353 and Arg394
and TAF-2 for this activity (92).
interact in tandem with the phenolic hydroxyl
The hER interacts strongly with (I), with
of (1). Hydrogen bonding and/or ion-dipole
an association constant of 5.5 x 109/M,corre-
sponding to a free energy of binding (AG) of bonding (Fig. (13.6a) are implicated in these
-13.5 kcdmol. Electrostatic, hydrophobic, associations. In addition, His524 interacts
and dispersional (van der Wads) interactions with (1)'s 17P-hydroxyloxygen by similar pro-
between (1)and ER contribute to AG, and the cesses. The hydrogen bond accepting Glu353
magnitude of specific bonding interactions has is very important in allowing ERa to discrim-
been estimated rationally (93). Thus, electro- inate between (1)and steroids bearing non-
static interactions involving the 3- and 17P- complementary 3-keto groups, especially an-
hydroxyl groups (Fig. 13.6a) contribute, in drogens (95). Lipophilic Phe, Tyr, Leu, Ile,
turn, -1.9 and -0.6 kcallmol to AG, and the Ala, Val, Met, and Ile units make up 40% of the
one involving (1)'s benzene ring 7~ electrons total of the LBD's amino acids (91a), and the
contributes -1.5 kcallmol. Interaction of (1) aliphatic or aromatic side chains of at least 15
with ER results in loss of ordered water mole- of these are complementary with the hydro-
cules surrounding the surfaces of the steroid carbon framework of (1)(961, and interact by
core and complementary hydrocarbon groups hydrophobic bonding.
of amino acid units proximate to the binding Analogously prepared complexes of dieth-
pocket. Thus, the entropy of binding is +22 ylstilbestrol (24) and raloxifene (29) were
entropy units, which contributes - 7.8 kcall found to exhibit the same electrostatic inter-
mol (at 30°C) to AG. The total dispersion en- actions with cysteine-carboxymethylated LBD
ergy, calculated from the sum of atomic polar- as did (1)(94,97). In addition, the protonated
izabilities of carbon, hydrogen, and oxygen in side-chain nitrogen of (29) interacted with the
(I),is -2.4 kcdmol. The sum of the estimated ionized carboxyl group of Asp351 by ionic
5 Physiology and Pharmacology

Glu 353

Arg 394

Asp 351

,
J .-H Figure 13.6. Electrostatic bonding in-
teractions seen in the crystalline LBD
Glu 353 (modified) of hERa liganded with (a)
17P-estradiol (1); (b) 4-hydroxytamox-
ifen (18) (94, 97). A water molecule as-
sociated with the phenolic hydroxyl of
each ligand is omitted. In complex b,
Thr347, Met421, and Met343 are inter-
NH2 positioned between the unsubstituted
Arg 394 phenyl ring of (18)and His524.

bonding (94). The phenolic hydroxyl of 4-hy- closely to the unsubstituted phenyl ring in
droxytamoxifen (18) interacted with Glu353 (l8),and presumably would not interact with
and Arg394 of cysteine-carboxymethylated this ring even if it was hydroxylated.
LBD in the same way as did that of (1)and, The LBD of the other hER isoform (hERp),
like (29), its (protonated) amino group inter- liganded with (29) or genistein (59), has also
acted ionically with the Asp351 carboxylate been crystallized (98). Electrostatic interac-
group of the modified LBD (Fig. 13.6b) (97). tions (bond lengths 3.3-3.8 A) in these com-
However, in contrast to complexes involving plexes were identical to those in the hERa
(11, (241, or (29), His524 was not positioned LBD-(29) complex.
648 Female Sex Hormones, Contraceptives, and Fertility Drugs

Unlike (I), (18), and (29) interact with sociation of estrogen antagonists from analo-
Asp351 in each hER isoform. Although such gous complexes was not affected.
bonding facilitates affinity of these nonsteroi- ER heterogeneity appears to play a role in
dal ligands for hER, it is not the basis for their modulation of responses of specific organs to
mixed agonist-antagonist effects (99) (see Sec- (1). ERP has been shown to inhibit transcrip-
tion 5.9). tional actvity of ERa at subsaturating levels of
(I), and therefore ERP can decrease overall
5.2 Ubiquitous Distribution of ER lsoforms cellular sensitivity to (1)(107).
ER has conventionally been associated with An observation consistent with the rate
organs of the female reproductive axis. ER has theory of drug action (108) has been demon-
also been associated with estrogen-stimulated strated in the interaction of liganded ER with
cancer, particularly of the breast and endome- ERE. Thus, (1)-ER exhibited an association/
trium. However, the number of organs now dissociation rate with ERE that was 1000
confirmed to contain ER, albeit at lower levels, times the rate observed when ER was com-
has expanded greatly in recent years, and is plexed with a "pure" estrogen antagonist, ici
not confined to females. Thus one or both of (ZN)182,780 (64) (109). It was hypothesized
the ER isoforms have been localized immuno- that the frequency of association of liganded
histochemically in the nuclei of cells in lung, ER with ERE determines the degree of agonist
liver, kidney, adrenal glands, colon, heart, efficacy of an ER ligand.
prostate, testis, most areas of the brain, and in
bone-remodeling cells (osteoblasts and oste-
oclasts) (100, 101). 5.4 Assessment of Estrogenic Activity
Estrogenic potency and efficacy have tradi-
5.3 Molecular Endocrinology of ER tionally been expressed in terms of uterotro-
When (1) interacts with (oligomeric)ER, helix phic effects in immature or OVX female ro-
12 of its LBD seals the occupied binding dents. Thus, daily administration of (1) to
pocket, thereby resulting in important 3-week-old female rats for 3 days results in a
changes in the association state and conforma- three- to fivefold increase in uterine weight
tion of ER (3,94).These include (1)conversion (110). This sensitive model for pharmacologic
of ERaIERP to homo- and heterodimeric characterization of estrogens can also be used
states; (2)exposure of regions in these dimers to identify and characterize estrogen antago-
proximate to TAF-2, and possibly to TAF-1 nists, wherein a maximally uterotrophic dose
also, which can bind to one of several coregu- of (1)is administered without and with the
lator proteins (102); and (3) "activation" of putative antagonist, to determine the maxi-
domain C. Domain C features two regions of mal degree and potency to which uterine
its peptide backbone extruded in fingerlike weight gain is prevented (110b).
fashion. Each finger is anchored through four Determination of ER affinity has provided
cysteine residues near its base by a zinc cation a way to identify substances with potential es-
(103). These zinc fingers interact strongly trogenic and antiestrogenic effects. Because
with complementary nucleotide sequences in ER is a soluble protein, uterine tissue homog-
DNA known as estrogen response elements enates from various animal sources provided a
(ERE4 (104). convenient source of cytosolic ER. Currently,
The (1-ER),-coregulator complex thus in- recombinant hERa and hERP variants are
teracts with ERE, ultimately resulting in preferred for these studies. The ability of pu-
changes in intracellular levels of various en- tative ER ligands to displace specifically
zymes, receptors, and other proteins, leading bound [3H]1 (radioreceptor assay) or fluores-
to control of cell activity and proliferation cein-linked (1) (fluorescence polarization
(105). An additional function of the coregula- assay) in these preparations is determined.
tor in this complex is to retard dissociation of Current therapeutic and noteworthy exper-
(1)(106). This was also the case with other imental estrogens and antiestrogens, and cer-
estrogen agonist ligands such as (241, but dis- tain environmental and dietary estrogens (see
5 Physiology and Pharmacology

Section 5.8) have RBAs ranging from at least as a transcriptional activator, but hPRA is less
0.05% to nearly 1000% (10 times) that of (1) active transcriptionally and can function as a
(see Tables 13.3-13.7). strong ligand-dependent repressor of tran-
Determination of estrogen agonist or an- scriptional activity. The ability of (30) li-
tagonist potencies and efficacies has been con- ganded with hPRB and hPRA to recruit, in
veniently carried out using lines of cultured turn, coactivator and corepressor proteins
neoplastic cells naturally endowed with ER, might account for this difference (117).
its coregulators, and EREs. Estimation of es- Both hPR and hER feature about 250
trogen-induced synthesis of enzymes or pre- amino acids in their LBDs. The crystal struc-
cursor RNAs, or determination of overall cell ture of the hPR LBD (amino acids 677-933)
proliferation rate or extent can be used to as- complexed with (30) revealed considerable
sess effects. Interpretation of results is usually similarity regarding electrostatic and hydro-
not complicated by biopharmaceutic factors, phobic/Van der Wads contact with that of the
such as biotransformation. However, experi- (1)-hER LBD complex (96, 120) (cf. Fig. 13.7
mental controls validating clonal integrity with Fig. 13.6a).Regarding electrostatic inter-
must always be run (111). MCF-7 human actions, the major difference is replacement of
breast cancer cells and Ishikawa human endo- Glu353 (H-bond acceptor) in hER with a ho-
metrial cancer cells are two lines that have mologous Gln725 (H-bond donor) in hPR.
become well established for these applications Otherwise, considerable homology exists be-
(112). tween the lipophilic amino acids (Leu, Ile,
Substances exhibiting significant ER affin- Met, Phe, Val), which line the binding pockets
ity can be evaluated in the OVX rat for other of these receptors. Crowding associated with
ER-mediated effects besides uterotrophic ac- interaction of hPR LBD with (30)in the region
tivity. The OVX rat has become firmly estab- of its 19-methyl group might explain in part
lished as a model for human disorders associ- why 19-norpregnane progestins (e.g., 94, 95)
ated with estrogen deprivation, among them interact somewhat more strongly with this re-
elevated serum cholesterol (113) and osteope- ceptor than does (30) (Table 13.2). However,
nia (bone density loss) (114). Also, progress in conceding that interactions of the lipophilic
development of models for disorders associ- amino acid units lining the binding pocket of
ated with CNS estrogen deficiency, using the hPR LBD might enhance PR ligand selectiv-
OVX rat, has been reported (115, 116). ity, the structure of hPR's complex with (30)
does not provide a clearcut basis for the selec-
5.5 Progesterone Receptors
tive affinity of hPR for (30) versus other A-
Most of the effects of (30) are mediated by ring enone steroid hormones such as testos-
interaction with PR. Cells that contain PR terone and cortisol, or why introduction of a
usually contain ER as well, and estrogens up- 17a-ethynyl group into testosterone results in
regulate PR in these cell types (117). There are a ligand with preferential affinity for hPR over
two isoforms of PR: PRB, a 933 amino acid hAR. Reasons for PR's ability to differentiate
peptide; and PRA, which is identical to PRB between progestins and estrogens on the basis
except that the N-terminal164 amino acid se- of electrostatic interactions with respective A-
quence in PRB is absent (118).Levels of PRA ring substituents (1)and (30) are, however,
and PRB generally are nearly equal in most evident. These are predominantly hydrogen
cell types that contain PR. Interaction of (30) bond-donating interactions in hPR LBD, but
with oligomeric P W R B leads to receptor the hER LBD interacts with the A-ring sub-
dimerization in which three dimers are pro- stituents by a combination of H-bond donor
duced. These are designated AA, AB, and BB. and acceptor interactions.
The two hormone-binding domains in each PRs are found in organs of the reproductive
dimer are both occupied by (30). These three system. Also, hPR mRNA has been localized in
dimers bind to DNA at specific progesterone vascular smooth muscle cells and in human
response elements on the promoters of proges- osteoblast-like cells (121). In human subjects,
terone-responsive genes, and regulate tran- regardless of gender, PR (and ER) has been
scription (119). In most cells, hPRB functions immunolocalized in normal and varicose sa-
Female Sex Hormones, Contraceptives, and Fertility Drugs

Gln 725

Figure 13.7. Electrostatic interactions of the


A-ring of (30) with the LBD of the PR. The
3-keto of (30) is a central hydrogen-bond accep-
tor in a network formed by Gln725, Arg766, and
a water molecule. Hydrogen bond lengths range
from 2.5 to 3.1 A. The 4,5-double bond of (30)
interacts with the ring of Phe778 by T-T bond-
ing.
Arg 766
c""
phenous veins, and levels of both receptors In the ovariectomized rat, simultaneous
seemed to be higher in the former (122). administration of estrone (2) and high doses of
medroxyprogesterone (34) results in a maxi-
5.6 Assessment of Activity of PR Ligands mal (30%)reduction in uterotrophic response
compared to that seen in animals receiving
Experimental characterization of progester-
only (2) (124). This animal model can be used
one mimetic activity was based classically on
to identify progestins having the ability to
the ability of analogs of (30) to stimulate an
counteract the reproductive tract estrogenic-
endometrial response in the estrogen-primed
ity of administered estrogens.
rabbit. The reader is referred to the Fourth
Edition of this series for specific details of this 5.7 Progestin and Antiprogestin Effects
and related assays used to characterize pro-
gestins, and literature references to these. Steroidal progestins are analogs of (30) that
Currently, putative PR ligands are evaluated interact with PR and activate it in ways simi-
initially for their ability to interact with lar to that of (30)itself. The primary applica-
hPRA, and with human androgen, glucocorti- tion of these drugs is in birth control (see Sec-
coid, and mineralocorticoid receptors to assess tion 5.10).
the degree of apparent selectivity of the test Progestins often have other effects distinct
compound for hPR, an important issue in from those of (30) as a result of differential
identification of new progestins (123). Com- interaction of progestins or their metabolites
pounds with noteworthy hPR affinity and se- with other steroid receptors besides PR (Table
lectivity can then be subjected to cell-based 13.2). Norethindrone (35) has been applied
assays to determine agonist and antagonist clinically as a bone-loss suppressant in post-
potency and efficacy. Mammalian cells trans- menopausal women (1251, but (34) is not ef-
fected with a plasmid containing hPR and a fective in this application (125a).Because (35)
reporter gene enabling induction of assayable is metabolized in patients to afford significant
enzyme activity have been used for this (123, levels of the potent estrogen (9) (Section 3.9),
124). Alternatively, human breast cancer cell it has been hypothesized that this metabolite
lines expressing hPR, such as T47D cells in accounts for the-observed effect on bone loss.
which progesterone mimetics induce the en- Partial conversion of an analog of (351, tibo-
zyme alkaline phosphatase, have been used to lone (54), to an estrogenic metabolite might
quantify potency and efficacy. similarly account for some of (54)'s effects. In
5 Physiology and Pharmacology

the OVX rat and in postmenopausal patients,


(54) suppressed bone loss without the degree
of uterine stimulation associated with estro-
gens (126). Whether the bone loss-suppressive tissues of the reproductive axis, which might
effect of (54) is mediated directly by this drug contribute to its anovulatory effect (129).
or indirectly by estrogenic metabolite(s1 (e.g., The most significant medical application of
see Section 3.9, estrogenic metabolites of (35) (55)is for its ability to counteract (30) regard-
and (39)is not yet known. ing preparation of the uterus for implantation
There are two types of progesterone antag- and retention of a fertilized egg. Thus, (55)
onists. Interaction of Type I antagonists with can be used sparingly, rather than on a daily
PR results in impairment of PR association basis, as is the case for conventional oral con-
with DNA. Interaction of Type I1 antagonists traceptives. However, because its contracep-
with PR does not prevent PR-DNA associa- tive mechanism in this application is post-
tion, but the conformation of PR associated conceptional, its routine medical use is ideo-
with DNA does not facilitate transcription logically intolerable in the perception of many
(127). Thus, Type I1 antagonists not only in- people. Another experimental and clinical ap-
terfere with interaction of (30) with PR, but plication of (55)and other antiprogestins is in
can prevent interaction of the (30)-PR com- suppression of breast cancers that contain PR
plex with DNA (128). (130). Growth-suppressive effects are believed
Mifepristone (RU486, 55) is the best to result from PR-mediated cytotoxic mecha-
known Type I1 antiprogestin. Type I antipro- nisms.

5.8 Environmental and Dietary Estrogens


and Progestins
Estrogenic effects can be expressed by numer-
ous natural products, as well as by certain syn-
thetic organic compounds introduced into the
soil and water. Of particular importance are
chlorinated aromatic hydrocarbons, phenolic
industrial chemicals, and flavonoid phy-
toestrogens. This section deals with three of
these, the pesticide methoxychlor (57) and fla-
vonoids (59) and (60).
An analog of DDT, methoxychlor (57) ex-
gestin onapristone (ZK98299, 56) resembles hibited uterotrophic effects in rodents (131)
(30) with respect to the configuration and and was subject to cytochrome P450-mediated
chain length of its C-17 alkyl substituent. 0-demethylation in vitro (132). Its bisphenolic
However, its C-18 angular methyl group is in a "metabolite" (58) exhibited contrasting ef-
novel a configuration. Both types contain fects in cells transfected with alternate ER iso-
identical llp-aryl substituents. In the female forms. Thus (58) was an estrogen agonist in
rat, (56) was shown to downregulate PR in human hepatoma cells transfected with ERa,
Female Sex Hormones, Contraceptives, and Fertility Drugs

trogen-responsive cells was completely pre-


vented by estrogen antagonists (138). In the
OVX rat at high doses, (59) had weak partial
uterotrophic effects, no observable effects on
tibial bone growth, and serum cholesterol was
reduced (137).
Reports of dietary phytoprogestins have
begun to appear. In particular, there is evi-
dence that extracts from selected herbs/spices
(e.g., oregano, thyme, turmeric) contain com-
ponents that interact with hPR. Furthermore,
those herb products suggested to contain com-
ponents with hPR affinity tended to antago-
nize the proliferation of hPR-containing neo-
plastic cells (140). The identity of these
phytoprogestins will undoubtably be reported
soon.
5.9 Tissue-Selective Estrogens and Progestins
In the presence of MCF-7 cells, and in the im-
mature rat uterus, tamoxifen (16a), ralox-
ifene (291, and other nonsteroidal ER ligands
exhibit dominant estrogen antagonist activity.
Contrasting results have been obtained re-
garding their effects on other tissues, in which
although in the same cell line transfected with these substances had full estrogen mimetic ef-
ERP, (58)was an antagonist of (1)(133). Fur- fects, and thus (16a) and (29) are the first
ther studies are clearly needed to clarify the examples of SERMs. In the OVX rat (Fig.
profile of estrogenic and antiestrogenic effects 13.81, currently known SERMs are approxi-
of (58). mately as efficacious (but not as potent) as
Human diets contain trace amounts of phy- ethynyl estradiol(9) on bone-loss suppression
toestrogens, various plant-derived compounds and serum cholesterol lowering. However, the
with weak estrogenic activity (134). Examples estrogenic efficacy in reproductive tissue of
are the isoflavonoids genistein (59) and daid- SERMs is much less than that of (9),although
zein (60). These substances and related ones (9) displays modest separation of skeletalilipid
have attracted attention because they might versus uterotrophic potencies. Furthermore,
be capable of preventing the development of (16a)and (29) antagonize the uterotrophic ef-
estrogen-related cancers, as well as blunting fect of (91, but not its skeletal and cardiovas-
the symptoms of menopause (135). Indeed, a cular effects.
significant reduction in severity and fre- These findings suggest an important phar-
quency of hot-flush episodes was experienced macodynamic difference expressed in SERMs
by menopausal patients whose diets were sup- relative to estrogen mimetics such as (9), or
plemented with soy extract or flour, both of for that matter with regard to respective ste-
which contain (59)and (60) (136). roidal and diarylethylene ER ligands (64) and
The hER RBA of (59) was 2% that of (I), (76) (below). Both of these "pure" estrogen
and in MCF-7 cells, (59) was a weak partial antagonists antagonized the bone-protective
agonist (67% efficacy) (137, 138). In human effect of (1) (142). Also, (64) partially pre-
hepatoma cells transfected with either ERa or vented the serum cholesterol-lowering effect
ERP, (59)and (60) were full agonists with (59) of (9) or (16a) (143).
being more potent; both isoflavonoids were Current SERMs have approximately the
more potent in the ERP-transfected cells same affinity for hERa and hERp. Also, no
(139). Growth stimulation by (59)in such es- noteworthy differences in levels of ER iso-
Physiology and Pharmacology

Ethynyl estradiol SERM

Figure 13.8. Depicted dose-


response relationships for
uterine weight retention,
bone density maintenance,
and serum cholesterol lower-
1 0.2 0.03 0.05 0.1 0.3
ing in the OVX rat for ethynyl
Oral dose, mglkglday
".
. . and a twical
estradiol (9)
SERM. This figure was com-
piled from selected data in
Refs. 113 and 141.

rms in bone-remodeling cells or hepatocytes, appear to be two distinct types of EREs. Stud-
lmpared to levels in uterine tissues, have ies in OVX-thyroidectomized (estrogen- and
!en reported. These findings indicate that thyroid hormone-deficient) rats suggested
3RM effects are not manifested by differen- that productive interaction (activation) of
d interaction or "activation" of one or the EREs with liganded ER in certain cells (hepa-
her of the ER isoforms. tocytes, osteoblasts) exerted a modulatory ef-
Rather, the molecular basis for tissue-se- fect on proximate thyroxine response ele-
letctive estrogenic effects expressed by (16a), ments (TREs) occupied by liganded thyroid
(29), and other SERMs arises from the dis- receptor. However, activation of EREs in uter-
ti1nct conformation they induce in ER. SERM- ine cells by interaction with liganded ER was
1ig~andedER results in a more "open" confor- independent of (occupied) TRE (143). With a
mation in the region of the LBD than when ER view toward the SERM profile of activity de-
is liganded with (1)(94, 97). Two mechanistic picted in Fig. 13.8, it can be argued that
PCssibilities have been proposed to account for SERM-ER complexes are more fully capable of
wlhat happens next. First, SERM-ER com- productive interaction with "TRE-coupled"
PI'exes could exhibit impaired ability to recruit ERE in osteoblasts and hepatocytes than with
co'regulator(s)in reproductive tissue (and neo- "TRE-independent" ERE in uterine cells.
pl;astic cells), as opposed to hepatocytes or os- Alternatively, SERMs might interact with
em
t oblasts. Assuming this to be correct, selec- EREs different from those with which steroi-
ti1Je ER modulation arises from differential dal estrogens might interact. Studies with
tirme-specific distribution of different coregu- yeast cells transfected with cDNA resulted in
la1tors. This notion, however, is not clearly identification of an ERE for (16a)-ER com-
SUpported at this time, although more studies plexes that was distinct from that which inter-
of ER coregulator distribution are needed (see, acted with (1)-ER (146). In addition, the
e4T., Ref. 144). Thus, expression levels of (29)-ER complex was found to interact in a
m.RNAs for each of six ER coregulators did not novel way with a transcription promoter
di:Ffer substantially across seven lines of ER- (147).
Palsitive neoplastic cells (145). A tissue-selective nonsteroidal progestin
The second possibility involves cell-specific has been characterized (124). Chromeno-
di.vergence in functions of ERE after interac- quinoline (61) had an hPR affinity equivalent
tic)n with liganded ER. In this regard, there to that of (30), but low affinity for other ste-
Female Sex Hormones, Contraceptives, and Fertility Drugs

levels subsequent to ovulation and during


pregnancy. These interact with respective ER
and PR in the hypothalamus to downregulate
GnRH production, ultimately reducing FSH
and LH levels during pregnancy (and during
the time when an egg is available for fertiliza-
tion), preventing further ovulation. This pro-
cess of feedback inhibition is mimicked by
orally administered estrogen-progestin combi-
nations.
Thus, in randomized, controlled clinical
trials, serum levels of FSH and LH were re-
duced in women administered oral contracep-
tives containing 0.025-0.05 mg of either
transdermal (1)or ethynyl estradiol (91, plus
relatively high doses (1-10 mg) of norethin-
roid receptors. In hPR-transfected cells, (61)
drone (35) or nomegestrol acetate (62), over
was a full agonist with potency approaching
that of (30). At high doses, (61) diminished by
30% the uterotrophic effect of (2) in the OVX
rat, as did medroxyprogesterone acetate (34).
However, in these same estrogen-"primed"
animals, (61)had a reduced stimulatory effect
on mammary gland differentiation (lobular al-
veolar bud proliferation), compared to that of
(34). These findings have interesting thera-
peutic implications. Estrogen-replacement
therapy with estrogentprogestin combinations
results in a decreased risk of endometrial can-
cer but an increased risk of breast cancer,
compared to use of estrogen alone (see Section
2.2). Combinations of estrogen and selective
women taking placebos (148). However, other
progestins with reduced mammary gland
studies using similar doses of (9) plus lower
stimulatory effects might result in reduction
progestin doses r0.15-0.5 mg of (35)or deso-
of both endometrial and breast cancer risk.
gestrel(38)l revealed serum level reduction of
FSH and LH in most, but not all, of the treat-
5.1 0 Modulation of Ovulation
ment cycles, despite no documented incidence
Orally effective suppression of ovulation is ac- of ovulation in treatment groups (149). These
complished by administration of progestintes- results suggested anticonceptive effects were
trogen combinations. This is the major appli- attributed not just to suppression of serum go-
cation of the progestin-estrogen mixtures nadotropin levels, but also to direct effects on
listed under progestins in Table 13.1. These uterine function (149), such as reduction in
combinations work primarily, but not exclu- cervical permeability and endometrial recep-
sively, at the level of the hypothalamus. tivity.
Coordination of synthesis/secretion of folli- Besides acting on the hypothalamus and
cle-stimulating hormone (FSH) and luteiniz- uterus, progestins, when administered with
ing hormone (LH) from the anterior pituitary estrogen, interfere with pituitary response to
originates in the hypothalamus. Releasing fac- GnRH (150, 151).
tors, such as gonadotrophin releasing hor- On the other hand, the ovulation-inducing
mone (GnRH),stimulate FSH and LH produc- drug clomiphene (23) elevates serum FSH and
tion, which facilitates ovulation. Endogenous LH levels. This drug interacts with ER in the
(1)and (30) are present in the blood at high hypothalamus to antagonize the (1)-mediated
6 History

feedback inhibition of GnRH secretion. In the rats treated with (16a) (159,160). Thus, (16a)
anterior pituitary, its constituent isomers appeared to express tissue-specific estrogenic-
might also have direct additive effects on ity. The molecular basis for SERM activity is
GnRH-stimulated FSH/LH production. In rat presented in Section 5.9.
pituitary cells, (23a) and/or (23b) enhanced These novel findings suggested a new di-
the potency of GnRH-mediated LH production mension in estrogen-replacement therapy: se-
(152). In ovine pituitary cells, only (23b) aug- lective estrogens useful in patients at risk for
mented the LH production stimulated by a breast cancer or other estrogen-dependent
GnRH agonist, and each isomer reversed the cancers, and in patients intolerant to side ef-
suppressive effect of (1) on FSH secretion fects associated with reproductive tract estro-
(153). Although direct pituitary effects of (23) genicity of nonselective estrogens.
in humans might vary from those in these an- Accordingly, in the early 1990s there
imal models, these in uitro studies imply that emerged a renewed interest in development of
both isomers of (23) are capable of interacting nonsteroidal antiestrogens as therapeutic
with ER in pituitary as well as the hypothala- agents. One such class of ER ligands had been
mus, ultimately leading to elevation of avail- discovered in the late 1970s-early 1980s by C.
able levels of FSH and LH (154). Thus, admin- David Jones and coworkers. The prototype of
istration of (23) at the beginning of the these, trioxifene (631, was noteworthy in that,
menstrual cycle maximizes the probability
that ovulation will occur in patients in whom
this process is impeded by inadequate levels of
these peptide hormones.

6 HISTORY

6.1 Discovery of SERMS, Including


Tamoxifen (16a)
In 1962 Michael Harper and Arthur Walpole
prepared the isomers of triarylethylene (16),
and found that the trans isomer, now known
as tamoxifen (16a),had antiestrogenic prop- in the rat, it exhibited greater antiuterotro-
erties (155). In the early 1970s,clinical studies phic efficacy and reduced uterotrophic efficacy
with (16a)showed that it could antagonize the compared to that of (16a) (161), as did ralox-
growth of ER-positive breast cancer (156). ifene (29), which was considerably more po-
Estrogen promotes maintenance of bone tentleffective (161b, 162, 163).
density and normal serum cholesterol in post- In the OVX rat, (29) was nearly as effective
menopausal women. Because (16a) was an es- as (1)in maintaining bone density and pre-
trogen antagonist in experimental breast can- venting excessive bone turnover (160, 164). It
cer models, and in reproductive tissue, it was was more effective than (1)in lowering serum
thought that expression of this antiestrogenic- cholesterol (159) and in patients, (29) ap-
ity in skeletal and hepatic tissues might result peared to have a reduced incidence of repro-
in bone density loss and serum lipid eleva- ductive tract side effects, including endometri-
tions. However, monitoring these parameters osis, compared to that of (16a). It was
in patients on extended therapy with (16a)for subsequently introduced for use in estrogen-
breast cancer treatmentlprevention revealed replacement therapy in 1998. Additionally,
no adverse effects (157). In fact, (16a) ap- the study of tamoxifen and raloxifene (STAR)
peared to prevent bone loss and promote cho- Phase I11 clinical trial, begun in 1999, is ongo-
lesterol lowering, while suppressing cancer ing to determine how (29) compares with
growth (157, 158). Bone loss suppression and (16a)as a breast cancer preventative in post-
cholesterol lowering were also seen in OVX menopausal women, with secondary focus on
Female Sex Hormones, Contraceptives, and Fertility Drugs

goH
(156). These physical and pharmacodynamic
properties have simplified interpretation of its
selective estrogenic effects, and, because (29)
formed a high affinity complex with ERa, crys-
tallographic studies of such complexes (see
Section 5.1) have provided a starting point for
HO
%
-' R establishing the molecular basis for its tissue-
dependent estrogenicity (see Section 5.9).
R
6.2 Other Discoveries
CH3
I Isolation and identification of endogenous ste-
(64) HZC"',' N - n , , - C H 3 roidal estrogens and progesterone, discovery
of nonsteroidal estrogens such as diethylstil-
0 bestrol(24), and development of progestides-
0 trogen combinations as birth control agents
II
(65) -s CF2CF3 have been covered in previous editions of this
H2C textbook (see also Ref. 155).

comparison of maintenance of skeletallcardio-


vascular systems and frequency
- and severity
, STRUCTURE-ACTIVITY

of side effects (165). 7.1 Steroidal Analogs of (1)


Furthermore., (29).
. ., unlike 4-hvdroxvtri-
" "

arylethylenes, cannot undergo geometric As suggested from the data in Table 13.3, the
isomerization, and it does not interact with a ER will accommodate sizable structural mod-
multitude of other receptors, enzymes, and ification of (1)at specific points in its B- or
regulatory proteins besides ER as (16a)does C-rings. Thus, addition of 7a- or llp-substitu-
7 Structure-Activity Relationships

ents, embodied in turn in (64), (65), and (66), widespread application in breast cancer ther-
resulted in analogs with high ER affinity. On apy, particularly if limited oral effectiveness
the other hand, removal, derivatization, or re- and side effects associated with estrogen an-
placement of either of (1)'s hydroxyl groups tagonism in other tissues (142a) do not
results in a considerable loss of affinity, with present serious drawbacks.
some exceptions (entries 5 and 9). Further- The molecular basis for the efficacious es-
more, inversion of hydroxyl group configura- trogen antagonist effects of (64) and (65) cen-
tion at C-17 of (1)or (9) resulted in large de- ters on the ways these compounds interact
creases in affinity (cf. 1 with 17-epi-1,9 with with ER. Specific electrostatic functional
17-epi-9) (166-169). ER affinities of more group and hydrophobic/dispersional bonding
comprehensive collections of steroidal ER li- interactions of (65) with the hERa LBD (Sec-
gands have been reported (93,170). tion 5.1) are similar to those seen with (1) as
ERa seems to predominate over ERP in rat ligand (Fig. 13.6) (crystal structure of the 65-
uterine tissues (101).Thus, RBAs obtained from hERa LBD to be reported). However, (65)'s
rat uterine tissue cytosols are a reasonable ap- lengthy 15-membered aliphatic chain overlaps
proximation of relative affinity for ERa. RBAs the region of the LBD in which TAF-2 is em-
obtained for a few steroids using hERa are in- bedded (Fig. 13.5), thereby interfering with
cluded for comparison in Table 13.3. dimerization and interaction of (65)-liganded
ER with coregulators (181). Although such
7.1.1 7a-Substitution. Addition of linear complexes are transcriptionally ineffective at
alkyl groups capped with polar end groups at TRE-independent EREs in uterine and breast
C-7 of (1)has resulted in analogs with no ob- cancer cells (see section 5.9), they appear to be
servable intrinsic estrogenicity in certain tis- partially effective in activating EREs in hepa-
sues and cells. Thus (64) (ICI 164,384) and tocytes and other cell types endowed with
(65) [ici (ZN) 182,7801, although clearly capa- TRE-dependent EREs (143).
ble of interaction with ER (Table 13.31, were A parallel solid-phase synthetic approach
not uterotrophic in the immature rat but an- has been used to make a library of reverse
tagonized completely the uterotrophic effect amide analogs of (64) with two levels of diver-
of (1) &benzoate ester (173). These com- sity (182). Thus (1)-17-tetrahydropyranyl
pounds were much more effective parenterally ether, bound to a polymeric resin at its 3-hy-
than orally, suggesting efficient presystemic droxyl group and bearing an o-arninoundecyl
metabolic inactivation. Regarding (641, minor group at its 7a position, was condensed with
variance in the length of the linear alkyl four activated N-protected amino acids, fol-
spacer or in the amide N-alkyl substituents lowed by deprotection and condensation of
often resulted in restoration of partial estro- each of these intermediates with five activated
genic activity (178). This suggested consider- carboxylic acids. Photolytic cleavage yielded
able overlap in structural requirements for ag- 20 compounds with varying degrees of ER af-
onistlantagonist effects in the alkylamide finity and estrogen antagonist activity.
region of these analogs. In MCF-7 cells, (65)
was more potent and effective than (16a) (be- 7.1.2 11 P-Substitution. Addition of 1lp
low) in suppressing growth. Furthermore, substituents to (1) or ethynyl estradiol(9) has
(65) suppressed growth of MCF-7 cells that given rise to analogs with noteworthy changes
had grown resistant to antiestrogens (1791, in ER affinity and intrinsic activity. Thus,
suggesting the possible clinical application of chloromethyl analog (66) had ER affinity al-
(65) for control of breast cancer that had ac- most 10 times greater than that of (1)(Table.
quired resistance to conventional antiestro- 13.3) (174). Interaction of (66) with ER was
gens. Indeed, a preliminary study showed that shown to be noncovalent, despite the potential
monthly intramuscular administration of (65) reactivity of its chloromethyl group. This was
resulted in a 69% response in patients with attributed either to steric encumbrance of the
breast tumors that had become refractory to chloromethyl group, or possibly to its lack of
other antiestrogen therapy (180). Thus, ste- proximity to nucleophilic substituents in the
roidal antagonists such as (65) might find ligand-binding site. The llp-methoxy analog
658 Female Sex Hormones, Contraceptives, and Fertility Drugs

(671, which also has a l7a-ethynyl group, in-


teracted strongly with ER as well. Both (66)
and (67) are potent estrogen agonists (172,
174). Addition of larger 11P-substituents,
some of which were similar to those in (64),
likewise has yielded antagonists with negligi-
ble intrinsic estrogenicity. Thus, N,N-dial-
kylundecylamides (68) and (69) exhibited
high ER affinity (Table 13.3), but had no ob-
servable uterotrophic effects. These analogs
had potent growth-suppressive effects on
MCF-7 cells and on a tamoxifen-resistant cell
line (175). The ethynyl analog (69) was signif- ER affinity of (70) is comparable to that of
icantly more potent than (68) when adminis- other analogs of (1)(Table 13.3). Androgenic
tered orally. receptor affinity, characteristic of these ana-
logs, is significant (176). Reversal of configu-
7.1.3 Comparison of Estrone (2) with Equi-
ration at C-2, or substitution of H for ethynyl
lin (52c). In uiuo reversible sulfate esters of in the D-ring, in turn resulted in loss of ER
(52a-c) together are the second most abun- affinity or greatly increased androgen recep-
dant components in conjugated estrogens. tor affinity. In the immature rat, (70) exhib-
Thus, studies have focused on their possible ited estrogenic properties divergent from
role in estrogen-replacement therapy (83). Af- those of nonsteroidal antiestrogens (177).
finity of (52c) for ER was similar to that of (2), A metabolite of gestodene (39), specifically
and (52b) was suggested to have greater ER (40b), whose structure is similar to that of
affinity than that of (1)(Tables 13.3 and 13.5) (701, also exhibited ER affinity and estrogenic-
(81).Also, (52b) and (52c) were comparable to ity in uitro and in uiuo (see Section 3.9).
(1)or (2) in terms of uterotrophic activity in
7.2 Two Caveats Concerning the Meaning
the rat.
of ER Affinity
However, no studies have yet appeared that
reveal a clear basis for pharmacodynamic dif- As is clear from the RBAs of steroidal ER li-
ferences between these B-ring dehydroge- gands in Table 13.3 in relation to their estro-
nated ER ligands, or the other ones in Table genic and antiestrogenic effects, and as will be
13.5, over (1)or (2). Thus the therapeutic ad- shown for nonsteroidal ER ligands below, the
vantage of conjugated equine estrogens over magnitude of ER affinity does not predeter-
(2) sulfate ester alone, or for that matter, over mine estrogenic or antiestrogenic efficacy.
some of the more potent semisynthetic estro- Other factors, such as the size and physico-
gens such as ethynyl estradiol(9) in estrogen- chemical character of the organic moiety
replacement therapy is not completely clear. It added to (I), determine whether the resulting
could be attributed in part to reduced repro- ER ligand is able to alter the conformation of
ductive tract estrogenicity, although this re- ER in ways favorable, or detrimental, to inter-
mains to be established experimentally. Per- action of the resulting complex with EREs.
haps novel modulating effects of the B-ring Furthermore, ER affinity does not always
dehydrogenated estrogen conjugates, particu- correlate well with potency. Substances with
larly 17-epi-(l),(52a), and/or (53a)(183), will relatively low RBAs can still exhibit agonist or
emerge in future endocrinologic studies. antagonist potencies equivalent to those of
their much higher affinity counterparts. For
7.1.4 Nonbenzenoid Steroid (-Like) ER Li- example, in Table 13.3, 17-deoxy (1)had an
gands. Anordiol (70) illustrates the impor- RBA of 79% that of (11, but its MCF-7 cell
tance of hydroxyl group spacing in steroidal mitogenic potency was only 1.2% of that of (1)
ER ligands, and provides evidence that ER will (170). Thus, particular structural characteris-
accommodate properly substituted steroid- tics, in addition to those responsible for ER
type ligands in which ring A is aliphatic. The affinity, can influence potency.
7 Structure-Activity Relationships 659

Table 13.6 ER Affinities of Selected Diarylethylenesa


Compound RBAb Source Reference
(24) (diethylstilbestrol) rat [hERal
(26) (meso-hexestrol) calf (rat) [hERal
(71) (dienestrol) rat [hERal
(72) calf (rat)
(73) (ZK 119,010) calf
(74) (ZK 169,978) calf
(76) (ZM189,154) rat
(29) (raloxifene) rat (human)
(77) (LY117018) lamb (rat)
(78) (arzoxifene) human
"Values were obtained using uterine cytosolic ER from the indicated sources, or using recombinant human ERa.
bRelativeto (I),RBA = 100.
'The dl-isomer of (26) had an RBA of 1.1 (188~).

Nevertheless, from comparison of ER li-


gand structures with respective numeric ER
RBAs there has emerged a recognizable struc-
tural pattern concerning substances whose bi-
ologic (pharmacologic) activities are mediated
by interaction with ER.

7.3 Nonsteroidal ER Ligands


It has long been known that the ER accommo-
dates a diverse array of natural and synthetic relative in vivo estrogenicities of (24) and its
aromatic structures that do not contain the geometric isomer have been difficult to assess
steroid nucleus. The ER affinities and estro- because of this isomerization.
genic effects of some of these (24), (261, (691, Estrogenicity of structural analogs incapa-
(60) have been summarized above (Sections ble, or less capable, of geometric isomerization
3.6 and 5.8). In addition, medicinal chemists have suggested that estrogenicity of (24) is far
have characterized a sizable and structurally greater than that of its cis isomer. Indeed, es-
diverse collection of therapeutic and experi- trogenic potency in the immature mouse of
mental nonsteroidal estrogen agonists and an- (24) dipropionate ester was 600 times that of
tagonists. These substances have generally its cis counterpart (185). (Both of these esters
been shown to interact at the same point in presumably undergo facile hydrolysis in vivo.)
the ER LBD as does (I), although the affinity, Also, meso-hexestrol (26), which assumes an
specific functional group interactions, and extended conformation resembling (241, was
consequences of such interactions can differ. 50 times more potent than dl-hexestrol(187),
which adopts conformations in which the
7.3.1 Vicinal Diarylethylenes. From exten- rings are in closer proximity to one another.
sive synthetic and endocrinologic studies fo- The ER affinities (Table 13.6) and/or estroge-
cused on substituted stilbene derivatives nicities of (24)and (26) have been suggested to
(184), trans-diethylstilbestrol (24) and (Z,Z)- arise from their stereochemical similarity to
dienestrol(71) emerged as potent, orally effec- (I),with particular reference to the spacing of
tive estrogens. Because of facile geometric hydroxyl groups (184b).
isomerization (185),(24)is accompanied in so- Both phenolic hydroxyls of vicinal diaryl-
lution by significant amounts of its cis isomer ethylenes are important for high potency. The
(186). Cytosolic ER interacts exclusively with monomethyl ether of (24) retains only 4% of
(24) in such mixtures (186b). However, the the estrogenic potency of (24) (184b). Analo-
Female Sex Hormones, Contraceptives, and Fertility Drugs

gously, relative ER affinities of various substi- found to have significant ER affinity (Table
tuted monoalkyl ethers of (26) were less than 13.6)' and was a partial estrogen agonist in
3% that of (26) (171b). These findings are con- MCF-7 cells (196) and in the immature female
sistent with the structure of the (24)-hERa mouse as its diacetate ester (189). Replace-
LBD complex, which features electrostatic in- ment of the N-ethyl substituent with linear
teractions of (24)'s hydroxyls with Glu353/ alkyl chains capped by polar amino or arnido
Arg394 and His524 of the LBD, respectively; groups afforded analogs with low residual es-
identical interactions were observed in the trogenicity. Thus, w-(N-pyrrolidiny1)-n-hexyl
complex involving (1)(see Fig. 13.6a, above). analog (73) had ER affinity 21% that of (I),
Another feature of (24) important to its ER and antagonized the uterotrophic effect of (2)
affinity and estrogenic potency is the confor- in the immature mouse and rat with greater
mation of its stilbene moiety. Crystallographic efficacy than did (72) (190). In addition, the
studies indicated that its rings are parallel but corresponding undecylamide analog (74) had
not coplanar, each ring being twisted 60" out ER affinity 7% that of (1); exhibited no utero-
of the plane of the ethylenic moiety (194). "Re- trophic effect in the mouse; and suppressed,
placement" of the ethyl groups of (24) with with modest potency compared to that of (64)
hydrogens, relieving the steric congestion and (65),the growth of MCF-7 cells (92a).
around >C==C< thus facilitating a more Introduction of a p-[2-(N-perhydroazepi-
nearly planar orientation of the stilbene moi- ny1)ethoxylbenzyl moiety as the indole ring N-
ety, had previously been found to result in a substituent gave (75) (TSE-424). This had ER
>99% loss of estrogenic potency (187). affinity comparable to that of (73) and (74). In
the OVX rat, (75) exhibited very low uterotro-
7.3.2 Diarylethylene Estrogen Antago- phic activity, and had serum cholesterol low-
nists. Structural alteration of the diarylethyl- ering and bone loss-suppressive effects at 0.1
ene (stilbene) pharmacophore to give sub- mg/kg doses (197). Note that, although (75)
stances retaining high ER affinity but lacking contains three phenyl rings, it retains the
in intrinsic activity has been the focus of many trans-p,pl-dihydroxystilbenebackbone that
investigations (195). Systematic structure-ac- originated in (24). Thus, it is likely that, like
tivity studies have been conducted on deriva- (24), (75) interacts with the ER LBD by spe-
tives of 2-phenylindole. Initially, (72) was cific hydrogen bonding of the indole and phe-
7 Structure-Activity Relationships

nolic hydroxyls in turn with Glu353lArg394 192), and (29) was shown to interact with the
and His524 (Fig. 13.61, and furthermore, that hERa LBD by hydrogen-bonding interactions
the (protonated) perhydroazepine ring nitro- of each of its phenolic hydroxyls, as well as
gen interacts with ionized Asp351. ionic interaction of its protonated ring nitro-
Another example of a purer antiestrogen gen (94) (Section 5.1). Members of this group
derived from the 1,2-diarylethylene pharma- were powerful suppressants of estrogen-stim-
cophore is (76). This ER ligand, with RBA of ulated uterine growth in rodents (162), and of
66 (Table 13.6), incorporates structural fea- MCF-7 cell proliferation (198). Molecular
tures previously found to give high ER affin- modeling studies on (77) suggested it to inter-
ity, and low residual estrogenicity [cf. with act with ER in a way that oriented thep-(sub-
hexestrol(26) and (65)l. In the immature rat, stituted)benzoyl group in a 7a-like position
(76) completely blocked the uterotrophic ef- relative to the interaction of (1)with ER (193).
fect of either (1)or tamoxifen (16a), and dis- Description of (29)'s experimental and clinical
played no intrinsic agonist activity of its own tissue-selective estrogenic effects can be found
(142b). In the OVX rat it had estrogen antag- above (see sections 2,5.9, and 6). However, the
onist effects on bone maintenance as well, un- oral potency of (29) appears to be limited by its
like (16a). susceptibility to glucuronide conjugation at
A therapeutically important class of het- the hydroxyl group on its pendant phenyl ring
erocyclic ER ligands in which the trans-p,pl- (Section 3.8). To circumvent this limitation,
dihydroxystilbene system is also embedded is (78),in which the structure of (29) was altered
the benzothiophenes. This group includes by (1) isosteric replacement of the carbonyl
raloxifene (29), LY117018 (771, and arzox- group with an ether oxygen and (2) O-methyl-
ifene (78). These were found to have ER afin- ation of the 4'-hydroxyl group, was character-
ities approaching that of (1)(Table 13.6) (191, ized experimentally (192b). Although (78)'s
differential estrogen efficacy was similar to
that of (29) in the OVX rat, it was 30-100
times more potent than (29) when given orally
(62). This potency increase could be attributed
to the inability of (78) to undergo metabolic
4'-0-glucuronidation, to which (29) is suscep-
tible. The 0-methyl group of (78) is resistant
to oxidative biotransformation, unlike those
of (101, (12) (Sections 3.1 and 3.51, and nafoxi-
dine (88,below), and its 4'-oxygen presumably
can still function as an H-bond acceptor, facil-
itating interaction of (78) with ER.

7.3.3 Triarylethylenes. This group includes


chlorotrianisene (121, tamoxifen (16a), clo-
662 Female Sex Hormones, Contraceptives, and Fertility Drugs

Table 13.7 Influence of p-Hydroxylor Side-Chain Substitution in Triarylethylenes on ER


Amity, and Estrogenic and Antiestrogenic Potency and Efficacy in MCF-7 Cellsa
Estrogenic Antiestrogenic
Compound RBAb ECsO (nM) Efficacy IC50 (nM) Efficacy
(79) CO.1 28 68" - Id
(80) 38.5 0.19 94 - 13
(81) 55 0.06 62 3.5 31
(1) 100 0.02 100 - 0
(16a) 1' 0.11 21 219 90
(18) 123 0.003 10 2.5 100
(16b) 0.1' 11 100 - 0
(82)f 1.5 5.3 79 5W 22
(83)f 2.5 - - 20 82
aData are from Refs. 112b and 199a except as indicated.
bDetermined using rat uterine cytosol.
"Percentage of the maximal proliferative effect of (1)in MCF-7 cells.
dPercentage by which the MCF-7 cell proliferative effect of 0.1 n M (1)was maximally antagonized.
"Data from Ref. 199b. Calf uterine wtosol ER RBAs of (Ma) and (16b) were in turn 13 and 0.17 (112b).
fData for this compound are from ~ e f200.
.
gMicromolar concentration.

miphene (23), and its positional isomer


toremifene (22). It also includes fused-ring an-
alogs centchroman (861, nafoxidine (881, and
lasofoxifene (89). Because of its structural
similarity to (86) and (89), (87) is also in-
cluded here. However, (87a) contains thep,pl-
dihydroxy-trans-stilbene moiety, and thus
structurally and pharmacologically is classi-
fied as a vicinal diarylethylene. The first four
of these have been described in terms of ther-
apeutic applications (Section 2) and biotrans-
formation (Section 31, with a view toward
structure-activity relationships. Further clar-
ification of the SAR of triarylethylenes is facil-
itated by consideration of the ER affinities and
in vitro agonistlantagonist properties of tri-
phenylacrylonitriles (79-81) compared with
those of tamoxifen (16a) and (1)(112b, 199). indicate a significant role for both of the p-
As shown in Table 13.7, introduction of a sin- oxygen substituents on the geminal phenyl
gle p-hydroxyl group to the weak ER ligand rings in binding to ER and modulating the
(79), afforded (80). In (80), there is a trans conformation of ER, resulting in antagonist
relationship between phenolic and phenyl activity in MCF-7 cells.
rings, and the phenolic ring is geminal to the "Etherification" of (81) with an N,N-di-
other unsubstituted phenyl ring. Its ER affm- methylaminoethyl group gives Chydroxyta-
ity and agonist potency are greatly increased moxifen (18).In analogy with the respective
over that of (79). Geminal bisphenol (81)ex- precursor phenol, the second oxygen substitu-
hibited a further 50% increase in affinity, and ent (hydroxyl) increased estrogen antagonist
had significant estrogen antagonist activity in potency and efficacy, although addition of the
the nanomolar concentration range. Thus, the basic ether side-chain also contributed to
secondp-hydroxyl conferred a shift toward an- (18)'s dominant estrogen antagonism (112b,
tiestrogenic activity. Together, these results 199). Removal of the second oxygen substitu-
i 7 Structure-Activity Relationships

ent, giving tamoxifen (16a), resulted in main- MCF-7 cells (200) (Table 13.7). In (84)
tenance of antagonist efficacy, but a reduction (GW5638) and (85) (GW7604) the role of ap-
in potency resulting from decreased ER aflin- (ether oxygen) in furnishing electron density
ity; its isomer (16b) had no antagonist efficacy on one of the geminal rings is provided instead
at all. Thus, both the second oxygen substitu- by the acrylate - . ER RBAs of (84) and
ent and an appropriately positioned basic side (85) were, in turn, 13% and 41% that of (1)
chain conferred maximal estrogen antago- (203).Mechanistic studies in estrogen-respon-
nism in MCF-7 cells. sive cells and with hERa suggested that (85)
Parallel effects with the isomers of (16) had less residual partial agonist efficacy than
were seen in the immature rat (110b). Thus, that of (18)(203),and that it alters the confor-
(16a)had a modest uterotrophic effect and an- mation of the receptor differently than do es-
tagonized that of (I),whereas its cis isomer trogen antagonists (16a), (29), or (65) (204).
(16b) had uterotrophic efficacy nearly equal to Acrylic acid derivative (84) was a potent an-
that of (I), although it was much less potent. tagonist of estrogen-supported proliferation of
Similar studies with the isomers of clomi- Ishikawa cells (92b). In the rat, both (83)and
phene (23) likewise indicated the trans isomer (84) exhibited endocrine profiles suggestive of
(23a)to have greater antiuterotrophic efficacy SERMs. Each was a weak partial agonist of
than that of its cis counterpart (23b). How- uterine weight maintenance or antagonized
ever, recent experimental findings extend an 17p-estradiol stimulated uterine weight gain.
earlier contention (201) (see also Section 5.10) Moreover, in OVX rats, each compound was as
that both isomers participate in the observed effective as (1)in suppressing bone loss and
endocrine effects of (23) (202). serum cholesterol (92b, 203, 205).
Replacement of the basic side chain of In female rodents, (16a) and many of its an-
(16a)/(l8)with acidic side chains has resulted alogs are effective postcoital contracep-
in ER ligands that have provided further def- tives (206). One such analog, centchroman
inition of the structure-activity relationship (levormeloxifene, 86), is in clinical use for this
in triarylethylene estrogens/antiestrogens.
Thus, oxyalkanoic acids (82) and (83) exhib-

application (206b). In the rat, (86) had an ER


RBA 16% that of (I),three times greater than
that of the mixture of corresponding d- and 1-
ited modest ER affinity, and estrogen mimick- isomers (207). It also exhibited potent antiestro-
ing or antagonist efficacy that pivoted on side- genic and weak estrogenic effects. Whether the
chain length: oxyacetic acid (82) exhibited antifertility effect of (86) and related com-
dominant agonist efficacy and oxybutyric acid pounds is attributed to residual estrogenic or es-
(83) had dominant antagonist efficacy in trogen antagonizing properties is not clear.
664 Female Sex Hormones, Contraceptives, and Fertility Drugs

Alteration of chroman ring substituents in maintenance seen with either (87a) or (29) (1
analogs of (86) has resulted in ER ligands mg/kg dose levels) was 63% that seen in intact
with novel antiestrogenic properties. Thus, controls (212). Moreover, (87a) (0.25 mg/kg)
chromene (87a, EM-652, SCH 57068) had an maximally lowered (50%) serum cholesterol
vs. OVX controls. It is not clear from these
studies what advantage (8%)might have over
its unesterified counterpart (87a): these two
substances appear to be equally potent and
effective in estrogen-responsive cells and in
laboratory animals.
The above chromene ring containing estro-
gen antagonist is an oxygen isostere of dihy-
dronaphthalene-based ER ligands, exempli-
fied by nafoxidine (88), which was originally

RBA for hER of 291. Its bis-(pivalate) ester


(8%,EM-800, SCH 57050) was an antagonist
with 100% efficacy (0%agonist effect) in T47D
cells, with an IC,, value of 0.14 nM.Similar
results were obtained with (87a). In the OVX
mouse, (8%)antagonized the uterine weight-
retaining effect of (2) with 86% efficacy, and
had an agonist efficacy 11% of (2), whereas
(16a), which is generally considered to be an
estrogen in mouse uterus, had respective an-
tagonist and agonist efficacies of 16 and 76%.
The (S)-(+)-isomers of (87) (shown) were
much more potent than the corresponding prepared and evaluated for its antifertility ef-
(R)-(-)-isomers in the above bioassays (208). fects in the rat (213). In the immature rat, (88)
Furthermore, (87a) was able to antagonize had uterine estrogenic and antiestrogenic ef-
the partial estrogen agonist effect of (18) or fects similar to those of (16a) (214). Recent
(29) in Ishikawa cells, and had no observable refinement of this structural type, by catalytic
agonist efficacy of its own (209). These prop- hydrogenation of (88), followed by O-demeth-
erties were attributed to the ability of (87a) to ylation and resolution of enantiomers, gave la-
interfere not just with TAF-2 of ERa and ERP sofoxifene (89)(215). The ER RBA of (89) was
(Fig. 13.5) as do (16a),(29), and other triaryl- 3.1%, 25 times greater than that of its enan-
ethylene ER ligands, but also with the func- tiomer, and one-half that of (88),in rat uterine
tioning of TAF-1 in each of the ER isoforms cytosol(215) [the hERa RBA of (89)was 320%
(210). However, in an ER-negative breast can- (141c)l.
cer cell line transfected with point-mutated Evidently because of its decreased molecu-
ERa (Asp351 + Tyr351), (87) exhibited an lar surface/volume ratio, (89)was less suscep-
estrogen-like effect on up-regulation of a spe- tible to metabolic glucuronidation than other,
cific mRNA, an effect not seen with steroidal more planar phenolic ER ligands such as its
estrogen antagonist (65) (211). This finding trans-diastereomer or raloxifene (29). Addi-
underscores a mechanistic difference in the tional support for this idea comes from bio-
estrogen antagonist properties of these struc- transformation studies with (86), in which the
turally divergent ER ligands. Finally, in OVX vicinal aryl rings are trans to each other, al-
rats, the maximal observed degree of bone lowing each to assume an equatorial orienta-
mineral density and trabecular bone volume tion in the chromane ring, and thus endowing
7 Structure-Activity Relationships

enes competitively inhibited interaction of ER


with (1) (220). Aziridine (go), which exhibited
ER affinity comparable to that of (16a), inter-

(86) with a high degree of planarity. In the rat,


(86) was subject to facile biliary elimination
solely as its 0-desmethyl-0-glucuronideand
as its N-glucuronide (216).
At any rate, due ostensibly to (89)'s de- acted covalently with Cys530 of ER (221). In
creased capability for metabolic conjugation, the LBD, this residue is in close proximity to
it exhibited sixfold higher bioavailability than Asp351, the residue with which the (proton-
that of its trans-diastereomer or (29). This ated) aziridine ring of (90) presumably makes
was implied to be the basis for its potent oral initial contact (see Fig. 13.6b) before nucleo-
activity in the OVX rat, in which ED,, values philic attack by the Cys530 sulkydryl. The
for bone loss suppression and serum choles- resulting covalent complex expressed estrogen
terol reduction were, in turn, <1and 10 kg antagonist effects similar to those of the non-
kg-' day-' (215). covalent one of ER and (16a) (222). An analog
7.3.3.1 Conformational Analysis, Like the of (26) bearing an aziridine ring linked to one
aryl rings of the stilbene moiety in (24), each end of its hex-3-ene backbone, and expressing
aryl ring of (l6a) is twisted 60" out of the plane full estrogen agonist efficacy, also interacted
of the ethylenic bond, based on X-ray crystal- covalently with cysteine 530 (221). These find-
lographic data and conformational energy cal- ings provided compelling evidence, before
culations (217). Similar results were obtained availability of crystal structures (Section 5.1),
with (23a) (218). Results of proton nuclear that considerable similarity exists in the re-
magnetic resonance studies demonstrated a gion of ER with which agonists and antago-
consequence of these conformational prefer- nists interact.
ences in solution. In (16a),the p-substituted
ring is in the shielding cone path of the vicind 7.3.4 Triarylheterocycles. 1,3,5-Triarylpyr-
unsubstituted ring, an interaction not occur- azoles such as (911, and 2,3,5triarylfurans
ring in (16b). Thus, aliphatic and aromatic
protons of thep-substituted ring in (16a) are
0.1-0.2 ppm upfield in its spectrum from cor-
responding ones in the spectrum of (16b)
(206a).Similar results were seen in triarylbu-
tane analogs with respective conformations
approximating the configurations of (16a) and
(16b) (219).
7.3.3.2 Affinity Labels for ER. Early studies
with ER had shown that the region in its LBD
with which (16a) and (23a) interact approxi-
mated that of (1)because these triarylethyl-
Female Sex Hormones, Contraceptives, and Fertility Drugs

(92) with substituted aryl rings [e.g., R, and


R, = OH and R, = p-(2-N-piperidinylethoxy)
group (R, = ethyl)] have been in some cases
found to have high isoform-selective ER affin-
ity (223). Diversity of aryl substituents in (92)
can be maximized using a combinatorial ap-
proach enabled by the facile condensation re-
actions used to assemble the furan ring sys-
tem.

7.4 Progesterone and Steroidal PR Ligands


Chemically, steroidal progestins in experi-
mental and clinical use are categorized as
pregnanes, 19-norpregnanes, and estranes. 17a-ethynyl group in these progestins con-
Steroid receptor affinities of progesterone (30) ferred potent oral activity. Other examples in
and other PR ligands are shown in Table 13.2. this category are norethynodrel(96), ethyno-
The pregnane derivative megesterol acetate diol diacetate (97), and 1-norgestrel (36). As
(93) has aMinity for PR about equal to that of indicated in Table 13.2, progestins are recog-

(301, but the 19-norpregnanes promegestone


(R-5020, 94) and ORG-2058 (95) had much
higher PR affinity. Regarding estrane-derived
progestins, the earliest member of this cate-
gory is norethindrone (norethisterone, 35).As
with steroidal estrogens, introduction of the
i
!
t
7 Structure-Activity Relationships
!

i
t
nized by other steroid receptors besides PR,
1 but generally not by ER.
Unlike the other steroidal progestins, (36)
i

was prepared synthetically from nonsteroidal


precursors (228).This enabled introduction of
a novel ethyl group at the C/D ring juncture. A
racemic mixture of (36) and its d-enantiomer
has been widely used clinically (Table 13.1).
However, the 1-enantiomer (36), now estab-
lished as the active component of this mixture,
is also in use.
The potential for androgenic side effects of
(36) and other estrane progestins is suggested
by the increased AR affinity of these relative to suggested that, as has been observed with ER
(30) and certain other pregnane progestins ligands (section 7.2), the degree of affinity for,
(94 and 95). The PRIAR selectivity of (36) has in this case GC receptors, does not always pre-
been improved by derivatization. Thus, AR af- dict (antagonist) potency. In T47D cells, (99)
finities of norgestimate (98a), the 3-oxime 17- was a full antagonist of (30)-induced alkaline
phosphatase, with potency and efficacy equiv-
alent to those of (55); (100)was a partial ago-
nist (20%efficacy) (128). Other analogs in this
series not endowed with llp-(p-dimethyl-
amino)phenyl moieties were full progesterone
mimetics in T47D cells.

7.5 Nonsteroidal PR Ligands


Cross-affinity of steroidal PR ligands for other
steroid receptors has stimulated efforts to
B X identify nonsteroidal ligands endowed with
CO-CH3 greater selectivity for PR. Such efforts have
(98a) HO-N
also been stimulated by the finding that differ-
(98b) HO-N OH
ent PR ligands can interact with different re-
gions of, and in different ways with, the hPR
acetate ester of (36), and its active metabolite LBD than does (30),resulting in selective PR
(98b) (229) are greatly reduced over that of modulation (231). An example of a nonsteroi-
(36), although high PR affinity is retained (Ta- dal PR ligand (61) with tissue-selective pro-
ble 13.2) (227). gestin agonist activity was described in Sec-
Antiprogestin (55)had a PR RBA equal to tion 5.9.
that of (94), and at least four times greater Nonsteroidal progestin mimetics and an-
than that of (30) (Table 13.2) (230). Its high tagonists have been identified from natural
affinity for GR accounts for its observed anti- sources. Thus (R,S)-cyclocymopolether (101)
glucocorticoid effects. It also exhibited AR af-
finity 10-25% that of testosterone, suggesting
some of its effects could be mediated by these
receptors.
Two 19-norpregnane analogs of (55), (991,
and (loo), each exhibited PR affinities 200%
that of (55), but only one-half the GC affinity
of (55). In a line of GC-receptor transfected
cells, the glucucorticoid antagonist potency of
(99) (and 100) was several orders of magni-
tude lower than that of (55). This observation
668 Female Sex Hormones, Contraceptives, and Fertility Drugs

was isolated from marine algae: its (R)-isomer


was a progesterone antagonist of modest po-
tency, but its (S)-isomer was a progesterone
agonist: EC,, = 35 nM, efficacy 84% of (30)
(225). These stereoisomers had respective
hPR RBA values of 0.7 and 1%that of (30).
Replacement of the trisubstituted aromatic
ring in (101)with ap-nitrophenyl moiety gave
(102), which had an hPR affinity of 6.5% of

phosphatase in T47D cells (90% efficacy), and


its EC,, (18 nM) indicated its potency was 2%
that of (34). COMFA analysis of (104) and its
(301, and a greatly reduced hGR and hAR af- analogs indicated that their "quinolone" rings
finity compared to those of (30). In T47D cells, might be analogous to the D-ring in (30) (124).
(102) was an antagonist with an IC,, value of If so, this would seem to indicate that (104)'s
186 nM (80% efficacy). Effects of the constitu- m-fluorostyryl moiety, and the m-trifluoro-
ent stereoisomers of (102) were not reported. methylphenyl moiety of (61), complete the H-
Synthesis of nonsteroidal PR ligand (103) bonding networks needed for interaction of
these ligands with hPR (Fig. 13.7) in a manner
like the enone A-ring of (30).
Further molecular studies with PR ligands
such as (102-104), aimed at elucidating the
anatomy (specific complementary functional
group interactions, overall conformation) of
their PR complexes, could reveal novel differ-
ences in the ways these ligands reshape PR
compared to when PR is liganded with (30).
The diversity of nonsteroidal structures ex-
(R = a-OH), produced by Penicillium obla-
hibiting PR affinity clearly indicates that PR,
tum, as well as those of several analogs, was
like ER, is capable of accommodatingaromatic
reported (232). Affinity of (103) for porcine PR
polycyclic substances, with subtle structural
was equal to that of (30), but replacement of
differences governing the balance between mi-
its hydroxyl group with a P-methyl group re-
metic and antagonist efficacy.
sulted in a sizable loss of PR affinity.
Chromenoquinolone (104) is a member of a
class of nonsteroidal progesterone mimetics 8 RECENT DEVELOPMENTS
that includes (61) (Section 5.9), which can ex- A N D THINGS T O COME
hibit high PR selectivity. Specifically, (104)
exhibited hPR/hAR and hPR/hGC ratios, in SERMs arzoxifene (78), lasofoxifene (89),and
turn, of 244 and 139, and its hPR affinity rel- SCH 57050 (87b)are in clinical trials, as are
ative to that of (34) was 7% (123). So, (104)'s steroidal estrogen antagonist ici (ZN)182,780
PR affinity was lower but its PR selectivities (65) and steroidal progestin tibolone (54).Sev-
were greater than those of (34) (compare eral nonsteroidal aromatase inhibitors, in-
(34)'s PR, GC, and AR RBAs in Table 13.2). cludingfadrozole (46) and vorozole (49),are in
Like (34) (and 301, (104) induced alkaline various stages of clinical development. Some
References

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Because of their diminished reproductive regard to structure-activity relationships
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daily administration of (89)prevented deteri- 10 ABBREVIATIONS
oration of bone histomorphometric parame-
ters and loss of bone density seen in controls ER estrogen receptor (note that human
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lesterol was also seen, but (89)had no effect on usually refers to ER from lysates of
prostate weight. Clearly, a more complete ER-positive neoplastic cells)
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animal model is warranted. hER human recombinant estrogen recep-
A need that is especially relevant to molec- tor
ular endocrinological studies would be avail- hPR human recombinant progesterone re-
ability of more ER ligands that are uniformly ceptor
selective for ERa or ERP binding, and whose LBD ligand-binding domain
effects are in turn expressed exclusively OVX ovariectomized
through one or the other of these ER isoforms. PR progesterone receptor
As summarized in Section 7.3.4, progress has RBA relative binding affmity
been made in achieving this goal, and it is an- SERM selective estrogen receptor modulator
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G. W. Duncan, Chem. Ind., 408 (1963); (b) D. and J. L. McGuire, Contraception, 41, 399
Lednicer, S. C. Lyster, and G. W. Duncan, (1990).
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214. E. R. Ferguson and B. S. Katzenellenbogen, Wendt, G. C. Buzby Jr., R. A. Edgren, J .
Endocrinology, 100, 1242 (1977). Fisher, T. Foell, B. Gadsby, D. Hartley, D.
215. R. L. Rosati, P. D a s i l v a k d i n e , K. 0. Cam- Herbst, A. B. A. Jansen, K. Ledig, B. J.
eron, D. D. Thompson, H. Z. Ke, S. M. Toler, McLoughlin, J. McMenamen, T. W. Pattison,
T.A. Brown, L. C. Pan, C. F. Ebbinghaus,A. R. P. C. Phillips, R. Rees, J. Siddall, J.Siuda, L. L.
Reinhold, N. C. Elliott, B. N. Newhouse, C. M. Smith, J. Tokolics, and D. H. P. Watson,
Tjoa, P. M. Sweetnam, M. J. Cole, M. W. Ar- J. Chem. Soc., 4472 (1964).
riola, J. W. Gauthier, D. T. Crawford, D. F. 229. J. L. McGuire, A. Phillips, D. W. Hahn, E. L.
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and G. T. Tkalcevic, J. Med. Chem., 41, 2928 Obstet. Gynecol., 163, 2127 (1990).
(1998). 230. J.-P. Raynaud and T. Ojasoo, J. Steroid Bio-
216. R. J. Mountfield, B. Kiehr, and B. A. John, chem., 25,811 (1986).
Drug Metab. Dispos., 28,503 (2000). 231. B. L. Wagner, G. Pollio, S. Leonhardt, M. C.
Wani, D. Y. Lee, M. 0. Imhof, D. P. Edwards,
217. B. T. Kilbourn and P. G. Owston, J.Chem. Soc.
C. E. Cook, and D. P. McDonnell, Proc. Natl.
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218. S. Ernst and G. Hite, Acta Crystallogr. Sect. B, 232. (a) Y. Tabata, M. Hatsu, Y. Kurata, K. Miya-
32,291 (1976). jima, M. Tani, T. Sasaki, Y. Kodama, T. Tsu-
219. R. McCague and G. Leclercq, J. Med. Chem., ruoka, and S. Omoto, J. Antibiot., 50, 309
30, 1761 (1987). (1997); (b) K. Kurihara, K. Tanabe, Y.
220. J. (R.) Skidmore, A. L. Walpole, and J. Wood- Yamamoto, R. Shinei, K. Ajito, and T.
burn, J. Endocrinol., 52, 289 (1972). Okonogi, Bioorg. Med. Chem. Lett., 9, 1837
(1999).
221. K. W. Harlow, D. N. Smith, J. A. Katzenellen- 233. R. J . Byers, J. A. Hoyland, and I. P. Braidman,
bogen, G. L. Greene, and B. S. Katzenellenbo-
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223. B. E. Fink, D. S. Mortensen, S. R. Stauffer, 235. H. Z. Ke, H. Qi, K. L. Chidsey-Frink, D. T.
Z. D. Aron, and J. A. Katzenellenbogen, Chem. Crawford, and D. D. Thompson, J.Bone Miner.
Biol., 6,205 (1999). Res., 16, 765 (2001).
CHAPTER FOURTEEN

Male Sex Hormones, Analogs,


and Antagonists
W. BRUEGGEMEIER
ROBERT
Division of Medicinal Chemistry and Pharmacognosy
The Ohio State University, College of Pharmacy
Columbus, Ohio

Contents
1 Introduction, 680
2 Historical, 680
3 Endogenous Male Sex Hormones, 681
3.1 Occurrence and Physiological Roles, 681
3.2 Biosynthesis, 682
3.3 Absorption and Distribution, 685
3.4 Metabolism, 686
3.4.1 Reductive Metabolism, 687
3.4.2 Oxidative Metabolism, 690
3.5 Mechanism of Action, 692
4 Synthetic Androgens, 697
4.1 Current Drugs on the Market, 697
4.2 Therapeutic Uses and Bioassays, 697
4.3 Structure-Activity Relationships
for Androgens, 699
4.3.1 Early Modifications, 699
4.3.2 Methylated Derivatives, 699
4.3.3 Ester Derivatives, 699
4.3.4 Halo Derivatives, 699
4.3.5 Other Androgen Derivatives, 700
4.3.6 Summary of Structure-Activity
Relationships, 700
4.4 Absorption, Distribution, and Metabolism,
703
4.5 Toxicities, 705
5 Anabolic Agents, 705
5.1 Current Drugs on the Market, 705
5.2 Therapeutic Uses and Bioassays, 705
5.3 Structure-Activity Relationships
for Anabolics, 707
5.3.1 19-Nor Derivatives, 707
5.3.2 Dehydro Derivatives, 707
Burger's Medicinal Chemistv and Drug Discovery 5.3.3 Alkylated Analogs, 708
Sixth Edition, Volume 3: Cardiovascular Agents and 5.3.4 Hydroxy and Mercapto Derivatives, 710
Endocrines 5.3.5 Oxa, Thia, and Aza Derivatives, 711
Edited by Donald J. Abraham 5.3.6 Deoxy and Heterocyclic-Fused Analogs,
ISBN 0-471-37029-0 02003 John Wiley & Sons,Inc. 712
679
680 Male Sex Hormones, Analogs, and Antagonists

5.3.7 Esters and Ethers, 714 6.2.2.1 Steroidal Agents, 718


5.3.8 Summary of Structure-Activity 6.2.2.2 Nonsteroidal Agents, 719
Relationships, 715 6.2.3 Absorption, Distribution, and
5.4 Absorption, Distribution, and Metabolism, Metabolism, 721
715 6.2.4 Toxicities, 721
5.5 Toxicities, 715 6.3 Enzyme Inhibitors, 722
6 Androgen Antagonists, 716 6.3.1 5a-Reductase Inhibitors, 722
6.1 Current Drugs on the Market, 717 6.3.2 17,20-Lyase Inhibitors, 724
6.2 Antiandrogens, 717 6.3.3 C,, Steroids as Aromatase Inhibitors,
6.2.1 Therapeutic Uses, 717 726
6.2.2 Structure-Activity Relationships 6.3.4 Other Agents, 728
for Antiandrogens, 718 7 Summary, 729

1 INTRODUCTION

Androgens are a class of steroids responsible


for the primary and secondary sex character-
istics of the male. In addition, these steroids
possess potent anabolic or growth-promoting
properties. The general chemical structure of
androgens is based on the androstane C,, ste-
roid, consisting of the fused four-ring steroid
nucleus (17 carbons atoms, rings A-D) and the (2) 5 a - dihydrotestosterne
two axial methyl groups (carbons 18 and 19) at
the N B and C/D ring junctions. The hormone
testosterone (1)is the predominant circulat- where. Modified androgens that have found
ing androgen and is produced mainly by the use as biochemical or pharmacological tools
testis. 5a-Dihydrotestosterone (2) is a 5a-re- also are included. More extensive presenta-
duced metabolite of testosterone, produced in tions of the topic of androgens, anabolics, and
certain androgen target tissues and is the androgens antagonists have appeared in sev-
most potent endogenous androgen. eral treatises published over the past two de-
cades (1-11).

2 HISTORICAL

The role of the testes in the development and


maintenance of the male sex characteristics,
and the dramatic physiological effects of male
castration have been recognized since early
time. Berthold (12) was the first to publish in
(1)testosterone 1849 a report that gonadal transplantation
prevented the effects of castration in roosters,
These two steroids and other endogenous suggesting that the testis produced internal
androgens influence not only the development secretions exhibiting androgenic effects. How-
and maturation of the male genitalia and sex ever, the elucidation of the molecules of testic-
glands but also affect other tissues such as kid- ular origin responsible for these actions took
ney, liver, and brain. This chapter discusses almost another century. The first report of the
the endogenous androgens, synthetic analogs, isolation of a substance with androgenic activ-
various anabolic agents, and the androgen an- ity was made by Butenandt (13, 14) in 1931.
tagonists employed in clinical practice or ani- The material, isolated in very small quantities
mal husbandry in the United States and else- from human male urine (15), was named an-
3 Endogenous Male Sex Hormones

drosterone (3) (16). A second weakly andro- male urine caused a marked retention of nitro-
genic steroid hormone was isolated from male gen when injected into dogs fed a constant
urine in 1934. This substance was named de- diet. Soon afterward testosterone propionate
hydroepiandrosterone (4) because of its ready was observed to produce a similar nitrogen-
chemical transformation and structural simi- sparing effect in humans (28). Subsequent
larity to androsterone (17). A year later La- clinical studies demonstrated that testoster-
queur et al. (18, 19) reported the isolation of one was capable of causing a major accelera-
the testicular androgenic hormone, testoster- tion of skeletal growth and a marked increase
one (I), which was 10 times as potent as an- in muscle mass (29-31).This action on muscle
drosterone in promoting capon comb growth. tissue has been referred to more specifically as
Shortly after this discovery, the first chemical the myotrophic effect.
synthesis of testosterone was reported by The first androgenic-like steroid used for its
Butenandt and Hanisch (20) and confirmed by anabolic properties in humans was testosterone.
Ruzicka (21, 22). Unfortunately, its use for this purpose was lim-
ited by the inherent androgenicity and the need
for parented administration. l7a-Methyltes-
tosterone (5)was the first androgen discovered
to possess oral activity, but it too did not show
any apparent separation of androgenic and ana-
bolic activity. The promise of finding a useful,
orally effective, anabolic agent free from andro-
genic side effects prompted numerous clinical
and biological studies.
(3) androsterone

(5) 17a - methyltestosterone

(4) dehydroepiandrosterone
3 ENDOGENOUS MALE SEX HORMONES
For many years it was believed that testos-
3.1 Occurrence and Physiological Roles
terone was the active androgenic hormone in
man. In 1968, however, research in two labo- The hormone testosterone affects many or-
ratories demonstrated that 5a-dihydrotestos- gans in the body. Its most dramatic effects are
terone (DHT, 2), also referred to as stanolone, observed on the primary and secondary sex
was the active androgen in target tissues, such characteristics of the male. These actions are
as the prostate and seminal vesicles, and was first manifested in the developing male fetus
formed from testosterone by a reductase when the embryonic testis begins to secrete
present in these tissues (23,241.Shortly there- testosterone. Differentiation of the Wolffian
after a soluble receptor protein was isolated ducts into the vas deferens, seminal vesicles,
and demonstrated to be specific for DHT and and epididymis occurs under this early andro-
related structures (25,26). gen influence, as does the development of ex-
The anabolic action of the androgens was ternal genitalia and the prostate (32). The re-
first documented by Kochakian and Murlin in ductive metabolism of testosterone to 5a-
1935 (27). In their experiments, extracts of dihydrotestosterone is critical for virilization
682 Male Sex Hormones, Analogs, and Antagonists

during this period of fetal development, as their 5P-metabolites (e.g., etiocholanolone)


dramatically demonstrated in patients with a markedly stimulate erythropoiesis, presum-
5a-reductase deficiency (33). ably by increasing the production of erythro-
At puberty, further development of the sex poietin and by enhancing the responsiveness
organs (prostate, penis, seminal vesicles, and of erythropoietic tissue to erythropoietin (37).
vas deferens) is again evident and under the The effects of androgens on carbohydrate me-
control of androgens. Additionally, the testes tabolism appear to be minor and secondary to
now begin to produce mature spermatozoa. their primary protein anabolic property. The
Other effects of testosterone, particularly on effects on lipid
- metabolism. on the other hand.
the secondary sex characteristics, are ob- seem to be unrelated to this anabolic property.
served. Hair growth on the face, arms, legs, Weakly androgenic metabolites such as andros
and chest is stimulated by this hormone dur- terone have been found to lower serum choles-
ing younger years; in later years, testosterone , terol levels when administered parenterally. A
is responsible for thinning of the hair and re- more detailed account of the biological actions
cession of the hairline. The larynx develops of androgens has been published (37).
and a deepening of the voice occurs. The
male's skin at puberty thickens, the sebaceous
3.2 Biosynthesis
glands proliferate, and the fructose content in The androgens are secreted not only by the
human semen increases. Testosterone influ- testis, but also by the ovary and adrenal cor-
ences sexual behavior, mood, and aggressive- tex. Testosterone is the principal circulating
ness of the male at the time of puberty. androgen and is formed by the Leydig cells
In addition to these androgenic properties, of the testes. Other tissues, such as liver and
testosterone also exhibits anabolic (myotropic) human prostate, form testosterone from pre-
characteristics. General body growth is initi- cursors, although this contribution to the an-
ated, including increased muscle mass and pro- drogen pool is minimal. Because dehydroepi-
tein synthesis, a loss of subcutaneous fat, and androsterone and androstenedione (see Fig.
increased skeletal maturation and rnineraliza- 14.1) are secreted by the adrenal cortex and
tion. This anabolic action is associated with a ovary, they indirectly augment the testoster-
marked retention of nitrogen brought about by one pool because they can be rapidly converted
an increase of protein synthesis and a decrease to testosterone by peripheral tissues.
of protein catabolism. The increase in nitrogen Plasma testosterone levels for men usually
retention is manifested primarily by a decrease range between 0.61 and 1.1 d l 0 0 mL and are
in urinary rather than fecal nitrogen excretion 5-100 times female values (39). The circulating
and results in a more positive nitrogen balance. level of DHT in normal adult men is about one-
For example, intramuscular administration of tenth the testosterone level (40). Daily testoster-
25 mg of testosterone propionate twice daily can one production rates have been estimated to be
produce a nitrogen retention to appear within 4-12 mg for young men and 0.5-2.9 mg for
1 3 days, reaching a maximum in about 5-8 young women (41). Although attempts have
days. This reduced level of nitrogen excretion been made to estimate the secretion rates for
may be maintained for at least a month and de- testosterone, these studies are hampered by the
pends on the patient's nutritional status and number of tissues capable of secreting andro-
diet (34). gens and the considerable interconversion of the
- influence skeletal maturation
Androgens steroids concerned (42,43).
and mineralization, which is reflected in an The synthesis of androgens in the Leydig
increase in skeletal calcium and phosphorus cells of the testes is regulated by the gonado-
(35). In various forms of osteoporosis, andro- tropic hormone, luteinizing hormone (LH),
gens decrease urinary calcium loss and im- also called interstitial cell-stimulating hor-
prove the calcium balance in patients. This mone (ICSH). The other pituitary gonadotro-
effect is not as noticeable in normal patients. pin, follicle-stimulating hormone (FSH), acts
Moreover, the various androgen analogs differ primarily on the germinal epithelium and is
markedly in their effects on calcium and phos- important for sperm development. Both of
phorus balance in man (36). Androgens and these pituitary gonadotropins are under the
3 Endogenous Male Sex Hormones

Mitochrondia
m CAMP + PP,
0 (+I
rregr~enolone Protein kinase A
I
0 (+)
Lipid droplet
n
cholesterol esterase
6------------
\!,-.17a + Cholesterol esters
3j?-HSD
Figure 14.1. Enzymatic con-
version of cholesterol to testos-
Endoplasmic reticulum terone.

regulation of a decapeptide hormone produced genic pathway, including cholesterol esterase


by the hypothalamus. This hypothalamic hor- and cholesterol side-chain cleavage (48). Cho-
mone is luteinizing hormone-releasing hor- lesterol esters (present in the cell as a storage
mone (LHRH), also referred to as gonadotro- form) are converted to free cholesterol by cho-
pin-releasing hormone (GnRH). In adult lesterol esterase, and free cholesterol is trans-
males, pulsatile secretion of LHRH, and sub- located to mitochondria. A cytochrome P-450
sequently LH and FSH, occurs at a frequency mixed-function oxidase system, termed cho-
of 8-14 pulses in 24 h (44). The secretions of lesterol side-chain cleavage, converts choles-
these hypothalamic and pituitary hormones terol to pregnenolone. Several nonmitochon-
are, in turn, regulated by circulating testoster- drial enzymatic transformations then convert
one levels in a negative feedback mechanism. pregnenolone to testosterone, which is se-
Testosterone will decrease the frequency and creted.
amplitude of pulsatile LH secretion (451, The conversion of cholesterol (6) to preg-
whereas both testosterone and a gonadal pep- nenolone (7) has been termed the rate-limit-
tide, inhibin, are both involved in suppressing ing step in steroid hormone biosynthesis. The
the release of FSH (46). reaction requires NADPH and molecular oxy-
Our understanding of steroidogenesis in gen and is catalyzed by a cytochrome P-450
the endocrine organs has advanced consider- enzyme complex termed cholesterol side-
ably during the past two decades, based chain cleavage. This enzyme complex is com-
largely on initial investigations with the adre- posed of three proteins: cytochrome P-45OsCc
nal cortex and subsequent studies in the testis (also called cytochrome P-450 llA), adreno-
and ovary as well (47). Figure 14.2 outlines the doxin, and adrenodoxin reductase. Three
following sequence of events known to be in- moles of NADPH and oxygen are required to
volved with steroidogenesis in the Leydig convert one mole of cholesterol into preg-
cells. LH binds to its receptor located on the nenolone (Fig. 14.1).
surface of the Leydig cell and, by way of a G Tracer studies have shown that two major
protein-mediated process, activates adenylyl pathways, known as the "4-ene" and "5-ene"
cyclase to result in an increase in intracellular pathways, are involved in the conversion of
concentrations of cyclic AMP (CAMP).CAMP pregnenolone to testosterone. Both these
activates a CAMP-dependent protein kinase, pathways and the requisite enzymes are
which subsequently phosphorylates and acti- shown in Fig. 14.1. Earlier studies tended to
vates several enzymes involved in the steroid0 favor the "4-ene" pathway, but more subse-
Male Sex Hormones, Analogs, and Antagonists

Cholesterol (6) Pregnenolone (7)

0
&'&
0
Progesterone (8)

HO
17a-Hydroxypregnenolone(9)

17a-Hydroxyprogesterone(10) Dehydroepiandrosterone (4)

Androstenedione (11) Androstenediol (12)

Testosterone (1)

Figure 14.2. Cellular events in steroidogenesis in Leydig cells.


Table 14.1 Mean Concentration of Steroids ment of two separate enzymes in the conver-
in Spermatic Venous Plasma from Five sion of C-21 to C-19 steroids existed until
Normal Males the purification of the proteins in the 1980s.
Concentration The 17a-hydroxylase/l7,20-lyasecytochrome
Compound (pg/100 mL) P-450 (abbreviated cytochrome P-450 17 or
cytochrome P-450,,,) was first isolated from
neonatal pig testis microsomes by Nakajin and
Dehydroepiandrosterone 2.2 Hall (53). This cytochrome P-45017, possessed
Androstenedione 2.5 both 17a-hydroxylase and 17,20-lyaseactivity
Testosterone 74.0 when reconstituted with cytochrome P-450 re-
Pregnenolone 4.8 ductase and phospholipid. Identical full-
17a-Hydroxypregnenolone 3.9 length human cytochrome P-45017, cDNA se-
17a-Hydroxyprogesterone 6.2 quences were independently isolated and
reported in 1987 (54,55). Extensive reviews of
the molecular biology, gene regulation, and
quent work has disputed this view and sug- enzyme deficiency syndromes have been pub-
gests that the "Bene" pathway is quantita- lished (48,56).
tively more important in man. Vihko and Two additional enzymes are necessary for
Ruokonen (49) analyzed the spermatic venous the formation of testosterone from dehydro-
plasma for free and conjugated steroids. The epiandrosterone. The first is the 3P-hydroxy
unconjugated steroids identified in normal steroid dehydrogenase/A4s5-isomerase com-
males are listed in Table 14.1 along with the plex, which catalyzes the oxidation of the 3P-
concentration in micrograms per 100 mL. All hydroxyl group to the 3-ketone and the
the intermediates of the "5-ene" pathway isomerization of the double bond from C,-C,
1i were identified, but progesterone (8), an im- to C,-C,. Again, these processes were origi-
I portant intermediate of the "4-ene" pathway, nally thought to involve two different en-
1 was not found. In addition, sulfate conjugates zymes, but purification of the enzymatic activ-
were present in significant quantities, espe- ity demonstrated that a single enzyme
cially androst-5-ene-3P,17P-diol3-monosul- catalyzes both reactions (57). The final en-
fate. The data strongly suggest that this inter- zyme in the pathway is the l7/3-hydroxy-
mediate and its unconjugated form constitute steroid dehydrogenase, which catalyzes the re-
an important precursor of testosterone in duction of the 17-ketone to the 17galcohol.
man. This view, however, was not supported
3.3 Absorption and Distribution
by a kinetic analysis of the metabolism of an-
drost-5-ene-3P,17P-diol(12) in man (50). Fur- Although considerable research has been de-
ther evidence that the predominant pathway voted to the biochemical mechanism of action
appears to be the "5-ene" pathway was pro- of the natural hormones and the synthesis of
vided by in vitro studies in human testicular modified androgens, little is known about the
tissues (51). absorption of these substances. It is well rec-
Another important step is the conversion of ognized that a steroid hormone might have
the C-21 steroids to the C-19 androstene deriv- high intrinsic activity but exert little or no bi-
atives. Whereas the enzymes for side-chain ological effects because its physicochemical
cleavage are localized in mitochondria, those characteristics prevent it from reaching the
responsible for cleavage of the C17-C,, bond site of action. This is particularly true in hu-
(C17-C,, lyase) reside in the endoplasmic re- mans, where slow oral absorption or rapid in-
ticulum of the cell. Early studies implicated activation may greatly reduce the efficacy of a
17a-hydroxypregnenolone (9) or 17a-hydroxy- drug. Even though steroids are commonly
progesterone (10) as an obligatory intermedi- given by mouth, little is known of their intes-
ate in testosterone biosynthesis (52), and the tinal absorption. One study in rats showed
CITC,, bond was subsequently cleaved by a that androstenedione (11)was absorbed bet-
second enzymatic process to produce the C-19 ter than testosterone or 17a-methyltestoster-
androstene molecule. This view of the involve- one, and conversion of testosterone to its ace-
Male Sex Hormones, Analogs, and Antagonists

tate enhanced absorption (58). Results with man SBG (67, 68, 74-79). The presence of a
other steroids indicated that lipid solubility 17P-hydroxylgroup is essential for binding to
was an important factor for intestinal absorp- SBG. In addition to testosterone, DHT, 5a-
tion. This may explain the oral activity of cer- androstane-3&17p-diol (19),and 5a-andro-
tain ethers and esters of testosterone. stane-3a,l7P-diol(20) bind with high affinity,
Once in the circulatory system by either se- and these steroids compete for a common
cretion from the testis or absorption of the ad- binding site. Binding to SBG is decreased by
ministered drug, testosterone and other andro- l7a-substituents such as l7a-methyl and 17a-
gens will reversibly associate with certain ethinyl moieties and by unsaturation at C-1or
plasma proteins, the unbound steroid being the C-6. Also, 19-nortestosterone derivatives have
biologically active form. The extent of this bind- lower affinity. SBG has been purified to homo-
ing is dependent on the nature of the proteins geneity by affinity chromatography by the use
and the structural features of the androgen. of a DHT-agarose adsorbent (80).
The first protein to be studied was albumin, Another extracellular carrier protein ex-
which exhibited a low association constant for hibiting high affinity for testosterone, found
testosterone and bound less polar androgens in seminiferous fluid and the epididymis, orig-
such as androstenedione to a greater extent (59- inates in the testis and is called androgen
61). a-Acid glycoprotein (AAG) was shown to binding protein (ABP) (81-83). This protein is
bind testosterone with a higher affinity than produced by the Sertoli cells on stimulation by
that of albumin (62,631.A third plasma protein FSH (84,851 and has very similar characteris-
that binds testosterone is corticosteroid-binding tics to those of plasma SBG produced in the
a-globulin (CBG) (64). However, under normal liver (84).
physiological conditions these plasma proteins The absorption of androgens and other ste-
are not responsible for extensive binding of an- roids from the blood by target cells was usually
drogens in plasma. assumed to occur by a passive diffusion of the
A specific protein, termed sex steroid bind- molecule through the cell membrane. How-
ing p-globulin (SBG)or testosterone-estradiol ever, studies in the early 1970s using tissue
binding globulin (TEBG), was found in plasma cultures or tissue slices suggested entry mech-
that bound testosterone with a very high af- anisms for the steroids. Estrogens (86, 87),
finity (65,661. The SBG-sex hormone complex glucocorticoids (88, 89), and androgens (90-
serves several functions, such as transport or 93) exhibit a temperature-dependent uptake
carrier system in the bloodstream, storage site into intact target cells, suggesting a protein-
or reservoir for the hormones, and protection mediated process. Among the androgens,
of the hormone from metabolic transforma- DHT exhibited a greater uptake than testos-
tions (67,681.SBG has been purified and con- terone in human prostate tissue slices (94),
tains high affinity, low capacity binding sites and it was found that estradiol or andro-
for the sex hormones (69). Dissociation con- stenedione interfered with this uptake mech-
stants of approximately 1 X lop9it4 have been anism (95, 96). In addition, cyproterone
reported for the binding of testosterone and competitively inhibited androstenedione, tes-
estradiol to SBG and are 2 orders of magni- tosterone, and DHT entry, whereas cypro-
tude less than values reported for the binding terone acetate enhanced the uptake of these
of the hormone to the cytosolic receptor pro- androgens (93). Little is known about the exit
tein (70-72). The plasma levels of SBG are of steroids from target cells; the only reported
regulated by the thyroid hormones (73) and research has dealt with an active transport of
remain fairly constant throughout adult life in glucocorticoids out of cells (94,95).
both the male and female (77). SBG is not
3.4 Metabolism
present in the plasma of all animals (67, 74).
For example, SBG-like activity is notably ab- For decades, the primary function of metabo-
sent in the rat, and testosterone may be bound lism was thought to be the inactivation of testos-
in the rat plasma to CBG. terone, the increase in hydrophilicity, and the
Numerous studies have been performed on mechanism for the excretion of the steroid into
the specificity of the binding of steroids to hu- the urine. However, the identification of metab-
3 Endogenous Male Sex Hormones

Testosterone (1)
5a-~eductase/ \ Aromatase

& dP
/

Figure 14.3. Enzymatic conversion of


0 HO testosterone to biologically active me-
H tabolites, 5a-dihydrotestosterone and
5a-Dihydrotestosterone(2) Estradiol (13) estradiol.

olites of testosterone formed in peripheral tis- 17phydroxysteroid dehydrogenases, and 5a-


sues and the potent and sometimes different bi- and 5P-reductases, capable of converting tes-
ological activities of these products emphasized tosterone to various metabolites. Prostatic
the importance of the metabolic transforma- carcinoma metabolizes testosterone more
tions of androgens in endocrinology. Two active slowly than does BPH or normal prostate
metabolites of testosterone have received con- (104). In addition, increased levels of andro-
siderable attention: the reductive metabolite 5a- gens are found in BPH (105). Voigt et al. (106-
dihydrotestosterone and the oxidative metabo- 108) have extensively studied in vivo meta-
lite estradiol (Fig. 14.3). bolic patterns of androgens in patients with
BPH. Tritiated androgens were injected intra-
3.4.1 Reductive Metabolism. The metabo-
venously into these patients 30 min before
lism of testosterone in a variety of in vitro and in
prostatectomy, and prostatic tissue, tissue
vivo systems has been reviewed (52,96-98). The
principal pathways for reductive metabolism of from surrounding skeletal muscle, and blood
testosterone in man appear in Figure 14.4. Hu- plasma were analyzed for metabolites. The
man liver produces a number of metabolites, in- major metabolite of testosterone found in
cluding androstenedione (111, 3P-hydroxy-5a- BPH tissues was DHT, with minor amounts of
androstan-17-one (16), 5a-androstane-3/3,17P- diols isolated. Skeletal muscle and plasma con-
diol (19), and 5u-androstane-3a,l7&diol (20) tained primarily unchanged testosterone.
(99,100). In addition, cirrhotic liver was shown Table 14.2 lists the urinary metabolites that
to produce more 17-ketosteroids than normal have been identified following the administra-
liver (101).Human adrenal preparations, on the tion of testosterone to humans (see Fig. 14.4).
other hand, gave llp-hydroxytestosterone as These products are excreted as such or in the
the major metabolite (102). Intestinal metabo- form of their glucuronide or sulfate conjugates.
lism of testosterone is similar to transforma- Androsterone (3)and etiocholanolone (18),the
tions in the liver (97); the major metabolite in major urinary metabolites, are excreted pre-
lung is androstenedione (103). dominantly as glucuronides, and only about
Studies on testosterone metabolism since 10% as sulfates (109). These conjugates are
the late 1960s have centered on steroid trans- capable of undergoing further metabolism.
formations by prostatic tissues. Normal pros- Testosterone glucuronide, for example, is me-
tate, benign prostatic hypertrophy (BPH), and tabolized differently from testosterone in
prostatic carcinoma all contain 3a-, 3P-, and man, giving rise mainly to 5P-metabolites
Male Sex Hormones, Analogs, and Antagonists

5a-androstane-3a,l7p-diol (21)// 5p-androstane3a,l7p-diol(23)

0
HO
Testosterone (1) 5p-dihydrotestosterone(14) H
3p-hydroxy-5p-androstane-17-one (18)

0
H
Androstenedione (11) 5p-androstane-3,17-dione (16) H
Eticholanolone (19)

0@(&\
5a-androstane-3,17-dione (15) HO H
3,9-hydroxy-5a-androstane-l7-one (17)
Ho,,,,@

Figure 14.4. Reductive metabolites of testosterone.


H

androsterone (3)

(110). Only a relatively small amount of the steroids (112). This explains why a significant
urinary 17-ketosteroids is derived from testos- increase in testosterone secretion associated
terone metabolism. In men at least 67% and in with various androgenic syndromes does not
women about 80% or more of the urinary 17- usually lead to elevated levels of 17-ketoster-
ketosteroids are metabolites of adrenocortical oid excretion.
3 Endogenous Male Sex Hormones

Table 14.2 Urinary Metabolites decreases after castration and can be restored
o f Testosteronea to normal levels of activity with testosterone
Approximate or DHT administration (126).
Metabolite Conversion (%) Early biochemical studies of 5a-reductase
Androsterone 25-50 were performed using a microsomal fraction
Etiocholanolone from rat ventral prostate. The irreversible en-
5p-Androstane-3a,17P-diol 2 zymatic reaction catalyzed by 5a-reductase re-
5a-Androstane-3a,17P-diol 1 quires NADPH as a cofactor, which provides
5a-Androstan-3P-ol-17-one 1 the hydrogen for carbon-5 (127). The 5a-re-
Androst-16-en-3a-01 0.4 ductase from rat ventral prostate tissues ex-
3a,l8-Dihydroxy-5P-androstan-
hibited a broad range of substrate specificity
17-one 0.3
3a,7p-Dihydroxy-5P-androstan- for various C,, and C,, steroids (101); this
17-one Trace broad specifity was also observed in inhibition
1lp-Hydroxytestosterone Trace studies (128). However, more detailed studies
6a-, 6P-Hydroxytestosterone Trace of the enzyme were limited because of the ex-
"Cf. Ref. 111 treme hydrophobic nature of the protein, its
instability upon isolation, and its low concen-
Although androsterone and etiocholanolone trations in androgen-dependent tissues (98).
are the major excretory products, the exact Investigations on the molecular biology of
sequence whereby these 17-ketosteroids arise 5a-reductase resulted in the demonstration of
is still not clear. Studies with radiolabeled an- two different genes and two different isozymes
drost-4-ene-3/3,17P-dioland the epimeric 3a- of the enzyme (129-131). The first cDNA iso-
diol in humans showed that oxidation to tes- lated and cloned that encoded 5a-reductase
tosterone was necessary before reduction of was designated Type 1, and the second was
the A-ring (113). Moreover, in rats 5P-andro- designated Type 2. The gene encoding Type 1
stane-3a,l7P-diol (22) was the major initial is located on chromosome 5, whereas the gene
liver metabolite. but this decreased with time encoding Type 2 is located on chromosome 2.
with the simultaneous increase of etiochol- The two human 5a-reductases have approxi-
anolone (114). This formation of saturated di- mately 60% sequence homology. These two
01s agrees with studies using human liver (99) isozymes differ in their biochemical proper-
and provides evidence that the initial step in ties, tissue location, and function (131, 132).
testosterone metabolism is reduction of the Type 1 5a-reductase exhibits an alkaline pH
a#-unsaturated ketone to a mixture of diols optimum (6-8.5) and has micromolar affini-
followed by oxidation to the 17-ketosteroids. ties for steroid substrates. On the other hand,
Until 1968, it was generally thought that Type 2 5a-reductase has a sharp pH optimum
the excretory metabolites of testosterone were at 4.7-5.5, has higher affinity (lower apparent
physiologically inert. Subsequent work has K,) for testosterone, and is more sensitive to
shown, however, that etiocholanolone has inhibitors than the Type 1 isozyme. The Type
thermogenic effects when administered to 2 isozyme is expressed primarily in androgen
man (115). Moreover, the hypocholester- target tissues, the liver expresses both types,
olemic effects of parenterally administered an- and the Type 1 form is expressed in various
drosterone have been described (116). peripheral tissues. Type 2 5a-reductase ap-
The conversion of testosterone to DHT by pears to be essential for masculine develop-
5a-reductase has major importance in the ment of the fetal urogential tract and the ex-
mechanism of action of the hormone. This en- ternal male phenotype, whereas the Type 1
zymatic activity has been found in the endo- isozyme is primarily a catabolic enzyme. In
plasmic reticulum (117, 118) and in the nu- certain cases of human male pseudohermaph-
clear membrane (119-125) of androgen- roditism, mutations in the Type 2 5a-reduc-
sensitive cells. In addition, the levels of tase gene are observed and results in signifi-
5a-reductase are under the control of testos- cant decreases in DHT levels needed for
terone and DHT (125); 5a-reductase activity virilization (133).
Male Sex Hormones, Analogs, and Antagonists

HO.

Androstenedione (11)

NADPH 02

NADPH

1 02

-
- HCOOH
HO
Estrone (27)

Peroxy enzyme
intermediate
Figure 14.5. Aromatization of androgens.

3.4.2 Oxidative Metabolism. Another met- cytochrome P-450 enzyme complex (135) and
abolic transformation of androgens leading to requires 3 moles of NADPH and 3 moles of
hormonally active compounds is their conver- oxygen per mole of substrate (136). Aromati-
sion to estrogens. Estrogens are biosynthe- zation proceeds through three successive
sized in the ovaries and placenta and, to a steps, with the first two steps being hydroxyl
lesser extent, in the testes, adrenals, and cer- ations. The observation by Meyer (137) that
tain regions of the brain. The enzyme complex 19-hydroxyandrostenedione (23) was a more
that catalyzes this biosynthesis is referred to active precursor of estrone (25) than the sub-
as aromatase, and the enzymatic activity was strate androstenedione led to its postulated
first identified by Ryan (134) in the microso- role in estrogen biosynthesis. This report and
ma1 fraction from human placental tissue. The numerous studies that followed led to the cur-
elucidation of the mechanism of the aromati- rently accepted pathway for aromatization, as
zation reaction began in the early 1960s and shown in Fig. 14.5. Relative percentages of
continues to receive extensive study. It is a substrate activity are presented in Table 14.3.
3 Endogenous Male Sex Hormones

Table 14.3 Relative Substrate Activity in Aromatization


Substrate Activity (%)
C,, Steroids
Androst-4-ene-3,17-dione
19-Hydroxyandrost-4-ene-3,17-dione
17P-Hydroxyandrost-4-en-3-one
3P-Hydroxyandrost-5-en-17-one
5a-Androst-1-ene-3,17-dione
5a-Androstane-3,17-dione
la-Hydroxyandrost-4-ene-3,17-dione
l7~-Hydroxy-la-methylandrost-4-ene-3-one
2p-Hydroxyandrost-4-ene-3,17-dione
2a-Hydroxyandrost-4-ene-3,17-dione
17/3-Hydroxy-2P-methylandrost-4-en-3-one
1 lp-Hydroxyandrost-4-ene-3,17-dione
lla-Hydroxyandrost-4-ene-3,17-dione
l7~-Hydroxy-l7a-methylandrost-4-en-3-one
6P-Hydroxyandrost-4-ene-3,17-dione
6a-Fluoro-l7~-hydmxyandrost-4-ene-3,l7-dione
6~-Fluoro-l7~-hydroxyandrost-4-ene-3,17-dione
9a-Fluoroandrosta-1,4-diene-3,17-dione
Androsta-1,4-diene-3,17-dione
Androsta-4,6-diene-3,17-dione
Androsta-l,4,6-triene-3,17-dione
Androst-4-ene-3,11,17-trione
C,, Steroids
Estr-4-ene-3,17-dione
17P-Hydroxyestr-4-en-3-one
17p-Hydroxy-5a,10P-estr-3-one
C,, Steroids
Pregn-4-ene-3,20-dione
17a,19,2l-Trihydroxypregn-4-ene-3,20-dione

The first two oxidations occur at the C,, into formic acid (145-147). These results have
position, producing the 19-alcohol (23) and led to the proposal that the last oxidation step
then the 19-gem-diol (24), originally isolated is a peroxidative attack at the C19position
as the 19-aldehyde (25) (138, 139). The exact (148-150).
mechanism of the last oxidation remains to be Incubation of a large number of testoster-
fully determined. The final oxidation results one analogs with human placental tissue
in the stereospecific elimination of the 1/3 and (151-153) has provided some insight into the
2P hydrogen atoms (140-142) and the con- structural requirements for aromatization
certed elimination of the oxidized Clgmoiety (see Table 14.4). Whereas androstenedione
as formic acid (139). Hydroxylation at the 2P- was converted rapidly to estrone, the l-dehy-
position was suggested as an intermediate in dro and 19-nor analogs were metabolized
this final oxidation, given that this substance slowly, and the 6-dehydro isomer and satu-
spontaneously aromatized to estrone (143). rated 5a-androstane-3,17-dione remained un-
However, investigations using ' '
0, and isoto- changed. Hydroxyl and other substituents at
pically labeled steroidal intermediates failed la, 2P, and l l p interfered with aromatization,
to show incorporation of the 2P-hydroxyl whereas similar substituents at 9a and l l a
group into formic acid under enzymatic or seemingly had no effect. Of the stereoisomers
nonenzymatic conditions (144) and did dem- of testosterone, only the 8P,9P,lOP-isomer is
onstrate that the oxygen atoms from the first aromatized, in addition to compounds having
and third oxidation steps are incorporated the normal configuration (8P, 9a, 10P). Thus,
692 Male Sex Hormones, Analogs, and Antagonists

Table 14.4 Comparison of the Androgenic and Myotrophic Activities of Testosterone


Derivatives in the Chick Comb and Castrated Male Rat Assays
after Subcutaneous Administration
Increase in Weight (%)
Rat Ventral Rat Levator
Testosterone Modification Chick Comb Prostate Ani
None
19-Nor
7a-Methyl
7a-Methyl-19-nor
14-Dehydro
14-Dehydro-19-nor
14-Dehydro-7a-methyl-19-nor

the substrate specificity of aromatase appears all three oxidation steps. Knowledge of the
to be limited to C,, steroids with the 4-en-3- molecular biology of aromatase has advanced
one system. Inhibition studies with various greatly in the past 5 years. A full-length cDNA
steroids have provided additional insights into complementary to mRNA encoding cytochrome
the structural requirements for the enzyme P-450,,, was sequenced and the open read-
(154-156); steroidal aromatase inhibitors are ing frame encodes a protein of 503 amino acids
described later in section . (158). This cDNA sequence was inserted into
Recent research in aromatase has focused COSl monkey kidney cells, and aromatase
on the biochemistry, molecular biology, and mRNA and aromatase enzymatic activity were
regulation of the aromatase protein. Aromatase detected in transfected cells. The entire hu-
is a membrane-bound cytochrome P-450 man cytochrome P-450,,, gene is greater
monooxygenase consisting of two proteins, than 70 kb in size (159,160) and is located on
aromatase cytochrome P-450 (P-450,,,) and chromosome 15 (161). Clones have been used
NADPH-cytochrome P-450 reductase. Cyto- to examine the regulation of aromatase in
chrome P-450,, is a heme protein that binds ovarian, adipose, and breast tissues (162,
the steroid substrate and molecular oxygen 163-166).
and catalyzes the oxidations. The reductase is The metabolism of androgens by the mam-
a flavoprotein, is found ubiquitously in endo- malian brain has also been investigated under
plasmic reticulum, and is responsible for in vitro conditions. Sholiton et al. (167) in
transferring reducing equivalents from 1966 first reported the metabolism of testos-
NADPH to cytochrome P-450,,. Purifica- terone in rat brain, and later studies demon-
tion of cytochrome P-450,,,, proved to be strated the conversion of testosterone to DHT.
very difficult because of its membrane-bound androstenedione, 5a-androstane3,17-dione, and
nature, instability, and low tissue concentra- 5a-androstane-3p,17p-diol (168-173). The
tion. The combination of hydrophobic chro- aromatization of androgens to estrogens was
matography usingphenyl Sepharose, nonionic also found to occur in the hypothalamus and
detergent, and the presence of micromolar the pituitary gland (174-179). The full signif-
amounts of substrate androstenedione yielded icance of these metabolites on various neu-
a highly purified and active cytochrome roendocrine functions, such as regulation of
p-45oar0,, with the highest specific content of gonadotropin secretion and sexual behavior, is
11.5 nmol of cytochrome P-450 per mg of pro- not yet fully understood (180, 181).
tein reported (157). Reconstitution of this cy-
3.5 Mechanism of Action
tochrome P-450,, with NADPH-cyto-
chrome P-450 reductase and phospholipid It would indeed be impossible to explain all the
resulted in complete conversion of andro- varied biological actions of testosterone by one
stenedione to estrone, thus demonstrating biochemical mechanism. Androgens, as well as
that one cytochrome P-450 protein catalyzes the other steroid hormones adrenocorticoids,
3 Endogenous Male Sex Hormones

DHEA Prostate

Autocrine & paracrine effects


Figure 14.6. Mechanism of action of 5m-dihydrotestosterone.

estrogens, and progestins exert potent physi- target cells such as blood and liver (197, 198).
ological effects on sensitive tissues and yet are With the availability of steroids with high spe-
present in the body in only extremely low con- cific activity, later studies demonstrated the
centrations (e.g., 0.1-1.0 nM). The majority of selective uptake and retention of androgens by
investigations concerning the elucidation of target tissues (23, 24, 199-201). In addition,
the mechanisms of action of androgens have DHT was found to be the steroidal form selec-
dealt with actions in androgen-dependent tis- tively retained in the nucleus of the rat ventral
sues and, in particular, the rat ventral pros- prostate (23, 200). This discovery led to the
tate. The results of these studies indicate that current concept that testosterone is converted
androgens primarily act in regulating gene ex- by 5a-reductase to DHT, which is the active
pression and protein biosynthesis by forma- form of cellular androgen in androgen-depen-
tion of a hormone-receptor complex, analo- dent tissues.
gous to the mechanisms of action of estrogens The rat prostate has been the most widely
and progestins. Extensive research activities examined tissue, and current hypotheses on
directed at elucidation of the general mecha- the mode of action of androgens are based
nism of steroid hormone action have been largely on these studies (see Fig. 14.6). The
performed for over three decades, and several lipophilic steroid hormones are carried in the
reviews have appeared on this subject (182- bloodstream, with the majority of the hor-
195). mones reversibly bound to serum carrier pro-
Jensen and Jacobson (1961, using radiola- teins and a small amount of free steroids. The
beled 17gestradio1, were the first to show that androgens circulating in the bloodstream are
a steroid was selectively retained by its target the sources of steroid hormone for androgen
tissues. Investigations of a selective uptake of action in target tissues. Testosterone, synthe-
androgens by target cells performed in the sized and secreted by the testis, is the major
early 1960s were complicated by low specific androgen in the bloodstream and the primary
activity of the radiolabeled hormones and the source of androgen for target tissues in men.
rapid metabolic transformations. Nonethe- Dehydroepiandrosteone (DHEA) and andro-
less, it was noted that target cells retained pri- stenedione also circulate in the bloodstream
marily unconjugated metabolites, whereas and are secreted by the adrenal gland under
conjugated metabolites were present in non- the regulation of adrenocorticotrophic hor-
Male Sex Hormones, Analogs, and Antagonists

Transcription DNA Ligand


activation binding binding
domain domain domain

H2N
COOH
1 559 624 919 a.a.

- Nuclear localization
- Dimerization
- Transactivation

Figure 14.7. Schematic diagram of androgen receptor.

mone (ACTH). DHEA and androstenedione with particular regions of the cellular DNA,
supplement the androgen sources in normal referred to as androgen responsive elements
adult men, but these steroids are the impor- (ARES), and with various nuclear transcrip-
tant circulating androgens in women. The free tional factors. Binding of the nuclear steroid-
circulating androgen steroids passively diffuse receptor complex to DNA initiates transcrip-
through the cell membrane and are converted tion of the DNA sequence to produce
to the active androgen 5a-DHT within the tar- messenger RNA (mRNA). Finally, the ele-
get cells. vated levels of mRNA lead to an increase in
The androgens act on target cells to regu- protein synthesis in the endoplasmic reticu-
late gene expression and protein biosynthesis lum; these proteins include enzymes, recep-
through the formation of steroid-receptor tors, andlor secreted factors that subsequently
complexes. Those cells sensitive to the partic- result in the steroid hormonal response regu-
ular steroid hormone (referred to as target lating cell function, growth, and differentia-
cells) contain high affinity steroid receptor tion.
proteins capable of interacting with the ste- Extensive structure-function studies on
roid (25, 202). The binding of DHT with the the androgen receptor (AR) have identified re-
receptor protein is a necessary step in the gions critical for hormone action. The andro-
mechanism of action of the steroid in the pros- gen receptor is encoded by the AR gene located
tate cell. Early studies suggested that the ste- on the X chromosome, and the AR gene is com-
roid receptor proteins were located in the cy- posed of 8 exons. The human AR contains ap-
tosol of target cells (196) and, after formation proximately 900-920 amino acids, and the ex-
of the steroid-receptor complex, the steroid- act length varies because of polymorphisms in
receptor complex translocated into the nu- the NH,-terminal of the protein. The primary
cleus of the cell. More recent investigations on amino acid sequences of AR, as well as of the
androgen action indicate that active, unoccu- various steroid hormone receptors, were
pied receptor proteins are present only in the deduced from cloned complementary DNAs
nucleus of the cell. This nuclear localization of (cDNAs) (191, 193). The calculated molecular
receptor is also observed for estrogens and weight of AR is approximately 98,000 kDa,
progestins, whereas the majority of the glu- based on amino acid composition; however,
cocorticoid receptor is located in the cyto- the AR is a phosphoprotein and migrates
plasm (190,203). In the current model, DHT is higher at approximately 110 kDa in SDS gel
formed in the cytoplasm, then enters the nu- electrophoresis. The steroid receptor proteins
cleus of the cell and binds to the nuclear ste- are part of a larger family of nuclear receptor
roid receptor protein. proteins that also include receptors for vita-
The binding of DHT to the nuclear andro- min D, thyroid hormones, and retinoids. The
gen receptor initiates a conformational overall structure of the androgen receptor
change or activation of the steroid-nuclear re- (shown in Fig. 14.7) has strong similarities to
ceptor complex and results in the formation of the other steroid hormone receptors, with the
a homodimer (191). The homodimer interacts proteins containing regions that bind to the
3 Endogenous Male Sex Hormones

DNA and bind to the steroid hormone ligand


(194,204,205).A high degree of homology (se-
quence similarities) in the steroid receptors is
found in the DNA binding region that inter-
acts with the HREs. The DNA binding region
is rich in cysteine amino acids and chelate zinc
ions, forming fingerlike projections called zinc
- that bind to the DNA. The hormone
fingers
binding domain (or ligand binding domain) is
located on the COOH-terminal of the protein.
Structure-function studies of cloned receptor
proteins also identify regions of the molecules
that are important for nuclear localization of
the receptor, receptor dimerization, interac-
tions with nuclear transcriptional factors, and
activation of gene transcription. Importantly,
two regions of the androgen receptor protein
are identified as transcriptional activation do-
mains. The domain on the NH,-terminal re-
gion may interact with both coactivators and
corepressors, and the COOH-terminal domain
initiates transcriptional activation only upon
binding of an agonist such as 5a-DHT. The
interactions necessary for formation of the
steroid-receptor complexes and subsequent
activation of gene transcription are compli-
cated, involve multiple protein partners re-
ferred to as coactivators, and leave many un-
answered questions.
Although the tertiary structure of the en- Figure 14.8. Ribbon diagram of androgen receptor
ligand binding domain (LBD) bound with 5a-dihy-
tire androgen receptor has not been deter-
drotestosterone. Atomic coordinates were obtained
mined, the crystallographic structure of the from the Protein Data Bank (PDB ID code 1137;
ligand binding domain (LBD) has recently www.rcsb.org) and displayed using ViewerLite 4.2,
been reported (206, 207). The AR LBD con- Accerlys Inc. 5a-Dihydrotestosterone is illustrated
sists of an a-helical sandwich, similar to the as a stick structure behind Helix 12.
LBDs reported for other nuclear receptors,
and AR LBD contains only 11 helices (no helix various forms of androgen resistance and ab-
2) and four short p-strands (Fig. 14.8). Minor normal male sexual development (194, 205,
differences in the two reported crystallo- 208-210). Two polymorphic regions have been
graphic structures are likely attributable to identified in the NH,-terminal region, encod-
limits of experimental resolution, differences ing a polyglycine repeat and a polyglutamate
in data interpretation, and the use of different tract. Currently, these polymorphic regions
ligands for crystallization. The endogenous li- have not been shown to significantly alter AR
gand DHT (2) interacts with helices 3 , 5 , and levels, stability, or transactivation (205).
11, and the DHT-bound AR LBD has a single, These repeats are useful in pedigree analysis
continuous helix 12 (207). Similar interac- of patients (194). Mutations in the androgen
tions are observed with metribolone (methyl- receptor have been identified in patients with
trienolone, 53); however, helix 12 is split into either partial or full androgen insensitivity
two shorter helical segments (206). syndrome (AIS),with the majority of the mu-
Additional information on receptor struc- tations identified in exons 4 through 8 encod-
ture-function has been obtained by analyzing ing the DNA binding domain and the hormone
androgen receptor mutations in patients with binding domain. Finally, studies with the hu-
696 Male Sex Hormones, Analogs, and Antagonists

man LNCaP prostate cancer cell line have pro- began to investigate the presence of cellular
vided interesting results regarding receptor receptor proteins in other androgen-sensitive
protein structure and ligand specificity. The tissues. Androgen receptors have been re-
LNCaP cells exhibited enhanced proliferation ported in seminal vesicles (232, 233), seba-
in the presence of androgens, but also these ceous gland (234-236), testis (235, 237), epi-
cells unexpectedly proliferated in the presence didymis (232, 238, 239), kidney (2401,
of estrogens, progestins, cortisol, or the anti- submandibular gland (241,242),pituitary and
androgen flutamide (211,212). Analysis of the hypothalamus (243-249), bone marrow (250,
cDNA for the LNCaP androgen receptor re- 251), liver (252), and androgen-sensitive tu-
vealed that a single base mutation in the mors (253, 254). Although DHT is the active
ligand binding domain was present and re- androgen in rat ventral prostate, it is not the
sulted in the increased affinity for progester-
only functioning form in other androgen-sen-
one and estradiol (213). The crystallographic
sitive cells. In ventral prostate and seminal
structures of the LBD with the T877A muta-
vesicles, DHT is readily formed. It is metabo-
tion confirm that the mutated AR LBD can
accommodate larger structures at the C-17 po- lized only slowly, however, and therefore can
sition (206, 207). accumulate and bind to receptors. Also, com-
The ultimate action of androgens on target parison of binding kinetics for testosterone
tissues is the stimulation of cellular growth and DHT demonstrated that testosterone dis-
and differentiation through regulation of pro- sociates faster, implying extended retention of
tein synthesis, and numerous androgen-in- DHT by the androgen receptor (255). In other
ducible proteins have been identified (205). tissues, such as brain or kidney, DHT is not
One of the prominent androgen-inducible pro- readily formed and is metabolized quickly
teins is prostate-specific antigen (PSA), a compared to testosterone. Species variations
serine protease expressed by secretory pros- have also been demonstrated. The most strik-
tate epithelial cells and used as blood test in ing example is the finding that 5a-androstane-
screening for possible prostate diseases such 3a,17a-diol interacts specifically with cyto
as prostate cancer. Three ARES have been solic receptor protein from dog prostate (256)
identified in the promoter regions of the PSA and may be the active androgen in this species
gene (214-216). Another androgen-regulated (257). Apparently the need for a 17@hydroxyl
gene examined extensively in rats is the gene is not essential in all species.
encoding the protein probasin (217,218),a 20- Thus, current findings indicate that andro-
kDa secretory protein from the rat dorsolat- gen receptor proteins vary in steroid specific-
eral prostate structurally similar to serum ity among different tissues from the same spe-
globulins. Other proteins induced by andro-
cies as well as among different species.
gens include spermine-binding protein (219),
Nevertheless, the basic molecular mechanism
keratinocyte growth factor (KGF or FGF-7)
of action of the androgens in androgen-sensi-
(220), androgen-induced growth factor (AIGF
or FGF-8) (221, 222), nerve growth factor tive tissues is consistent with the results of the
(223), epidermal growth factor (2241, c-myc studies on rat ventral prostate.
(225), protease D (2261, p-glucuronidase The manner whereby the androgens exert
(227), and a,,-globulin (228-230). Studies of their anabolic effects has not been as exten-
these proteins suggest that the androgens act sively studied. The conversion of testosterone
by enhancing transcription andlor translation to DHT was been shown to be insignificant in
of specific RNAs for the proteins. Also, the an- skeletal and levator ani muscles, suggesting
drogen receptor represses gene expression of that the androgen-mediated growth of muscle
certain proteins such as glutathione S-trans- is ascribed to testosterone itself (258, 259).
ferase, TRMP-2 involved in apoptosis, and cy- Classical steroid receptors for testosterone are
tokines such as interleukin-4, interleukin-5, found in the cytoplasm of the levator ani and
and y-interferon (205,231). quadriceps muscles of the rat (260,261). Un-
Although most biochemical studies focused like prostate receptor protein, DHT had a
on the rat ventral prostate, some researchers lower affinity than that of testosterone for this
4 Synthetic Androgens

protein. Androgen receptors have also been 4.2 Therapeutic Uses and Bioassays
identified in other muscle tissues as well, in-
cluding cardiac muscle (262-267). The primary uses of synthetic androgens are
the treatment of disorders of testicular func-
Fj 4 SYNTHETIC ANDROGENS tion and of cases with decreased testosterone
I production. Several types of clinical conditions
L
4.1 Current Drugs on the Market result from testicular dysfunction. Informa-

Generic Name Chemical


(structure) Trade Name U.S. Manufacturer Class Dose
Testosterone Delatestryl BTG Pharm Androstane Injection:
enanthate (31) 200 mg/mL
Testosterone Depo-Testosterone Pharmacia Androstane Injection:
cypionate (33) 100 mg/mL
200 mg/mL
Testosterone Testopel Bartor Pharmacal Androstane Pellets: 75 mg
pellets (1)
Testosterone Testoderm Alza Androstane Transdermal:
transdermal 10 mg
system (1) 15 mg
Testoderm TTS Alza Androstane Transdermal:
328 mg
Testoderm with Alza Androstane Transdermal:
Adhesive 15 mg
Androderm Watson Pharma Androstane Transdermal:
12.2 mg
24.3 mg
Testosterone gel AndroGel 1% Unirned Pharm. Androstane Gel: 1%testosterone
(1)
Methyltestosterone Methyltestosterone Various suppliers Androstane Tablets:
(5) 10 mg
25 mg
Tablets (buccal):
10 mg
Testred ICN Pharm Androstane Capsules: 10 mg
Android ICN Pharm Androstane Capsules: 10 mg
Virilon Star Androstane Capsules: 10 mg
Virilon IM Star Androstane Injection:
200 mg/mL
Fluoxymesterone Halotestin Pharmacia Androstane Tablets:
(36) 2 mg
5 mg
10 mg
Fluoxymesterone Various suppliers Androstane Tablets: 10 mg
Danazol Various suppliers Androstane Capsules:
50 mg
100 mg
200 mg
Danocrine Sanofi-Synthelabo Androstane Capsules:
50 mg
100 mg
200 mg
Testolactone (38) Teslac Bristol-Myers Squibb Androstane Tablets: 50 mg
Male Sex Hormones, Analogs, and Antagonists

tion on the biochemistry and mechanism of vated LH levels are found. Partially deficient
action of testosterone that has accumulated androgen receptors are present in these pa-
over the past 20 years has greatly aided in the tients (270, 272). In most cases of male
elucidation of the underlying pathophysiology pseudohermaphroditism, androgen replace-
of these diseases. Two reviews describe in ment has little or no effect and thus steroid
greater detail the mechanisms involved in dis- treatment is not recommended.
orders of testicular function and androgen re- Deficiencies of circulating gonadotropins
sistance (268, 269). lead to secondary hypogonadism. This condi-
Hypogonadism arises from the inability of tion can be caused by disorders of the pituitary
the testis to secrete androgens and can be and/or hypothalamus resulting in diminished
caused by various conditions. These hypogo- secretions of neurohormones. The lack of
nadal diseases can, in many cases, result in stimulation of the seminiferous tubules and
disturbances in sexual differentiation and the Leydig cells attributed to the low levels of
function and/or sterility. Primary hypogonad- these neurohormones decreases androgen
ism is the result of a basic disorder in the tes- production. Drugs such as the neuroleptic
tes, whereas secondary hypogonadism results phenothiazines and the stimulant marihuana
from the failure of pituitary and/or hypotha- can also interfere with release of gonadotro-
lamic release of gonadotropins and thus di- pins. The use of androgens in secondary hypo-
minished stimulation of the testis. Usually gonadism is symptomatic.
primary hypogonadism is not recognized in Synthetic androgens have also been used in
early childhood (with the exception of cryptor women for the treatment of endometriosis, ab-
chidism) until the expected time of puberty. normal uterine bleeding, and menopausal
This testosterone deficiency is corrected by symptoms. However, their utility is severely
androgen treatment for several months, at limited by the virilizing side effects of those
which time the testes are evaluated for possi- agents. Two weak androgens, calusterone and
ble development. Long-term therapy is neces- l-dehydrotestolactone, are used clinically in
sary if complete testicular failure is present. the treatment of mammary carcinoma in
Patients with Klinefelter's syndrome, a dis- women. The mode of action of these drugs in
ease in which a genetic male has an extra X the treatment of breast cancer is unknown.
chromosome, have low testosterone levels and and is not simply related to their androgenic-
can also be treated by androgen replacement. ity (276). More recent evidence on the ability
Male pseudohermaphroditism are disor- of these compounds to inhibit estrogen biosyn-
ders in which genetically normal men do not thesis catalyzed by aromatase suggests that
undergo normal male development. One type, they effectively lower estrogen levels in vivo
testicular feminization, is observed in patients (276).
who have normal male XY chromosomes, but The various analytical methods used to es-
the male genitalia and accessory sex glands do tablish the androgenic properties of steroidal
not develop. Rather, the patients have female substances have been reviewed by Dorfman
external genitalia. These patients are unre- (277). Traditionally, androgens have been as-
sponsive to androgens and have defective an- sayed by the capon comb growth method and
drogen receptors (270-272). Another type of by the use of the seminal vesicles and prostate
male pseudohermaphroditism results from a organs of the rodent. Increases in weight
deficiency of the enzyme 5a-reductase (273, and/or growth of the capon comb have been
274). Because DHT is necessary for early dif- used to denote androgenic activity after injec-
ferentiation and development, the patients tion or topical application of a solution of the
again develop female genitalia; later, some test compound in oil (278). A number of minor
masculinization can occur at the time of pu- modifications of this test have been described
berty because of elevated testosterone levels (279-281). The increase in weight of the sem-
in the blood. A third disorder is Reifenstein inal vesicles and the ventral mostate of the
syndrome, an incomplete pseudohermaphro- immature castrated male rat has provided
ditism. In these patients, the androgen levels another measure of androgenic potency (282-
are normal, 5a-reductase is present, and ele- 285). The test compound is administered ei-
4 Synthetic Androgens

ther intramuscularly or orally, and the weight 4.3.2 Methylated Deriv


of the target organs is compared with those of ery that C-l7a-methylation conferred oral ac-
control animals. In vitro evaluation of the rel- tivity on testosterone prompted the synthesis
ative affinity of potential androgens for the of additional C-17a-substituted analogs. In-
androgen receptor has also become an impor- creasing the chain length beyond methyl in-
tant tool in assessing biological activity of an- variably led to a decrease in activity (297). As a
drogens (128,286). result of these studies, however, l7a-methyl-
androst-5-ene-3P,17P-diol (methandriol, 29)
4.3 Structure-Activity Relationships was widely evaluated in humans as an ana-
for Androgens bolic agent. Early biological studies with
methandriol had shown a separation of ana-
4.3.1 Early Modifications. Most of the early bolic and androgenic properties, although this
structure-activity relationship studies con- was not confirmed by subsequent studies
cerned minor modifications of testosterone (284). In addition, clinical studies showed no
t and other naturally occurring androgens. advantage of methandriol over l7a-methyl-
Studies in animals (287) and humans (288) testosterone (5) (298).
showed the 17p-hydroxylfunction to be essen-
tial for androgenic and anabolic activity. In
certain cases esterification of the l7P-hy-
droxyl group not only enhanced but also pro-
longed the anabolic and androgenic properties
(289).The early statement (290) that the 1-de-
hydro isomer of testosterone (28) was a weak
androgen was subsequently disproved. This
isomer and related compounds are potent an-
drogenic and anabolic steroids (291).

4.3.3 Ester Derivatives. As early as 1936 it


was known that esterification of testosterone
markedly prolonged the activity of this andro-
gen when it was administered parenterally
(299). This modification enhances the lipid
solubility of the steroid and, after injection,
permits a local depot effect. The acyl moiety is
usually derived from a long-chain aliphatic or
arylaliphatic acid such as propionic, heptanoic
(enanthoic), decanoic, cyclopentylpropionic
(cypionic), or P-phenylpropionic acid (30)
Reduction of the A-ring functional groups (30-34).
has variable effects on activity. For example,
conversion of testosterone to DHT has little 4.3.4 Halo Derivatives. In general, the
effect or may increase potency in a variety of preparation of halogenated testosterone de-
bioassay systems (292, 293). On the other rivatives has been therapeutically unreward-
hand, changing the AIB trans stereochemistry ing. 4-Chloro-17~-hydroxyandrost-4-en-3-one
of known androgens such as androsterone (3) (chlorotestosterone, 35) and its derivatives
and DHT to the All3 cis-etiocholanolone (18) are the only chlorinated androgens that have
and 17p-hydroxy-5p-androstan-3-one (131,re- been used clinically, albeit sparingly (300).
spectively, drastically reduces both the ana- The introduction of a 9a-fluoro and an l l p -
bolic and androgenic properties (294-296). hydroxy substitutents (analogous to synthetic
These observations established the impor- glucocorticoids) gives 9a-fluoro-llp,l7P-dihy-
tance of the All3 trans ring juncture for activ- droxy-17a-methylandrost-4-en-3-one (flu-
ity. oxymesterone, Halotestin, 36), which is an
Male Sex Hormones, Analogs, and Antagonists

(30) propionate R = CH2CH3

(31) heptanoate (enanthate) (CH2)5CH3

(32) decanoate (Cb.)aCH3

(33) cyclopentylpropionate (cypionate) CH2CH2


-@
(34) P - phenylpropionate

orally active androgen exhibiting approxi- tivity are being utilized in patients. 7P,17a-
mately fourfold greater oral activity than that Dimethyltestosterone (calusterone, 37) and
of 17a-methyltestosterone. Early clinical 1-dehydrotestolactone (Testlac, 38) are very
studies with fluoxymesterone indicated an an- weak androgenic agents that have been used
abolic potency of 11 times that of the unhalo- in the treatment of advanced metastatic
genated derivative (301-303). Nitrogen bal- breast cancer (307-309).
ance studies, however, revealed an activity of
only 3 times that of 17a-methyltestosterone
(304). Because of the lack of any substantial
separation of anabolic and androgenic activ-
ity, halotestin is used primarily as an orally
effective androgen, particularly in the treat-
ment of mammary carcinoma (305,306).

(37) calusterone

(35) chlorotestosterone

(38) 1 - dehydrotestolactone

4.3.6 Summary of Structure-Activity Rela-


tionships. As with other areas of medicinal
chemistry, the desire to relate chemical struc-
ture to androgenic activity has attracted the
attention of numerous investigators. Al-
though it is often difficult to interrelate biolog-
ical results from different laboratories, andro-
(36) fluoxymesterone genicity data from the same laboratory afford
useful information. In evaluating the data,
4.3.5 Other Androgen Derivatives. Several one must be careful to note not only the ani-
synthetic steroids having weak androgenic ac- mal model employed, but also the mode of ad-
4 Synthetic Androgens

ministration. For example, marked differ- bard set the stage for a more thorough analy-
ences in androgenic activity can be found sis of 3-deoxy testosterone analogs by Syntex
when compounds are evaluated in the chick scientists (312,313). The relative androgenic-
comb assay (local application) as opposed to ity of the isomeric A-ring olefins of 3-deoxy
the rat ventral prostate assay (subcutaneous testosterones was the order A' > A2 > A 3 > A4.
or oral). The chick comb assay measures "local The A2-isomer displayed the greatest anabolic
androgenicity" and is believed to minimize activity and the best anabolic-to-androgenic
such factors as absorption, tissue distribution, ratio.
and metabolism, which complicate the inter- On the basis that sulfur is bioisosteric with
pretation of in vivo data in terms of hormone- C H G H , Wolff and coworkers (314) synthe-
receptor interactions. sized the thia, seleno, and tellurio andro-
Furthermore, although the rat assays cor- stanes, which displayed androgenic activity.
relate well with what one eventually finds in When the heteroatom was oxygen, however,
humans, few studies of comparative pharma- the compound (41) was essentially devoid of
cology have been performed. Indeed, DHT androgenicity (315). The oxygen analog was
may not be the principal mediator of androge- said to be inactive because oxygen is isosteric
nicity in all species. For example, a cytosol re- with CH, rather than with CH,=CH2. Thus a
ceptor protein has been found in normal and minimum ring size was found to be required
hyperplastic canine prostate that is specific for for activity. When the oxygen atom was intro-
5a-androstane-3a,17a-diol(256). duced as part of a six-membered A-ring, an
Because the presence of the l7P-hydroxyl active androgen resulted (315).
group was demonstrated very early to be an As with the case of the double-bond iso-
important feature for androgenic activity in mers, the position of the oxygen atom was
rodents, most investigators interested in -
found to be important. The substitution of ox-
structure-activity relationships maintained ygen at C-2 gives rise to the most active com-
this function and modified other parts of the pound, and the order of activity was 2 > 3 S- 4.
testosterone molecule. Three observations can As pointed out by Zanati and Wolff (315),
be made based on these studies: (1)The l-de- these and earlier results are consistent with
hydro isomer of testosterone is at least as the concept that "the activity-engendering
active as testosterone. (2) The 1- and 4-keto group in ring A is wholly steric and that, in
isomers of testosterone and DHT have vari- principle, isosteric groups of any type could be
able activity. (3) The 2-keto isomers of testos- used to construct an androgenic molecule."
terone and DHT consistently lack appreciable Further support for this idea has been ob-
activity. tained from X-ray crystallographic structure
The first attempt to ascertain the minimal determinations (316). That even the full ste-
structural requirements for androgenicity roid nucleus is not essential for activity was
was by Segaloff and Gabbard (310). Whereas shown by Zanati and Wolff (317) in the prep-
the oxygen function at position 3 could be re- aration of 7a-methyl 1,4-seco-2,3-bisnor-5a-
moved from testosterone with little reduction androstan-17P-ol(42),having 50% of the ana-
in androgenic activity, removal of the hy- bolic activity of testosterone.
droxyl group from position 17 sharply reduced
the androgenicity. As a continuation of these
studies, the hydrocarbon nucleus, 5a-andro-
stane (39),was synthesized (310). It too was
found to possess androgenicity when applied
topically or given intramuscularly in the chick
comb assay, albeit at high doses. On the other
hand, it was learned later that the 19-nor an-
alog, 5a-estrane (401, had less than 1%of the
androgenic activity of testosterone propionate
in castrated male rats (311).
Nonetheless, the work of Segaloff and Gab-
Male Sex Hormones, Analogs, and Antagonists

The effects of either 7a-methyl or 14-dehy-


dro modification are more pronounced for 19-
nortestosterone than for testosterone. The 14-
dehydro modification had a greater effect on
local androgenicity, whereas 7a-methylation
had a more positive effect on systemic andro-
genicity. A marked synergism resulted when
both the 14-dehydro and 7a-methyl modifica-
tions were present. The resultant compound,
7a-methyl-14-dehydro-19-nortestosterone,
represents one of the most potent androgens
reported to date.
The characterization of a specific receptor
protein in androgen target tissues has made it
possible to directly analyze the receptor affin-
ity of various testosterone analogs. Liao and
coworkers (320) were the first to employ this
parameter for comparison with systemic an-
drogenicity. Table 14.5 shows the receptor af-
finity and androgenic activity of a variety of
androgens relative to DHT. The ability of the
various steroids to be retained by prostate nu-
Studies by Segaloff and Gabbard (318) clei is also indicated.
illustrated the marked enhancement of andro- As would be expected, the receptor affinity
genicity achieved when a double bond was in- data did not necessarily correlate with the sys-
troduced at C-14. Both 14-dehydrotestoster- temic androgenicity. In some cases, such as
one and the corresponding 19-nor analog were with 7a-methyl-19-nortestosterone, there was
found to be potent androgens when applied
good agreement. Such was not the case, how-
topically. An extension of this series ascer-
ever, for 19-nortestosterone. Receptor binding
tained the effect of introducing a 7a-methyl
(319). The results of this study are listed in analysis of androgens was also performed by
Table 14.4, in terms of percentage increases in other groups (128, 321). Table 14.6 summa-
the weights of chick combs, rat ventral pros- rizes their findings. Whereas the importance
tates, and rat levator ani induced by the test of the All3 trans ring fusion and 17&hydroxyl
compounds as related to a similar dose of tes- prevailed, the data failed to demonstrate the
tosterone, the responses to the latter being de- potency previously noted for 7a-methyl-14-
scribed as 100%. dehydro-19-nortestosterone. Moreover, 19-nor-

Table 14.5 Relative Androgenicity and Receptor Binding Capacity of Various Androgens
Androgenicity Cytosol Nuclear
Steroid in Rat (s.c.) Receptor Retention
DHT
Testosterone (T)
5a-Androstanedione
19-NorDHT
19-NorT
7a,CH3-DHT
7d3H3-T
7P-CH3-T
7a-CH3-19-NorDHT
7a-CH,-19-NorT
4 Synthetic Androgens

Table 14.6 Binding Affinity of Various commercial preparations of testosterone pro-


Androgens for Rat Ventral Prostate pionate are administered every 2 or 3 days.
Receptor Protein Increasing the size of the ester functionality
Steroid KB (M-'1 enables the testosterone esters such as the
ethanate or cypionate to be given in a depot
5a-DHT
5P-DHT injection lasting 2-4 weeks.
17P-Testosterone Once absorbed, the steroids are trans-
17a-Testosterone ported in the circulation primarily in a pro-
Androstenedione tein-bound complex. Testosterone and other
5a-Androstanedione androgens are reversibly associated with cer-
19-Nortestosterone tain plasma proteins and the unbound frac-
14-Dehydrotestosterone tion can be absorbed into target cells to exert
14-Dehydro-19-nortestosterone its action. The structure-binding relationships
7a-CH,-14-Dehydro-19- of the natural and synthetic androgens to sex
nortestosterone steroid binding globulin (SBG) have been
extensively investigated (65-69, 74-79). A
l7a-hydroxyl group is essential for binding
testosterone displayed a receptor affinity
and the presence of a 17a-substituentsuch as
greater than that of DHT, yet its androgenicity
the l7a-methyl moiety decreases its affinity.
is much less than that of DHT.
The 5a-reduced androgens bind with the high-
These differences in correlations between
est affinity. A much smaller quantity of the
receptor assays and in vivo data should not
androgen is bound to other plasma proteins,
cloud the importance of the receptor studies.
principally albumin and corticosteroid-bind-
The receptor assays measure affinity for the
ing globulin or transcortin (CBG).
receptor protein, and this property is shared
The metabolism of the synthetic androgens
by both androgens and antiandrogens. More-
is similar to that of testosterone. The intro-
over, such assays cannot predict the disposi-
duction of the l7a-methyl group greatly re-
tion and metabolic fate of an androgen after
tards the metabolism, thus providing oral ac-
administration. Figure 14.9 contains a sum-
tivity. Reduction of the 4-en-3-one system in
mary of the structure-activity relationships
synthetic androgens to give the various a- and
for androgens.
p-isomers occurs in vivo (322, 323). Finally,
aromatization of the A-ring can also occur
4.4 Absorption, Distribution,
(151-153). The metabolism of modified andro-
and Metabolism
gens or anabolic steroids is summarized in Ta-
Numerous factors are involved in the absorp- ble 14.7. One analog that demonstrates an
tion, distribution, and metabolism of the syn- alternate metabolic pattern is 4-chlorotestos-
thetic androgens and the physicochemical
properties of these steroids greatly influence 17pesters
the pharmacokinetic parameters. The lipid enables parenteral use
solubility of a synthetic steroid is an impor-
,OH J
9a-fluoro
tant factor in its intestinal absorption. The increases activity ,
acetate ester of testosterone demonstrated en-
hanced absorption from the gastrointestinal
tract over that of both testosterone and 17a-
methyltestosterone (58). Injected solutions of
testosterone in oil result in the rapid absorp-
tion of the hormone from the injection site;
however, rapid metabolism greatly decreases
5a-reduction
the biological effects of the injected testoster- increases activity
one. The esters of testosterone are much more
nonpolar and, when injected intramuscularly, Figure 14.9. Summary of structure-activity rela-
are absorbed more slowly. As a result, the tionships for androgens.
Table 14.7 Metabolism of Modified Androgens
Substrate Products Test System Species Reference
17P-Hydroxyestra-4-en-3-one 5a-Estran-3a-01-17-one In vivo Human
5P-Estran-3a-01-17-one
plus
5a,5P-Estran-3a-ol-17-one Liver Female
5a-Estran-3,3-01-17-one homogenate rat
5a-Estrane-3a,
17P-diol
5a-Estrane-3P,
17P-diol
Estra-4-ene-3,17-dione Prostate slices Human
5a-Estran-17P-01-3-one
2-Methoxyestrone
acetate
la-Methyl-17P-hydroxyestra-4-en-3-one la-Methyl-5a-estran-3a-01-17-one In vivo Human
la-Methyl-5P-estran-3a-01-17-one
17a-Methyl-17P-hydroxyestra-4-en-3-one 17a-Methyl-5a-estran-17P-01-3-one Liver Female
17a-Methyl-5a-estrane-3a, 17P-diol homogenate rat
1701Methyltestostertone 17a-Methyl-5a-androstane-3a, 17p-diol In vivo Human
V
o
P
17a-Methyl-5P-androstane-3a, 170-diol
17a-Methyl-5a-androstane-3P, l7P-diol
17a-Methylandrost-5-ene-3P,l7p-&ol 17a-Methyltestosterone Adrenal Male
lip-Hydroxy-17a-methyltestosterone rat
17a-Methyl-l7~-hydroxyandrosta-l,4-dien-3-one l7a-Methyl-6P,17P-dihydroxyandrosta-1,4- In vivo Human
&en-3-one
la-Methyl-l7P-hydroxy-5a-androst-1-en-3-one la-Methyl-5a-androstan-3a-01-17-one In vivo Human
acetate la-methvl-5a-androst-1-ene-3.17-dione
" In vivo Human
la-methyl/compoundsabove plus
1-Methylene-5a-androstan-3a-01-17-one
la-Methyl-5a-androstane-3,17-dione In vivo Human
acetate
4-Chloro-17P-hydroxyandrost-4-en-3-one 4-Chloro-3a-hydroxyandrost-4-3en-17-one In vivo Human
17P-Hydroxy-9P,lOa-androst-4-en-3-one 9~,10a-androst-4-ene-3,17-dione Placenta Human
(retrotestosterone)
~-Homo-17a~-hydroxyandrost-4-en-3-one ~-Homo-5P-androstane-3a,
17ap-diol In vivo Human
(D-homotestosterone)
la-Methyl-5a-androstan-17P-01-3-one la-Methyl-5a-androstan-3a-01-17-one In vivo Human
la-Methyl-5a-androstan-3a,17Ppdiol
5 Anabolic Agents

terone, which in humans gave rise to an allylic 5.2 Therapeutic Uses and Bioassays
alcohol, 4-chloro-3a-hydroxyandrost-4-en-17-
Many synthetic analogs of testosterone were
one (336). A number of other halogenated tes-
prepared to separate the anabolic activity of
tosterone derivatives subsequently were
found to take this abnormal reduction path in the C,, steroids from their androgenic activ-
vitro (337). It was proposed that fluorine or ity. Although the goal of a pure synthetic ana-
chlorine substituents at the 2-, 4-, or 6- posi- bolic that retains no androgenic activity has
tion in testosterone interfere with the usual not been accomplished, numerous prepara-
+unsaturated ketone resonance so that the tions are now available on the market that
have high anabolic/androgenic ratios. Exten-
C-3 carbonyl electronically resembles a satu-
sive reviews on anabolic agents have been pub-
rated ketone.
lished (4, 10).
4.5 Toxicities The primary criterion for assessing ana-
bolic activity of a compound is the demonstra-
The use of androgens in women and children tion of a marked retention of nitrogen. This
can often result in virilizing or masculinizing nitrogen-retaining effect is the result of an in-
side effects. In boys an acceleration of the sex- crease of protein synthesis and a decrease in
ual maturation is seen, whereas in girls and protein catabolism in the body (344). Thus,
women growth of facial hair and deepening of the urinary nitrogen excretion, particularly
the voice can be observed (338, 339). These urea excretion, is greatly diminished. The cas-
effects are reversible when medication is trated male rat serves as the most sensitive
stopped; however, prolonged treatment can animal model for nitrogen retention, although
produce effects that are irreversible. Inhibi- other animals have been used (345-347). An-
tion of gonadotropin secretion by the pituitary other bioassay for anabolic activity involves
can also occur in patients receiving androgens. examination of the increase in mass of the le-
Both males and females experience salt and vator ani muscle of the rat upon administra-
water retention resulting in edema. This tion of an anabolic agent (284,285). This mea-
edema can be treated by either maintaining a sure of myotrophic effect correlates well with
low salt diet or by using diuretic agents. Liver the nitrogen-retention bioassay and the two
problems are also encountered with some of are usually performed in the determination of
the synthetic androgens. Clinical jaundice and anabolic activity.
cholestasis can develop after the use of the Anabolic steroids exert other effects on the
l7a-alkylated products (340-343). Various body as well. Skeletal mineralization and bone
clinical laboratory tests for hepatic function maturation are enhanced by androgens and
such as bilirubin concentrations, sulfobromo- anabolics (348). These agents will decrease
phthalein retention, and glutamate transami- calcium excretion by the kidney and result in
nase and alkaline phosphatase activities are increased deposition of both calcium and phos-
affected by these androgen analogs. phorous in bone. Androgenic and anabolic
agents also can influence red blood cell forma-
5 ANABOLIC AGENTS tion. Two mechanisms of action of this eryth-
5.1 Current Drugs on the Market ropoiesis involving increased erythropoietin

U.S. Chemical
Generic Name (structure) Trade Name Manufacturer Class Dose
Nandrolone decanonate (43) Deca-Durabolin Organon Estrane Injection (in oil):
100 mglml
200 mglmL
Oxandrolone (65) Oxandrin Gynex Androstane Tablets: 2.5 mg
Oxymetholone (63) Anadrol-50 Syntex Androstane Tablets: 50 mg
Stanozolol(75) Winstrol Winthrop Pharm Androstane Tablets: 2 mg
706 Male Sex Hormones, Analogs, and Antagonists

production and enhanced responsiveness of sional athletes use anabolic steroids (374),
the tissue have been described (349). which are readily available "on the street."
These various biological activities of the The use of these steroids for increasing
anabolics have prompted the use of these strength and power is banned in intercolle-
agents in treatment protocols, with varying giate and international sports, and very sensi-
success. Clinical trials have demonstrated the tive assays (RIA, GC-MS) have been developed
effectiveness of the anabolic steroids in induc- for measuring anabolic levels in urine and
ing muscle growth and development in some blood (362). Anabolic steroids also have the
diseases (38).Anabolic steroids are effective in ability to lower serum lipid levels in vivo (360,
the symptomatic treatment of various mal- 375-377). The most widely studied agent is
nourished states because of their ability to in- oxandrolone, which dramatically lowers se-
crease protein synthesis and decrease protein rum triglycerides and, to a lesser extent, cho-
catabolism. Treatment of diseases such as lesterol levels at pharmacological doses (378-
malabsorption, anorexia nervosa, emaciation, 380). The proposed mechanism of this
and malnutrition as a result of psychoses in- hypolipidemic effect includes both an inhibi-
cludes dietary supplements, appetite stimu- tion of triglyceride synthesis (381) and an in-
lants, and anabolics (350-355). Improved creased clearance of the triglycerides (382).
postoperative recovery with adjunctive use of The androgenic side effects of the anabolics
anabolic agents has been demonstrated in nu- and the lack of superiority over conventional
merous clinical studies (350, 356-360). How- hypolipidemic agents have curtailed its use for
ever, their usefulness in other diseases such as treatment of these conditions.
muscular dystrophies and atrophies and in ge- The stimulation of erythropoiesis by ana-
riatrics has not been observed. bolic~has resulted in the use of these agents
In addition, the myotrophic effects have for the treatment of various anemias (383-386).
also led to the use and the abuse of these Anemias arising from deficiencies of the bone
agents by athletes (361-364). Conflicting re- marrow are particularly responsive to phar-
ports on the effectiveness of anabolics to in- macological doses of anabolic agents. Treat-
crease strength and power in healthy males ment of aplastic anemia with anabolics and
have resulted from clinical trials. Several corticosteroids has proved effective (383-386).
groups reported no significant differences be- Secondary anemias resulting from inflamma-
tween groups of male college-age students re- tion, renal disease, or neoplasia are also re-
ceiving anabolics and weight training and sponsive to anabolic steroid administration
those groups receiving placebo plus the weight (349, 386-3901. Finally, synthetic anabolics
training in double-blind studies (365-368). have been prescribed for women with osteopo-
Other reports cited some improvement in rosis (348) and for children with delayed
strength and power, although they used small growth (381). These applications have pro-
numbers of subjects or were only single-blind duced limited success; however, the virilizing
studies (369-372). A recent study on "supra- side effects severely limit their usefulness,
physiologic" doses of anabolics has demon- particularly in children.
strated enhancement of muscle size and The methods employed to determine the
strength (373). Overall, the consensus of these anabolic or myotrophic properties of steroids
various studies are that anabolic steroids pro- have been reviewed (391). Generally, these are
vide only very limited improvement in based on an increase in nitrogen retention
strength and power in healthy males. Anabolic and/or muscle mass in various laboratory ani-
steroids also exhibit an anticatabolic effect, mals. The castrated male rat is presently the
that is, reversing the catabolic effects of glu- most widely used and most sensitive labora-
cocorticoids released in response to stress. tory animal for nitrogen balance studies (345).
Such effects would enable individuals to re- Dogs and ovariectomized monkeys have also
cover more quickly after strenuous workouts. been employed (346, 347). Although it is gen-
Even though clinical studies do not indicate erally agreed that variations in urinary nitro-
the efficacy of anabolics for healthy males, an gen excretion relate to an increase or decrease
alarming percentage of amateur and profes- in protein synthesis, nitrogen balance assays
5 Anabolic Agents

are not without their limitations (392). This is


partly because such studies fail to describe the
shifts in organ protein and measure only the
overall status of nitrogen retention in the an-
imal (393).
The easily accessible levator ani muscle of
the rat has provided a valuable index for mea-
suring the myotrophic activity of steroidal
hormones (283). By comparing the weight of (43) nandrolone R=H
levator ani muscle, seminal vesicles, and ven- (44) normethandrone R = CH3
tral prostate with controls, one can obtain a (45) norethandrone R = CH2CH3
ratio of anabolic-to-androgenic activity (283,
285). There also appears to be some correla-
tion between the levator ani response and uri- Nonetheless, these studies did stimulate
nary nitrogen retention (283). A modification the synthesis of other norsteroids. Interest-
of this muscle assay uses the parabiotic rat ingly, both l&nortestosterone (406) and
(394, 395) and allows for the simultaneous 18,19-bisnortestosterone (407) were essen-
measurement of pituitary gonadotrophic inhi- tially devoid of both androgenic and anabolic
bition and myotrophic activity. The suitability properties (408). Contraction of the B-ring led
of the levator ani assay has been questioned on to B-norsteroids, which were also lacking in
the possibility that its growth is more a result androgenicity, but unlike the foregoing, this
of androgenic sensitivity than of any steroid- modification led to compounds with antian-
induced myotrophic effect (395398). Thus drogenic activity.
this assay is usually performed in conjunction Of the number of homoandrostane deriva-
with nitrogen balance studies or acceleration tives (those having one or more additional
of body growth (399). methylene groups included in normal tetracy-
clic ring system) that have been synthesized,
5.3 Structure-Activity Relationships only B-homo (409, 410) and D-homodihy-
for Anabolics drotestosterone (411,412) have shown appre-
ciable androgenic activity. A D-bishomo ana-
5.3.1 19-Nor Derivatives. Animportant step log (46) was reported to be weakly androgenic
toward developing an anabolic agent with (413).
minimal androgenicity was taken when Hersh
berger and associates (285), and later others
(400,401), found 19-nortestosterone (l7P-hy-
droxyestr-4-en-3-one, nandrolone, 43) to be as
myotrophic but only about 0.1 as androgenic
as testosterone. This observation prompted
the synthesis and evaluation of a variety of
19-norsteroids, including the l7a-methyl
(normethandrone, 44) (402) and the l7a-ethyl
(norethandrolone, 45) (403) homologs of

Nandrolone in the form of a variety of es-


ters (such as decanoate and P-phenylpropi- 5.3.2 Dehydro Derivatives. The marked en-
onate) and norethandrolone have been widely hancement in biological activity afforded by
used clinically. The latter, under the name Ni- introduction of a double bond at C , of corti-
levar, was the first agent to be marketed in the sone and hydrocortisone prompted similar
United States as an anabolic steroid. Andro- transformations in the androgens. The ace-
genic (404) and progestational (405) side ef- tate of 17P-hydroxyandrosta-1,4-dien-3-one
fects, however, led to its eventual replacement (47) (414) was as myotrophic as testosterone
by other agents. propionate but was much less androgenic.
708 Male Sex Hormones, Analogs, and Antagonists

Furthernore, l7a-methyl-l7P-hydroxyandro- bond (51)increased androgenicity when com-


sta-1,4-dien-3-one (methandrostenolone, 48) pared with testosterone by local application in
had 1-2 times the oral potency of l7a-methyl the chick comb assay. On the other hand,
testosterone in the rat nitrogen retention there was a 25% decrease in androgenicity
(415,416) and levator ani muscle assays (417, when measured by the rat ventral prostate af-
418). In clinical studies, methandrostenolone ter subcutaneous administration. Conversion
produced a marked anabolic effect when given to the 19-nor analog (52) increased androge-
orally at doses of 1.25-10 mg daily and was nicity, but the anabolic activity was signifi-
several times more potent than 17a-methyltes- cantly enhanced (319).
tosterone (419). Of a variety of triene analogs of testoster-
one that have been tested, only 17a-methyl-
17fl-hydroxyestra-4,9,ll-trien-3-one (methyl-
trienolone, 53) showed significant activity in
rats. Surprisingly, this compound had 300
times the anabolic and 60 times the andro-
genic potency of 17a-methyltestosterone
when administered orally to castrated male
rats (426). In this instance, however, the po-
tent hormonal properties on rats did not cor-
(47) R=H relate with later studies in humans (427-429).
(48) methandrostenolone R = CH3 One study in patients with advanced breast
cancer found methyltrienolone to have weak
In contrast with the 1-dehydroanalogs, in- androgenicity and to produce severe hepatic
troduction of an additional double bond at the dysfunction at very low doses (429).
6-position (49) markedly decreased both an-
drogenic and myotrophic activity in the rat
(414, 420). Moreover, removal of the Cl,-
methyl (421),inversion of the configuration at
C, and Clo (422) and at C, and C,, (4231, and
reduction of the C,-ketone failed to improve
the biological properties (424).

On the other hand, introduction of unsat-


uration into the B, C, and D rings has given
rise to compounds with significant androgenic
or anabolic activity. Ethyldienolone (50), for
example, displayed an anabolic-to-androgenic 5.3.3 Alkylated Analogs. An extensive ef-
ratio of 5 and was slightly more active than fort has been directed toward assessing the
methyltestosterone when both were given physiological effect of replacing hydrogen with
orally (425). Segaloff and Gabbard (318) alkyl groups at most positions of the steroid
showed that introduction of a 14-15 double molecule. Although methyl substitution at C,,
5 Anabolic Agents

(54) methenolone

C,, C,, C,, C,,, and C,, has generally led to


compounds with low anabolic and androgenic
activity, similar substitutions at C,, C,, C,,
and C,, have afforded derivatives of clinical
significance.
1-Methyl-l7P-hydroxy-5a-androst-1-en-3-
one (54) as the acetate (methenolone acetate)
was about 5 times as myotrophic, but only 0.1
as androgenic, as testosterone propionate in
animals (430). In addition, this compound or
the free alcohol represented one of the few in- (55) mesterolone
stances of a C,, nonalkylated steroid that pos-
sessed significant oral anabolic activity in an- (441,443,444). The most interest in drostan-
imals (431) and in man (432). This effect may olone has been in relation to its potential as an
be related to the slow in vivo oxidation of the antitumor agent with decreased masculiniz-
17P-hydroxylgroup when compared with tes- ing propensity.
tosterone (331).At a daily dose of 300 mg, me-
thenolone acetate caused little virilization
(433) or BSP retention (434). By contrast, the
dihydro analog, la-methyl-l7P-hydroxy-5a-
androstan-&one (mesterolone, 55) was found
to possess significant oral androgenic activity
in the cockscomb test (435) and in clinical as-
says (436). A comparison of the anabolic and
androgenic activity of 54 with its A-ring con-
geners revealed that the double bond was nec-
essary at C, for anabolic activity. For example,
1a-methyl-l7~-hydroxyandrost-4-en-3-one
had a much lower activity (437). Furthermore,
either reduction of the C, carbonyl group of 55
(438) or removal of the C,, methyl group (439, 7a,17a-Dimethyltestosterone (bolaster-
440) greatly reduced both anabolic and andro- one, 58) had 6.6 times the oral anabolic po-
genic activity in this series. tency of 17a-methyltestosteronein rats (445).
Among the C,-alkylated testosterone ana- Similar activity was observed in man at 1-2
logs, 2a-methyl-5a-androstan-l7~-01-3-onemglday without many of the usual side effects
(drostanolone, 56) and its l7a-methylated ho- (446). Moreover, the corresponding 19-nor de-
molog (57) have displayed anabolic activity rivative was 41 times as active as l7a-methyl
both in animals (441) and in man (442). In testosterone as an oral myotrophic agent in
contrast, 2,2-dimethyland 2-methylenetestos- the rat (447). Segaloff and Gabbard (319)
terone or their derivatives showed only low found 7a-methyl-14-dehydro-19-nortestoster-
anabolic or androgenic activity in animals one (59) to be approximately 1000 times as
710 Male Sex Hormones, Analogs, and Antagonists

active as testosterone in the chick comb assay drogenic properties. Two striking exceptions
and about 100 times as active as testosterone to this, however, are 4-hydroxy- and lip-
in the ventral prostate assay. hydroxytestosterones.4-Hydroxy-17a-methyl-
testosteronk (oxymesterone, 61), for instance,
had 3-5 times the myotrophic and 0.5 times
the androgenic activity of l7a-methyltestos-
terone in the rat (451). In clinical studies,
oxymesterone produced nitrogen retention in
adults at a daily dose of 20-40 mg, and no
adverse liver function was observed (452,
453). Introduction of an llp-hydroxyl group
in many instances resulted in a favorable ef-
fect on biological activity. llp-Hydroxy-17a-
methyltestosterone (62) was more anabolic in
the rat than was 17a-methyltestosterone
(4541, and 1.5 times as myotrophic in humans
(455).
One of the most widely studied anabolic
steroids has been 2-hydroxymethylene-17a-
methyl-5a-androstan-17P-01-3-one (oxy-
metholone, 63).In animals it was found to be 3
times as anabolic and 0.5 times as androgenic
as 17a-methyltestosterone (456,457). Clinical
studies confirmed these results (452,
456-458).
Certain totally synthetic 18-ethylgonane
derivatives possessed pronounced anabolic ac-
tivity. Similar to other 19-norsteroids,
13P,17a-diethyl-l7P-hydroxygon-4-en-3-one
(norbolethone, 60) was found to be a potent
anabolic agent in animals and in man (449,
450). Because it is prepared by total synthesis,
the product is isolated and marketed as the
racemic DL-mixture. The hormonal activity re-
sides in the D-enantiomer.

5.3.4 Hydroxy and Mercapto Derivatives.


Testosterone has been hydroxylated at virtu-
ally every position on the steroid nucleus. For
the most part, nearly all these substances pos- The substitution of a mercapto for a hy-
sess no more than weak myotrophic and an- droxyl group has generally resulted in de-
5 Anabolic Agents

thesized and biologically evaluated. Of these


derivatives, 17~-hydroxy-17a-methyl-2-oxa-
5a-androstan-Bone (oxandrolone, 65) (464)
was 3 times as anabolic and only 0.24 times as
androgenic as 17a-methyltestosterone in the
oral levator ani assay (465). By contrast, only
minimal responses were obtained followingin-
tramuscular administration. The 2-thia (466)
and 2-aza (467) analogs were essentially de-
void of activity by both routes. The 3-aza-A-
homoandrostene derivative 66 displayed only
5% the anabolic-to-androgenic activity of
creased activity. However, the introduction of methyltestosterone (468).
a thioacetyl group at C, and C, of l7a-meth-
yltestosterone afforded la,7a-bis(acety1thio)-
17a-methyl-17~-hydroxyandrost-4-en-3-one
(thiomesterone, 64), a compound with signifi-
cant activity. Thiomesterone was 4.5 times as
myotrophic and 0.6 times as androgenic as
17a-methyltestosterone in the rat (459) and
has been used clinically as an anabolic agent
(460).

Moreover, numerous 7a-alkylthio andro-


gens have exhibited anabolic-androgenic ac-
tivity similar to that of testosterone propi-
onate when administered subcutaneously The clinical anabolic potency of oxan-
(461, 462). Even though no clinically useful drolone was considerably more active than
androgen resulted, similar 7a-substitutions 17a-methyltestosterone and provided percep-
were advantageous in the development of ra- tible nitrogen sparing at a dose as low as 0.6
dioimmunoassays now employed in clinical mglday (469). Moreover, at dosages of 0.25-
laboratories (463). In addition, certain 7a- 0.5 mglkg, oxandrolone was effective as a
arylthioandrost-4-ene-3,17-diones are effec- growth-promoting agent without producing
tive inhibitors of estrogen biosynthesis (see the androgenically induced bone maturation
Section 6.2.3). (470). Because of this favorable separation of
anabolic from androgenic effects, oxandrolone
5.3.5 Oxa, Thia, and Aza Derivatives. A has been one of the most widely studied ana-
number of androgen analogs in which an oxy- bolic steroids. Its potential utility in various
gen atom replaces one of the methylene clinical hyperlipidemias was discussed in Sec-
groups in the steroid nucleus have been syn- tion 5.1.
712 Male Sex Hormones, Analogs, and Antagonists

The significant hormonal activity noted for in animals (477) and was effective in humans
estra-4,9-dien-3-ones such as 50 (see section at a daily dose of 3-5 mg (478-480). In addi-
5.2.2) prompted the synthesis of the 2-oxa bio- tion. 17a-methyl-5a-androst-2-en-17~-ol (71)
isosteres in this series. Despite the lack of a offered a good separation of anabolic from an-
17a-methyl group, (67) had 93 times the oral drogenic activity (474,481).
anabolic activity of 17a-methyltestosterone;it
was also 2.7 times as androgenic. As might be
expected, the corresponding l7a-methyl de-
rivative, 68, was the most active substance in
this series. It had 550 times the myotrophic
and 47 times the androgenic effect of 17a-
methyltestosterone (471). These two com-
pounds differed dramatically in progesta-
tional activity, however. The activity of 67 was
only 0.1 times, whereas the activity of 68 was
100 times, that of progesterone in the Clau
berg assay (471). The pronounced oral activity
of 67 suggests that it is not a substrate for the
l7p-alcohol dehydrogenase and represents an
interesting finding.

5.3.6 Deoxy and Heterocyclic-Fused Ana-


logs. Early studies by Kochakian (472) indi-
cated that the 17P-hydroxyl group and the
3-keto group were essential for maximum an-
drogenic activity. Based on this observation,
the C, oxygen function was removed in the
hope of decreasing the androgenic potency
while maintaining anabolic activity (473). Un-
fortunately, the results failed to substantiate
the rationale, and 17a-methyl-5a-androstan-
17p-o1(69)was found to be a potent androgen Because sulfur is considered to be isosteric
in animals (474) and humans (475). However, with -CH=CH-, Wolff and Zanati (334) rea-
Wolff and Kasuya showed that this substance soned that 2-thia-A-nor-5a-androstane deriv-
is extensively metabolized to the 3-keto deriv- atives such as 72 should have androgenic ac-
ative by rabbit liver homogenate (476). Other tivity. Indeed, this compound possessed high
deoxy analogs of testosterone have been syn- androgenic and anabolic activity and served to
thesized and tested. A 19-nor derivative, 17a- verify that steric rather than electronic factors
ethylester-4-en-17P-01 (estrenol, 70) had at are important in connection with the struc-
least 4 times the anabolic and 0.25 times the tural requirements at C-2 andlor C-3 in andro-
androgenic activity of 17a-methyltestosterone gens (482). Interestingly, the selenium and
5 Anabolic Agents

tellurium isosteres in the same series were activity, as well as some beneficial effects in
found to have good androgenic activity (483, the treatment of mammary carcinoma (488).
484). Moreover, experiments with a 75Se-la- Other heterocyclic androstane derivatives
beled analog have shown 73 to selectively bind have included the pyrazoles. Thus l7p-hy-
with the specific cytosol receptor for DHT in droxy-17a-methylandrostano-(3,2-c)-pyrazole
rat prostate (485). (stanazolol, 75) was 10 times as active as 17a-
The high biological activity noted for the methyltestosterone in improving nitrogen re-
3-deoxy androstanes prompted numerous tention in rats (489). The myotrophic activity,
investigators to fuse various systems to the however, was only twice that of l7a-methyl-
A-ring. The simplest such changes were 2,3- testosterone (490). Stanazolol at a dose of 6
mglday produced an adequate anabolic re-
epoxy, 2,3-cyclopropano, and 2,3-epithioan-
sponse with no lasting adverse side effects
drostanes. The 2a3a-cyclopropano-5a-andro-
(491,492).
stan-17/3-01 was as active as testosterone The high activity of the pyrazoles insti-
propionate as an anabolic agent (486). Al- gated the synthesis of other heterocyclic-fused
though the epoxides had little or no biological androstane derivatives including isoxazoles,
activity, certain of the episulfides possessed thiazoles, pyridines, pyrimidines, pteridines,
pronounced anabolic androgenic activity oxadiazoles, pyrroles, indoles, and triazoles.
(487). For example, 2,3a-epithio-l7a-methyl- One of the most potent was l7a-methylandro-
5a-androstan-l7p-1 (74) was found to have stan-17p-01-(2,3-dl-isoxazole (androisoxazol,
approximately equal androgenic and 11 times 76), which exhibited an oral anabolic-to-
the anabolic activity of methyltestosterone af- androgenic ratio of 40 (493). The correspond-
ter oral administration to rats. The 2,3-p- ing l7a-ethynyl analog (danazol, 77) has been
episulfide, on the other hand, was much less of most interest clinically. This compound has
active. 2,3a-Epithio-5a-androstan-17P-01 has impeded androgenic activity and inhibits pitu-
been shown to have long-acting antiestrogenic itary gonadotropin secretion (494). Because it
depresses blood levels of androgens and go-
nadotropins, it has been studied as an antifer-
tility agent in males (495). At doses of 200 or
600 mg daily, danazol lowered plasma testos-
terone and androstenedione levels, and this
effect was dose related. In addition to an inhi-
bition in gonadotropin release, a direct inhibi-
tion of Leydig cell androgen synthesis was ob-
served. Other studies have shown danazol to
be effective for the treatment of endometrio-
sis, benign fibrocystic mastitis, and precocious
puberty (496). Several reports have appeared
relating to its disposition and metabolic fate
(496,497).
Male Sex Hormones, Analogs, and Antagonists

Other factors must be involved, however,


given that the potency and prolongation of
action vary markedly with the nature of the
esterifying acid.
Studies by James and coworkers (506,507)
have shed the most light on this problem.
They studied the effect of various aliphatic es-
ters of testosterone on rat prostate and semi-
nal vesicles and correlated androgenicity with
lipophilicity and rate of ester hydrolysis by
liver esterase. The peak androgenic response
was observed with the butyrate ester, which
was also the most readily hydrolyzed. The
5.3.7 Esters and Ethers. Because esterifica-
tion of testosterone markedly prolonged its ac- more lipophilic valerate ester was slightly less
tivity, it was only natural that this approach to androgenic in a quantitative sense, but its ac-
drug latentiation would be extended to the an- tion was longer lasting. It was concluded that
abolic steroids. The acyl moiety is usually de- the ease of hydrolysis controls the weight of
rived from a long-chain aliphatic or arylali the target organs, whereas lipophilicity was
phatic acid such as heptanoic (enanthoic), responsible for the duration of the androgenic
decanoic, cyclopentylpropionic, and P-phenyl- effect. These results explain the low andro-
propionic. For example, no less than 12 esters genic activity previously noted for hindered
of 19-nortestosterone (nandrolone) have been trimethylacetate (pivalate) esters, which
used clinically as long-acting anabolic agents would be expected to be resistant to in vivo
(498,499). hydrolysis.
In the case of nandrolone, the duration of The effect of etherification on anabolic or
action and the anabolic-to-androgenic ratio in- androgenic activity has been studied less rig-
creased with the chain length-of the ester orously. Replacement of the 17P-OH with
group (500, 501). The decanoate and laurate 17P-OCH, markedly reduced androgenic ac-
esters, for instance, were active 6 weeks after tivity but did not affect greatly the ability to
injection. Clinically, nandrolone decanoate ap- counteract cortisone-induced adrenal atro-
peared to be the most practical, in that a dose phy in male rats (508). A series of 17p-ac-
of 25-100 mglweek produced marked nitrogen etals (509, 510), alkyl ethers (5111, and
retention (502, 503). 3-en01 ethers (512, 513), however, showed
Because the l7a-alkyl group has been im-
significant activity when given orally (514-
plicated as the cause of the hepatotoxic side
516). The cyclohexyl en01ether of l7a-meth-
effects of oral preparations, the effect of ester-
ification on oral efficacy has attracted atten- yltestosterone, for example, was orally 5
tion. For example, esterification of dihydrotes- times as myotrophic as 17a-methyltestoster-
tosterone with short-chain fatty acids resulted one (516).
in oral anabolic and androgenic activity in rats Other ethers such as the tetrahydropyra-
(504). Moreover, esters of methenolone pos- no1 (517, 518) and trimethylsilyl (519) have
sessed appreciable oral anabolic activity (505). oral anabolic and androgenic activity in ani-
Unfortunately, follow-up studies in humans mals. The trimethylsilyl ether of testosterone
have not been reported. (silandrone) had protracted activity after in-
The manner bv " which the steroid esters jection (520) and orally had twice the anabolic
evoke their enhanced activity and increased and androgenic acivities of 17a-methyltestos-
duration of action has puzzled investigators terone (519). Solo et al. (521) evaluated a vari-
for many years. The classical concept has ety of ethers that would not be expected to be
been that esterification delays the absorp- readily cleaved in vivo and found them to be
tion rate of the steroid from the site of injec- almost devoid of anabolic and androgenic ac-
tion, thus preventing its rapid destruction. tivity. This provides additional support for the
5 Anabolic Agents

necessity of a free 17p-hydroxyl for andro- 5.4 Absorption, Distribution,


genic activity. and Metabolism
The absorption, distribution, and metabolism
5.3.8 Summary of Structure-Activity Rela- of the various anabolic steroids is quite similar
tionships. Synthetic modifications of C,, ste- to those pharmacokinetic properties of the en-
roids have resulted in the enhancement of an- dogenous and synthetic androgens discussed
abolic activity, even though a pure synthetic earlier in the chapter (522). Again, lipid solu-
anabolic agent that retains no androgenic ac- bility is critical for the absorption of these
tivity has not been accomplished. Structural agents after oral or parented administration.
changes in two regions of the testosterone The 1 'la-methyl group retards the metabolism
molecule have resulted in the greatest en- of the compounds and provides orally active
hancement of the anaboliclandrogenic ratio. agents. Other anabolics such as methenolone
are orally active without a l7a-substituent, in-
The first region is the C-17 position of the tes-
dicating that these steroids are poor sub-
tosterone molecule. Introduction of the 17a-
strates for 17a-hydroxysteroid dehydrogenase
alkyl functionality, such as a l7a-methyl or a
(523,524). Reduction of the 4-en-3-one system
l7a-ethyl group, greatly increases the meta-
in synthetic anabolics to give the various a-
bolic stability of the anabolic and decreases in
and p-isomers occurs in viuo (322). The 3-de-
vivo conversion of the 17p-alcohol to the 17-
oxy agent 17a-methyl-5a-androstan-l7a-01
ketone by 17Shydoxysteroiddehydrogenases.
was shown to be extensively converted to the
In addition, esterification of the 17p-alcohol
3-keto derivative by liver homogenate prepa-
enhances the lipid solubility of the steroids
rations (525). The metabolic fates of stanzolol
and provides injectable preparations for depot
and danazol have been reported (526, 5271,
therapy. with the major metabolites being heterocycle
The A-ring of testosterone is the second re-
ring-opened derivatives and their deaminated
gion in which structural modifications can be
products. Finally, both the unchanged ana-
made to increase anabolic activity. Removal of bolic~and their metabolites are primarily ex-
the C-19 methyl group results in the 19-nor-
creted in the urine as the glucuronide or sul-
testosterone analogs, which have slightly fate conjugates.
higher anabolic activity. A major impact on
the structure-activity relationships of ana-
5.5 Toxicities
bolic~can be observed with modifications at
the C-2 position. Bioisosteric replacement of The major side effect of the anabolic steroids is
the carbon atom at position 2 with an oxygen the residual androgenic activity of the mole-
provides a threefold increase in anabolic activ- cules. The virilizing actions are undesirable in
ity, as is seen with oxandrolone. Finally, the adult males as well as in females and children.
greatest effects were observed with the addi- In addition, many anabolic steroids can sup-
tion of heterocyclic rings fused at positions 2 press the release of gonadotropins from the
and 3 of the A-ring. The two heterocycles that anterior pituitary and lead to lower levels of
have led to the greatest changes are the pyr circulating hormones and potential reproduc-
azole and the isoxazole rings, as seen in stana- tive problems. Headaches, acne, and elevated
20101 and androisoxazole, respectively. In blood pressure are common in individuals tak-
these anabolics, the 3-ketone of testosterone is ing anabolics. The salt and water retention
replaced by the bioisosteric 3-imine. Stanazo- induced by these agents can produce edema.
101, which contains the pyrazole ring at C-2 The most serious toxicities resulting from
and C-3, shows the greatest increases when the use of anabolic steroids are subseauent
compared to testosterone. Table 14.8 com- liver damages. Liver damage including jaun-
pares the anabolic activities of nitrogen reten- dice and cholestasis can occur after use of the
tion and myotrophic activity for several com- l7a-alkylated C,, steroids (340-343). Also, in-
mon anabolics. Figure 14.10 contains a dividuals who have received anabolic agents-
summary of the structure-activity relation- over an extended period have developed he-
ships for anabolic agents. patic adenocarcinomas (528-530). Such clini-
716 Male Sex Hormones, Analogs, and Antagonists

Table 14.8 Comparison of Anabolic Activities


Anabolic Activity
Trade Nitrogen Myotrophic
Compound Number Names Retention Activity
Testosterone 1 Android-T 1.0 1.0
Malestrone
Oreton
Primotest
Virosterone
19-Nortestosterone nandrolone Nerobolil
Nortestonate
Normethandrone Methalutin
Orgasteron
Norethandrolone Nilevar
Solevar
Methandrostenolone methandienone Danabol
Dianabol
Nabolin
Nerobil
Drostanolone Drolban
Masterone
Oxymetholone Adroyd
Anadrol
Anadroyd
Anapolon
Anasterone
Nastenon
Protanabol
Synasteron
Oxandrolone Anavar
Provita
Estrenol Duraboral-0
Maxibolin
Orabolin
Orgaboral
Orgabolin
Stanazolol 75 Stanozol 10.0 7.5
Winstrol
Tevabolin
Androisoxazole 76 Androxan 1.5 1.7
Neo-ponden

cal reports serve to underscore the inherent defined as a substance that antagonizes the
risks associated with anabolic steroid use in actions of testosterone in various androgen-
amateur athletes for no demonstrable bene- sensitive target organs and, when adminis-
fits. tered with an androgen, blocks or diminishes
the effectiveness of the androgen at various
6 ANDROGEN ANTAGONISTS androgen-sensitive tissues. Androgen antago-
nists may act to block the action of testoster-
A majority of the recent research efforts in the one at several possible sites. First, such com-
area of androgens has concentrated on the pounds could interfere with the entrance of
preparation and biological activities of andro- the androgen into the target cell. A second site
gen antagonists. An androgen antagonist is of action of androgen antagonists may be to
6 Androgen Antagonists

17p-esters
Removal of 19-methyl enables parenteral use
increases activity J
lsosteric 0
increases activity
17a-alkylation
Addition of imparts oral activity
heterocycle
increases
activity
5a-reduction Figure 14.10. Summary of structure-ac-
increases activity tivity relationships for anabolic agents.

block the conversion of testosterone to its 6.2 Antiandrogens


more active metabolite dihydrotestosterone.
Third, competition for the high affinity bind-
6.2.1 Therapeutic Uses. Antiandrogens are
ing sites on the androgen receptor molecule
agents that compete with endogenous andro-
may account for antiandrogenic effects. Fi-
gens for the hormone binding site on the andro-
nally, certain agents such as LHRH agonists
can act in the pituitary to lower gonadotropin gen receptor. These agents have therapeutic po-
secretion by way of receptor downregulation tential in the treatment of acne, virilization in
and thus diminish the production of testoster- women, hyperplasia and neoplasia of the pros-
one by the testis. The substances described in tate, baldness, and male contraception,and clin-
this section act through at least one of these ical studies have demonstrated the potential
mechanisms. Several reviews on the androgen- therapeutic benefits of the antiandrogens. The
antagonists are available (531-537). applications of antiandrogens for the treatment
of prostatic carcinoma and for the treatment of
6.1 Current Drugs on the Market benign prostatic hypertrophy (BPH) have also

Antiandrogens
Generic Name U.S. Chemical
(structure) Trade Name Manufacturer Class Dose
Flutamide (91) Eulexin Schering-Plough Nonsteroidal Tablets: 125 mg
Bicalutamide (97) Casodex AstraZeneca Nonsteroidal Tablets: 50 mg
Nilutamide (93) Nilandron Aventis Nonsteroidal Tablets:
50 mg
150 mg
Cyproterone acetate (78) Androcur Schering AG Pregnane

Sa-ReductaseInhibitors
Generic Name U.S. Chemical
(structure) Trade Name Manufacturer Class Dose
Finasteride (104) Proscar Merck Androstane Tablets: 5 mg
Propecia Merck Androstane Tablets: 1 mg
71 8 Male Sex Hormones, Analogs, and Antagonists

been investigated. Antiandrogens are effective drogen receptor are chlormadinone acetate
for the treatment of prostate cancer when com- (79),medroxyprogesterone acetate (go),medro-
bined with androgen ablation, such as surgical gesterone (81), A-norprogesterone (82), and
(orchiectomy) or medical (LHRH agonist) cas- gestonorone capronate (83)(570-574). In ad-
tration. dition, medrogesterone exerts antiandrogenic
effects by inhibiting 5a-reductase and thus
6.2.2 Structure-Activity Relationships preventing the formation of DHT (575, 576).
for Antiandrogens Gestonorone capronate interferes with the up-
take process in target cells (574).
6.2.2.1 Steroidal Agents. Several steroidal
and nonsteroidal compounds with demon-
strated antiandrogenic activity have been
used clinically (533). The first compounds
used as antiandrogens were the estrogens and
progestins (538). Steroidal estrogens and di-
ethylstilbestrol are used in the treatment of
prostatic carcinoma (539-542) and exert their
action by suppression of the release of pitu-
itary gonadotropins. Progestational com-
pounds have also been used for antiandrogenic
actions with limited success (543). The inher-
ent hormonal activities of these compounds
and the development of more selective antian-
drogens have limited the clinical applications
of estrogens and progestins as antiandrogens.
A modified progestin that is a potent anti-
androgen and has minimal progestational ac-
tivity is the agent cyproterone acetate (78).
This compound was originally prepared in
search of orally active progestins, but was
quickly recognized for its ability to suppress
gonadotropin release (544-552). It was later
demonstrated that this compound also bound
with high affinity to the androgen receptor
and thus competed with DHT for the binding
site (552-555). Cyproterone acetate has re-
ceived the most clinical attention in antian-
drogen therapy (556-567). Cyproterone ace-
tate has produced quite satisfactory results in
the treatment of acne, seborrhea, and hirsut-
ism (556-562). Therapeutic effectiveness of
this agent in the treatment of prostatic carci-
noma has been reported (563-567). Cypro-
terone acetate was reported to be a good alter-
native to estrogens for the treatment of
prostate cancer when combined with andro-
gen ablation (568, 569). However, this combi-
nation did not improve disease-free survival or
overall survival when compared to castration
alone. Several androstane derivatives demon-
Other pregnane compounds that exhibit strate antiandrogenic properties. 17a-Methyl-
antiandrogenic actions by binding to the an- B-nortestosterone (84) was prepared and
6 Androgen Antagonists

A-ring antiandrogens such as zanoterone


(WIN 49,596) (88)further support these con-
clusions on the structure-activity relation-
ships of steroidal antiandrogens. Additional
A-ring heterocycles identified as novel antian-
drogens are the thiazole (89)and oxazole (90)
(592, 593). The optimal substitutions on the
A-ring heterocyclic androstanes for in vivo an-
tiandrogenic activity are the methylsulfonyl
group at the N-1' position and a l7a-substitu-
ent (e.g., l7a-methyl or l7a-ethinyl).

tested in 1964 for antihormonal activity (577).


Within the next decade, several other andro-
stane analogs were prepared and found to
possess antiandrogenic activity (578-5841, in-
cluding BOMT (85),R2956 (861, and oxend-
olone (TSAA291,87). As expected, the mech-
anism of antiandrogenic action of these
synthetic steroids is the competition with an-
drogens for the binding sites on the receptor
molecule (585-589). Numerous A- and B-ring-
modified steroids were examined for antian-
drogenic activity and the ability to bind to the 6.2.2.2 Nonsteroidal Agents. The absolute
androgen receptor (590, 591), demonstrating requirement of a steroidal compound for in-
that the structural requirements of a receptor teraction with the androgen receptor was
binding site can accommodate some degree of invalidated when the potent nonsteroidal
flexibility in the A- andlor B-rings of antian- antiandrogen flutamide (Eulexin, 91) was in-
drogenic molecules. Heterocyclic-substituted troduced (594, 595). Subsequent receptor
Male Sex Hormones, Analogs, and Antagonists

activity relationships of flutamide and analogs


are the presence of an electron-deficient aro-
matic ring and a powerful hydrogen bond do-
nor group.

Flutamide has been extensively evaluated


for the treatment of prostate cancer. Large
double-blind studies in prostate cancer pa-
tients using a combination of flutamide with
an LHRH agonist (as a medical castration) re-
sulted in an increased number of favorable re-
sponses and increased overall survival when
compared to an LHRH agonist or surgical cas-
tration (599,600).
Nilutamide (Anandron, 93), related nilut-
amide analogs (94-96)(94)(95)(96), and bica-
lutamide (Casodex, 97) are other nonsteroidal
antiandrogens with a similar electron-defi-
cient aromatic ring and have been shown to
interact with the androgen receptor to varying
degrees (538, 601). Nilutamide and bicaluta-
mide are pure antiandrogens and are effective
in suppressing testosterone-stimulated cell
proliferation (602). Both nilutamide and bica-
lutamide demonstrate effectiveness against
prostate cancer (603-607).

studies (589,596,597) demonstrated that this


compound competed with DHT for the bind-
ing sites. The side chain of flutamide allows
sufficient flexibility for the molecule to as-
sume a structure similar to that of an andro-
gen. In addition, a hydroxylated metabolite
(92) has been identified, is a more powerful Other aryl substituted nonsteroidal com-
antiandrogen in vivo, and has a higher affinity pounds have also been identified as antiandro-
for the receptor than for the parent compound gens. DIMP (98) is a phthalimide derivative
(589,598). Important factors in the structure- that showed weak affinity for the androgen
6 Androgen Antagonists

receptor and poor in vivo activity (589,608). A


series of tetrafluorophthalimides such as (99)
demonstrated moderate activity as antiandro-
gens in cell proliferation assays (609).
6.2.3 Absorption, Distribution, and Metabo-
lism. The steroidal antiandrogens exhibit
pharmacokinetic properties similar to those of
the androgens and anabolic agents. The li-
pophilicity of the compounds influences ab-
sorption both orally and from injection sites
(611-615). Reduction of the 3-ketone and 4,5
double bond are common routes of metabo-
lism (611). An unusual metabolite of cypro-
(98) DIMP terone acetate, 15a-hydroxycyproterone ace-
tate, was isolated and identified in both
animals and man (616). The nonsteroidal an-
tiandrogen flutamide is rapidly absorbed and
extensively metabolized in vivo (617,618). As
described earlier, the hydroxy metabolite (92)
of flutamide is a more potent antiandrogen
(589, 598). The major metabolite of bicaluta-
mide is the sulfone, which has comparable in
vivo activity (619). Finally, the antiandrogens
are primarily excreted as the glucuronide and
sulfate conjugates in the urine.

A series of 1,2-dihydropyridono[5,6-glquino- 6.2.4 Toxicities. Side effects of these


lines were identified as novel nonsteroidal anti- agents have been identified from various clin-
androgens based on a cell-based screening ical trials. Testicular atrophy and decreased
approach (610). Several analogs (100-103) spermatogenesis have been observed during
demonstrated excellent in vivo activity, reduc- treatment with cyproterone acetate (620,
ing rat ventral prostate weight without af!fec.ting 621). Antiandrogens can also impair libido and
serum gonadotropins and serum testosterone result in impotency (622). Certain antiandro-
levels. gens such as cyproterone acetate and medro-
Male Sex Hormones, Analogs, and Antagonists

gesterone also exhibit inherent progestational 6.3.1 5a-Reductase Inhibitors. The most
activity, suppress corticotropin release, and extensively studied class of 5a-reductase in-
have some androgenic effects (623-625). No hibitors is the 4-azasteroids (629), which in-
hormonal activities were observed for the non- clude the drug finasteride (Proscar, 104). Fin-
steroidal antiandrogens, such as flutamide asteride is the first 5a-reductase inhibitor
(618). On the other hand, many nonsteroidal approved in the United States for the treat-
antiandrogens exhibit other endocrine side ef- ment of benign prostatic hyperplasia (BPH).
fects, such as elevated serum gonadotropins This drug has approximately a 100-fold
and serum testosterone levels. Gynecomastia, greater affinity for Type 2 5a-reductase than
nausea, diarrhea, and liver toxicities have for the Type 1 enzyme, demonstrating an IC,,
been observed in patients on nonsteroidal an- value of 4.2 nM for Type 2 5a-reductase (630).
tiandrogens (626,627). Also, resistance to an- In humans, finasteride decreases prostatic
tiandrogen therapy has been observed in pros- DHT levels by 70-90% and reduces prostate
tate cancer patients (628). size (6311, whereas testosterone tissue levels
increased. Clinical trials demonstrated sus-
tained improvement in BPH disease and
6.3 Enzyme Inhibitors
reduction in PSA levels (632,633). Related an-
Enzymes involved in the biosynthesis and me- alogs (105-107) have also demonstrated effec-
tabolism of testosterone are attractive targets tiveness in vitro and in vivo (634-636). These
for drug design and drug development. Sup- agents were originally designed to mimic the
pression of the synthesis of androgenic hor- putative 3-enolate intermediate of testoster-
mones and androgen precursors is a viable one and serve as transition-state inhibitors
therapeutic approach for the treatment of var- (535, 536). Subsequently, finasteride was
ious androgen-mediated disease processes and shown to produce time-dependent enzyme in-
an important endocrine treatment for pros- activation (637) and function as a mechanism-
tate cancer. Potent inhibition of Type 2 5a- based inactivator. The structure-activity rela-
reductase in androgen target tissues and the tionships for the Cazasteroids illustrate the
resultant decrease in DHT levels will provide stringent requirements for inhibition of hu-
selective interference with androgen action man Type 2 5a-reductase (638). The 5a-re-
- tissues and no alterations
within those target duced azasteroids are preferred, a 1,2-double
of other effects produced by testosterone, bond can be tolerated, and the nitrogen can be
other structurally related steroids, and other substituted with only hydrogen or small li-
hormones such as corticoids and progester- pophilic groups. Lipophilic arnides or ketones
one. The cytochrome P-45O1,, enzyme com- are preferred as substituents at the C-17p
plex displays two enzymatic activities: l7a-hy- position.
droxylation, to produce 17a-hydroxysteroids;
and Cl,-C,, bond cleavage (17,204yase activ-
ity), to produce androgens. In the male, this
enzyme is found in both testicular and adrenal
tissues, with these organs providing circulat-
ing androgens in the blood. Effective inhibi-
tion of this microsomal enzyme complex would
eliminate both testicular and adrenal andro-
gens and remove the growth stimulus to an-
drogen-dependent prostate carcinoma. Syn-
thetic androgen analogs that inhibit the
oxidative metabolism of androgens testoster- (104) finasteride
one and androstenedione to estrogens estra-
diol and estrone can serve as potential thera- Several 8azasteroids, such as (108) and
peutic agents for controlling estrogen- (1091, were prepared as extended mimics of the
dependent diseases such as hormone- enolate transition state and have also demon-
dependent breast cancer. strated potent inhibition of 5a-redudase (639).
6 Androgen Antagonists

Lipophilic groups
are preferred
,

3-keto-4-aza can
be replaced with
3-ene-3-COOH
\
0
7j3-methyl tolerated
R --- H or small alkyl groups
are preferred
Figure 14.11. Summary of structure-activity rela-
tionships for 5a-reductase inhibitors.

The 6-azasteroids are more effective inhibitors


of Type 2 5a-reductase, but some analogs also
exhibit good inhibition of Type 2 5a-reductase.
Alkylation of the nitrogen can be tolerated; how-
ever, a l,&double bond decreases inhibitory ac-
tivity in this series. The best inhibitors contain Androstadiene 3-carboxylicacids (110)and
large lipophilic substituents at the C-17p posi- (111)were recently designed as transition-
tion. Figure 14.11 contains a summary of the state inhibitors and have demonstrated potent
structure-activityrelationships for steroidal 5a- uncompetitive inhibition of Type 2 5a-reduc-
reductase inhibitors. tase (640, 641). Epristeride (SK&F 105,657,
724 Male Sex Hormones, Analogs, and Antagonists

110)has demonstrated the ability to lower se-


rum DHT levels by 50% in clinical trials (642,
643). Other analogs with acidic functionalities
at the C-3 position include other androstene
carboxylic acids (112, 113) and estratriene
carboxylic acids (114) (644). The allenic sec-
osteroid (115) has demonstrated potent ir-
reversible inhibitor of 5a-reductase, even
though it was originally developed as an irre-
versible inhibitor of 3P-hydroxysteroid dehy- HOOC
dr~genase/A~,~-isomerase (645-647). Finally, H
selective and potent inhibitors of Type 1 5a-
reductase were developed based on the 4-aza- (113)
cholestane MK-386 (116) (648).

6.3.2 17,204yase Inhibitors. Both nonste-


roidal and steroidal agents have been exam-
ined as inhibitors of 17a-hydroxylase/l7,20-
lyase. The nonsteroidal agents studied most
extensively are aminoglutethimide (120) and
Several nonsteroidal 5a-reductase inhibi- ketoconazole (121), both in vitro and in clini-
tors have developed based on the azasteroid cal trials. Objective response rates for treat-
molecule or from high throughput screening ment of prostate cancer in relapsed patients
methods (535,536). Examples of these nonste- were observed with high doses of aminoglu-
roidal inhibitors include the benzoquinolin- tethimide (652) and high doses of ketocona-
one (117), an aryl carboxylic acid (118),and zole (653), but both agents produce frequent
FK143 (119)(649-651). side effects. A third nonsteroidal agent that
6 Androgen Antagonists

and pyridylacetic acid esters (658). In general,


high doses of nonsteroidal agents are needed
to produce significant in vitro or in vivo activ-
ity. Another potential problem with these
agents is nonspecific inhibition of other cyto-
chrome P-450 enzymes involved in either ste-
roidogenesis or liver metabolism.

(120) aminoglutethimide

cl'
(121) ketoconazole

(122) liarozole

A few studies of steroidal inhibitors of 17a-


hydroxylase/l7,20-lyase have been reported.
An extensive analysis of the specificity of ste-
roid binding to testicular microsomal cyto-
chrome P-450 identified several steroids ex-
hibiting binding aMinity (659). One of these,
has received extensive preclinical evaluation promegestrone (123), has been used in kinetic
is the benzimidazole analog, liarozole (1221, analysis of purified cytochrome P-450,,,
which produced reduction in plasma testoster- (660). An affinity label inhibitor, 17-bromoace-
one and androstenedione levels in vivo (654). toxyprogesterone (124), alkylates a unique
Other nonsteroidal agents reported to exhibit cysteine residue on purified cytochrome
17a-hydroxylase/l7,20-lyaseinhibitory activ- P-450,,, (661). %Potentialmechanism-based
ity in vitro include other imidazole analogs inhibitors include 17p-(cyclopropy1amino)-5-
(6551, nicotine (6561, bifluranol analogs (657), androsten-3p-01 (125; (662)) and l7pvinyl-
Male Sex Hormones, Analogs, and Antagonists

progesterone (126); (663). To date, all of these


inhibitors exhibit apparent Ki' values in the
micromolar ( p M l range, whereas the apparent
K, for progesterone is 140 nM.The 17P-aziri-
dinyl analog (127) and 17ppyridyl derivative
(128)also exhibited similar inhibitory activity
(664,665).

of aromatase has been an attractive approach


for examining the roles of estrogen biosynthe-
sis in various physiological or pathological
processes. Furthermore, effective aromatase
inhibitors can serve as potential therapeutic
agents for controlling estrogen-dependent dis-
eases such as hormone-dependent breast can-
6.3.3 C,, Steroids as Aromatase Inhibitors. cer. Investigations on the development of aro-
Aromatase is the enzyme complex that cata- matase inhibitors began in the 1970s and have
lyzes the conversion of androgens into the es- expanded greatly in the past two decades. Re-
trogens. This enzymatic process is the rate- search summaries of steroidal and nonsteroi-
limiting step in estrogen biosynthesis and .
dal aromatase inhibitors have been resented
converts C,, steroids, such as testosterone at the three international aromatase confer-
and androstenedione, into the C18 estrogens, ences (666 -668) and several reviews have also
estradiol and estrone, respectively. Inhibition been published (669-676).
6 Androgen Antagonists

Steroidal inhibitors that have been devel-


oped to date build on the basic androstenedi-
one nucleus and incorporate chemical sub-
stituents at varying positions on the steroid.
These inhibitors bind to the aromatase cyto-
chrome P-450 enzyme in the same manner as
that of the substrate androstenedione. Even
though the steroidal aromatase inhibitors are
C,, steroids, these agents exhibit no signifi-
cant androgenic activity. A limited number of
effective inhibitors with substituents on the
A-ring have been reported. Several steroidal
aromatase inhibitors contain modifications at
the C-4 position, with 4-hydroxyandrostenedi-
one (129) (4-OW,forrnestane) being the pro-
totype agent. Initially, 4-OHA was thought to
be a competitive inhibitor, but was later dem-
onstrated to produce enzyme-mediated inacti-
vation (677-679). In vivo, 4-OHA inhibits
reproductive process (680) and causes regres-
sion of hormone-dependent mammary rat tu-
mors (681, 682). 4-OHA is effective in the
treatment of advanced breast cancer in post-
menopausal women (683-685), and this drug More extensive structural modifications
is approved in the United Kingdom for breast may be made on the B-ring of the steroid nu-
cancer therapy. Thus, the spacial require- cleus. Bulky substitutions at the C-7 position
ments of the A-ring for binding of the steroidal of the B-ring have provided several very po-
inhibitor to aromatase are rather restrictive, tent aromatase inhibitors (687). 7~144'-
permitting only small structural modifications Amino)phenylthio-4-androstene-3,17-dione
to be made. Incorporation of the polar hy- 116 (7a-APTA)is a very effective competitive
droxyl group at C-4 enhances inhibitory activ- inhibitor, with an apparent Ki of 18 nM. This
ity. 1-Methyl-1,4-androstadiene-3,17-dione inhibitor has also demonstrated effectiveness
(130) is a potent inhibitor of aromatase in in inhibiting aromatase in cell cultures (689,
vitro and in vivo (686); on the other hand, 690) and in treating hormone-dependent rat
bulky substitutents at the la-position are mammary tumors (690, 691). Evaluation of
poor inhibitors (687). At the C-3 position, re- various substituted aromatic analogs of 7a-
placement of the ketone with a methylene pro- APTA provided no correlation between the
vided effective inhibitors (688). electronic character of the substituents and
inhibitory activity (692).Investigations of var-
ious 7-substituted 4,6-androstadiene-3,17-
dione derivatives (693, 694) suggest that only
those derivatives that can project the 7-aryl
substitutent into the 7a pocket are effective
inhibitors. Overall, the most effective B-ring-
modified aromatase inhibitors are those with
7a-aryl derivatives, with several analogs hav-
ing 2-10 times greater affinity for the enzyme
than that of the substrate. These results sug-
gest that additional interactions occur be-
tween the phenyl ring at the 7a-position and
amino acids at or near the enzymatic site of
aromatase, resulting in enhanced affinity.
728 Male Sex Hormones, Analogs, and Antagonists

Numerous modified androstenedione ana-


logs have been developed as effective mecha-
nism-based inhibitors of aromatase. The first
compound designed as a mechanism- based in-
hibitor of aromatase was lO-propargyl-4-
estrene-3,17-dione (132) (PED; MDL 18,962);
it was synthesized and studied independently
by three research groups (695-697). MDL
18,962 has an electron-rich alkynyl function
on the C-19 carbon atom, the site of aro-
matase-mediated oxidation of the substrate.
Although the identity of the reactive interme-
diate formed is not known, an oxirene and a
Michael acceptor have been suggested. This
agent is an effective inhibitor in vitro and in
vivo (697-702). Other approaches to C-19 sub-
stituted mechanism-based inhibitors contain-
ing latent chemical groups have provided a
limited number of inhibitors. These agents in-
clude the difluoromethyl analog (133) (703)
and a thiol(134) (704, 705). Another series of
mechanism-based inhibitors have developed
from more detailed biochemical investigations
of several inhibitors originally thought to be
competitive inhibitors. These inhibitors can
be grouped into the general categories of
4-substituted androst-4-ene-3,17-diones such
as 4-hydroxy-androstenedione (129) (679),
substituted androsta-1,4-diene-3,17-diones
such as 7a-(4'-amino)-phenylthioandrosta-4,6-
diene-3,17-dione (7a-APTADD; 135)(706), and
6-methyleneandrost-4-ene-3,17-dione (exemes-
tane; 136) (707). Exemestane (Aromasin) is
marketed as second-line therapy for the treat-
ment of breast cancer patients who failed ta-
moxifen and is effective as first-line therapy in
women with advanced breast cancer.

els in vitro is the nonsteroidal agent gossypol.


Gossypol, (2,2'-binaphthalene)-8,8'-dicarbox-
a1dehyde-1,1',6,6',7,7'-hexahydroxy-5,5'-di-
isopropyl-3,3'-dimethyl (137) is a polyphe-
nolic compound contained in the pigment of
6.3.4 Other Agents. An interesting natural cottonseed. This natural product has been
product that lowers circulating androgen lev- used in fertile men in China as an effective
7 Summary

male contraceptive agent for many years (708, the testes under the stimulation of the gonad-
709); its antifertility effects on reproductive otropin LH. A critical aspect of testosterone
endocrine tissues are observed at 1000 times and its biochemistry is that this steroid is con-
lower doses than its toxic effects in other tis- verted in various cells to other active steroidal
sues. Gossypol has been shown to disrupt agents. The reduction of testosterone to dihy-
spermatogenesis by inhibiting lactate dehy- drotestosterone is necessary for the andro-
drogenase-X (LDH-X) (710, 711), to interfere genic actions of testosterone in androgen tar-
with steroidogenesis in testicular Leydig cells get tissues such as the prostate. On the other
(712-714), and to hinder the function of pri- hand, oxidation of testosterone by the enzyme
mary cultures of Leydigand Sertoli cells (715). aromatase to yield the estrogens is crucial for
In addition, gossypol is also capable of altering certain CNS actions. Investigations of these
steroid biosynthesis in the female reproduc- enzymatic conversions of circulating testos-
tive systems (716-719). The antisteroidogenic terone continues to be a fruitful area of bio-
effect of gossypol in cultured bovine luteal chemical research on the roles of the steroid
cells involves suppression of the activities of hormones in the body. Additionally, the eluci-
adenylate cyclase and 3P-hydroxysteroid de- dation of the mechanism of action of the an-
hydrogenase (3P-HSD) (720). Reproductive drogens in various target tissues receives on-
endocrine tissues are also sensitive to metab- going emphasis. The androgenic actions of
olites of gossypol, such as gossypolone. Gossy- testosterone are attributed to the binding of
polone, a major metabolite formed by gossypol dihydrotestosterone to its nuclear receptor,
oxidation, inhibits both 3p-HSD and cyto- followed by dimerization of the receptor com-
chrome P-450s,, activities in cultured bovine plex and binding to a specific DNA sequence.
luteal cells (721) and suppresses adrenocorti- This binding of the homodimer to the andro-
cotropic hormone-induced corticosterone se- gen response element leads to gene expres-
cretion in cultured rat adrenocortical cells at a sion, stimulation of the synthesis of new
similar potency as gossypol (722). Gossypol mRNA, and subsequent protein biosynthesis.
metabolites have also demonstrated inhibi- Other actions of testosterone, particularly the
tory action on hCG-induced testosterone pro- anabolic actions, appeared to be mediated
duction in young male rats (718). The major through a similar nuclear receptor-mediated
side effects of gossypol therapy are fatigue, mechanism. Many of the intricate biochemical
gastrointestinal upset, weakness, and hypoka- events that occur during the action of the an-
lemia, which thus limit its therapeutic useful- drogens in their target cells remain for further
ness (723). clarification. Nevertheless, receptor studies of
new agents are an important biological tool in
CHO OH OH CHO the evaluation of the compounds for later, in-
depth pharmacological testing.
The synthetic androgens and anabolics
were prepared to impart oral activity to the
HO androgen molecule, to separate the andro-
genic effects of testosterone from its ana-
bolic effects and to improve on its biological
activities. These research efforts have pro-
vided several effective drug preparations for
7 SUMMARY the treatment of various androgen-deficient
diseases, for the therapy of diseases charac-
The steroid testosterone is the major circulat- terized by muscle wasting and protein catab-
ing sex hormone of the male and serves as the olism, for postoperative adjuvant therapy,
prototype for the androgens, the anabolic and for the treatment of certain hormone-
agents, and androgen antagonists. The endog- dependent cancers. The synthetic anabolics
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lesterol in various tissues in the body; the ma- agents in athletics. Finally, the most recent
jority of the circulating androgens are made in area of research emphasis is the develop-
Male Sex Hormones, Analogs, and Antagonists

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CHAPTER FIFTEEN

Anti-Inflammatory Steroids
MITCHELL A. AVERY
JOHN R. WOOLFREY
University of Mississippi-University
Department of Medicinal Chemistry, School of Pharmacy
University, Mississippi

Contents
1 Introduction, 748
2 Clinical Use of Anti-Inflammatory Steroids, 751
2.1 Topical Corticosteroids Currently Available,
751
3 Adverse Effects Associated with the Use of Anti-
Inflammatory Steroids, 751
4 Effects on Absorption, 756
4.1 Intestinal Absorption, 756
4.2 Percutaneous Absorption, 759
4.2.1 16aJ7a-Ketals, 761
4.2.2 Esters, 761
4.3 Intra-Articular Administration, 767
4.4 Water-Soluble Esters, 768
4.5 Corneal Penetration, 771
5 Effects on Drug Distribution, 771
6 Glucocorticoid Biosynthesis and Metabolism, 773
7 Mechanism of Action of
Anti-Inflammatory Steroids, 776
7.1 Glucocorticoid Receptor Structure, 776
7.2 Mechanism of Action, 776
8 Effects on Drug Receptor Affinity, 780
9 Effect of Structural Change
on Pharmacological Action, 786
9.1 Pharmacological Tests, 786
9.2 QSAR Analyses of Pharmacological Action,
786
9.2.1 Use of De Nouo Constants, 787
9.2.2 Hansch-Type Analyses, 788
9.2.3 Use of Neural Networks to Predict
Corticoid Properties, 791
9.3 Some Steric and Electronic Factors Affecting
Anti-Inflammatory Activity, 792
Burger's Medicinal Chemistry and Drug Discovery 9.3.1 Effect of Structural Change on Steroid
Sixth Edition, Volume 3: Cardiovascular Agents Conformation, 792
and Endocrines 9.3.2 Effects of Structural Change on
Edited by Donald J. Abraham Electronic Characteristics of the
ISBN 0-471-37029-0 O 2003 John Wiley & Sons, Inc. Steroid, 793
747
748 Anti-Inflammatory Steroids

9.3.3 3D-Quantitative Structure-Activity 10.8 Alterations at C-7,808


Relationships of Steroids, 793 10.9 6,7-DisubstitutedCompounds, 811
9.4 Summary of the Results 10.10 Alterations at C-8,811
of Theoretical Studies, 796 10.11 Alterations at C-9,811
10 Effect of Individual Structural Changes on 10.12 Alterations at C-10,811
Anti-Inflammatory Activity, 796 10.13 Alterations at C-11,811
10.1 Overview of Major Changes 10.14 Alterations at C-12,813
in Anti-Inflammatory Steroids, 796 10.15 Alterations at C-15,816
10.2 Skeletal Changes in the Cortisol Molecule, 10.16 Alterations at C-16,816
796 10.17 Alterations at C-17,820
10.3 Alterations at C-1,804 10.18 Alterations at C-20,826
10.4 Alterations at C-2,804 10.19 Alterations at C-21,826
10.5 Alterations at C-3, 804 11 Antiglucocorticoids, 836
10.6 Alterations at C-4,806 12 Nonsteroidal Glucocorticoids, 844
10.7 Alterations at C-6,807 13 Acknowledgments,845

1 INTRODUCTION

Since the introduction of cortisone (1948) and


hydrocortisone (1951),anti-inflammatory ste-
roids have remained an important and unre-
placed drug class. Although not without
adverse effects, these compounds have contin-
ued to be the "drug of choice" in the treatment
of afflictions ranging from the moderate skin
rash to severe acute inflammatory disorders.
The adrenal cortex releases both mineralo-
corticoids and glucocorticoids, the latter of
which includes corticosterone, cortisol (2),
by the adrenal cortex, the primary endoge-
nous function of glucocorticosteroids is in in-
fluencing carbohydrate and protein metabo-
lism.
Glucocorticoids and their metabolites (1)
were recognized early as possessing powerful
anti-inflammatory and immunomodulatory
properties. Even before 1950, reports of the
antiarthritic properties of cortisone (1) by
Hench and coworkers (2) indicated the poten-
tial for these com~ounds
* to reduce the suffer-
ing of patients with inflammatory diseases.
This, combined with the first synthesis of nat-
urally occurring glucocorticoids (ll-desoxy-
corticosterone), led not only to the massive in-
crease in research in the area of steroid
synthesis and physiology, but to a Nobel prize
and cortisone (1).The generation of glucocor- in 1950 for early steroid pioneers Hench,
ticosteroids is intricately balanced: when the Reichstein, and Kendall.
production and regulatory process malfunc- Progress in the field took an additional leap
tions, the result is either an excess (e.g., Cush- forward with the developement of synthetic
ing's syndrome) or a deficiency (e.g., Addison's steroids exhibiting activities far greater than
disease) in glucocorticoid levels. Once released those of the natural hormones. The synthesis
1 Introduction

the GR-drug complex, the synthesis of anti-


inflammatory proteins is either inhibited or
stimulated.
Although limited structural information on
the glucocorticoid and other hormone recep-
tors is available, quantitative structure-activ-
ity relationship (QSAR) studies have at-
tempted to define a pharmacophore model for
the anti-inflammatory activity. Approaches
that have been applied are the use of conven-
tional Hansch as well as de novo constants for
regression analysis, neural networks, compar-
ative molecular field analysis (CoMFA), and
principal-component (PC) analysis with PLS
of 9a-fluorocortisol (a), described in 1953 by (partial least squares) analysis. These studies
Fried and Sabo (3), opened the way for many have provided predictive models for glucocor-
more highly active anti-inflammatory agents, ticoid binding that could in principle be used
and indeed some of todav's " most active anti- in drug design efforts.
inflammatory agents have some semblance to Comprehensive reviews dealing with the
this 9a-fluorinated steroid. pharmacological structure-activity relation-
-

We know today that structural changes in ships of anti-inflammatory steroids have been
steroids can bring about potency alterations in published (4-6) as well as reviews of the
animal pharmacology through a number of mechanism of action (7, 8). Almost all anti-
mechanisms. The processes that are affected inflammatory steroids are based on the preg-
include pharmacokinetic parameters such as nane (4) and 17-substituted androstane ring
drug absorption and drug distribution (dis- system.
cussed elsewhere), as well as drug metabolism
to more or less active metabolites (discussed
elsewhere). There is now a large body of liter-
ature dealing with these phenomena that
makes predictive thinking possible in these ar-
eas. Other mechanisms that are involved in
the effect of structural changes on pharmaco-
logical activity in steroids include the effect of
steroid structure on receptor affinity and the
manner in which changes in the steroid ago-
nist affect intrinsic activity through alter-
ations in gene expression. Steroid-binding
proteins and receptors have been studied in
detail over the last two decades but have
eluded crystallographic analysis. The glu- Abbreviated names for compounds em-
cocorticoid receptor (GR) is a cytoplasmic pro- ployed in this chapter are as follows:
tein of complex structure that is associated
with various heat shock proteins (e.g., hsp90). Cortisone (1):17,21-Dihydroxypregn-4-ene-
When activated by agonistic binding of drug to 3,11,20-trione
the C-terminal region of the GR, the GR-drug Cortisol or hydrocortisone (2): 1lp,17,21-Tri-
complex undergoes translocation to the nu- hydroxypregn-4-ene-3,20-dione
cleus. The active form of the GR-drug complex Hydrocortisone esters (2a-20: llp,21-Dihy-
is thought to be dimeric in its binding to the droxy-17-(1-oxoalky1oxy)-prep-4-ene-
DNA sequence, termed the glucocorticoid re- 3,20-dione
sponse element (GRE). Upon binding to the 9a-Fluorocortisol(3):9-Fluoro-11p, 17,2141-i-
GRE of the DNA by the "zinc finger" region of hydroxypregn-4-ene-3,20-dione
750 Anti-Inflammatory Steroids

Betamethasone (5):9-Fluoro-llp,l7,21-trihy- Aclomethasone dipropionate(18): 7a-Chloro-


droxy-16P-methylpregna-l,4-diene-3,20- 11P-hydroxy- l6a-methyl-17a,2l-bis(l-oxo-
dione propanoxy)-pregna-1,4-diene-3,20-dione
Clobetasol propionate(6):21-Chloro-9-fluoro- Desonide(19): 11/3,21-Dihydroxy-16a, 17-[(I-
11~-hydroxy-16~-methyl-17-(l-oxoprop- methylethylidene)bis(oxy)l-pregna-1,4-
om)-pregna-l,4-diene-3,20-dione diene-3,20-dione
Diflorasone diacetate (7): 17,21-Bis(1-oxoeth- Dexamethasone:9-Fluoro-lla,17,21-trihydroxy-
anoxy)-6a,9-difluoro-1lp-hydroxy-l6P-methyl- 16a-methylpregna-1,4diene-3,2@dione
pregna-1,4-diene-3,20-dione Prednisolone : 1 lp,l7,21-Trihydroxypregna-
Halobetasol propionate(8): 21-Chloro-6a,9- 1,4-diene-3,20-dione
difluoro-1lp-hydroxy-16gmethyl-17-(1-0x0- Fluoromethalone(22): 9-Fluoro-1 lp,17-dihy-
propoxy)-pregna-l,4-diene-3,20-dione droxy-6a-methylpregna-1,4-diene-3,20-dione
Amcinonide(9): 16,17a-[Cyclopentylidene- Rimexolone(23): 16a,17-Dimethyl-1 lp-hy-
bis(oxy)l8-fluoro-llp-hydroxy-21-(1-oxoeth- droxy-17P-(1-oxopropy1)-andro-
oxy)-pregna-l,4-diene-3,20-dione sta-1,4-dien-3-one
Desoximetasone(10): llp,21-Dihydroxy-9- Medrysone(24): llp-Hydroxy-6a-methyl-
fluoro-16gmethylpregna-l,4-diene-3,20- pregna-l,4-diene-3,20-dione
dione Beclomethasone dipropionate (25):9-Chloro-
Fluocinolone(a): 6a,9-Difluoro-1 lp,l6a,17,21- 11p-hydroxy-16p-methyl-17,2l-bis(1-oxopro-
tetrahydroxypregna-1,4-diene-3,20-dione panoxyl-pregna-l,4-diene-3,20-dione
Fluocinolone acetonide (llb): 6a,9-Difluoro- Flunisolide(26): 6a-Fluoro-llp,21-dihy-
11P,21-dihydroxy-l6a,17-[(I-methylethyl- droxy-16a,17-[(I-methylethylidene)bis(oxy)]-
idene)bis(oxy)]-pregna-1,4-diene-3,20- pregna-1,4-diene-3,20-dione
dione Budesonide(27): 16a,17-[Butylidine-
Fluocinonide (llc): 6a,9-Difluoro-llp-hy- bis(oxy)]-llP,21-dihydroxy-pregna-l,4-diene-
droxy-16a,17-[(l-methylethy1idene)bis 3,20-dione
(oxy)-21-(1-oxoethoxy)l-pregna-1,Cdiene- 6a-Methylprednisolone (28): 6a-Methyl-11P,
3,204one 17,21-trihydroxypregna-l,4-diene-3,20-
Halcinonide(12) : 21-Chloro-9-fluoro-llp-hy- dione
droxy-16a,17-[(l-methylethy1idene)bis Prednisone (29): 17,2l-Dihydroxypregna-1,4-
(oxy)l-pregna-l,4-diene-3,20-dione diene-3,11,20-trione
Triamcinolone acetonide (13):9-Fluoro-llp, Dichlorisone(30): llp-Dichloro-17,21-dihy-
21-dihydroxy-16a,17-[(l-methylethylidene) droxypregn-4-ene-3,2O-dione
bis(oxy)]-pregna-l,4-diene-3,20-dione Corticosterone(31): llp,21-Dihydroxypregn-
Triamcinolone (13a): 9-Fluoro-1 lp,16a,l7,21- 4-ene-3,20-dione
tetrahydroxypregna-1,4-diene-3,20-dione 6a-Fluorocortisol(32): 6a-Fluoro-llp,l7,21-
Clocortolone pivalate(14): 9-Chloro-6a-fluoro- trihydroxypregn-4-ene-3,20-dione
1lp-hydroxy-16a-methyl-21-[l-oxo(2,2- Deoxycorticosterone(33): 21-Hydroxypregn-
dimethyl) propy1oxy)l-pregna-l,4-diene- 4-ene-3,20-dione
3,20-dione Cortexolone(34): 17,21-Dihydroxypregn-4-
Flurandrenolide(15): 6a-Fluoro-llp,2l-dihy- ene-3,20-dione
droxy-16a,17-[(l-methylethy1idene)- Progesterone(35): Pregn-4-ene-3,20-dione
bis(oxy)]-pregn-4-ene-3,20-dione 21-Deoxydexamethasone(36): 9-Fluoro-llp,
Fluticasone propionate(16): 6a,9-Difluoro- 17-dihydroxy-l6a-methylpregna-1,4-diene-
11~-hydroxy-l6a-methyl-3-oxo-l7a-(l-oxo- 3,20-dione
propoxy)-androsta-1,4-diene- l7P-carbo- Paramethasone(37): 6a-Fluoro-llp,l7,2l-trjhy-
thioic acid,S-fluoromethyl ester droxy-16a-methylpregna-1,4-diene-3,20-d
Mometasone furoate(17): 9a,2l-Dichloro-17- 6a-Fluoro-16a-hydroxycortisol(38):6a-
[(2-furanylcarbonyl)oxy]-1lp-hydroxy-16a- Fluoro-llp,l6a,l7,21-tetrahydroxypregn-
methyl-pregna-l,4-diene-3,20-dione 4-ene-3,20-dione
3 Adverse Effects Associated with the Use of Anti-Inflammatory Steroids 751

9a-Fluoroprednisolone (39):9-Fluoro-llp,l7, nous glucocorticosteroids. In general, exoge-


21-trihydroxypregna-l,4-diene-3,20-dione nous anti-inflammatory corticosteroid use is
Flumethasone (40): 6a,9a-Difluoro-16a- for the suppression of symptoms, including in-
methyl-1lp, 17,2l-trihydroxy-1,4- flammation, occurring in a particular disease
pregnadiene-3,20-dione state; these compounds are rarely considered
Structures for many of the above steroids curative in their usage.
appear in Figs. 15.1 and 15.2. Many disease states, however, do respond
well symptomatically to treatment with corti-
2 CLINICAL USE OF costeroid therapy. Some of these are listed in
ANTI-INFLAMMATORY STEROIDS the following table; an asterisk (*) denotes
those states in which the anti-inflammatory
The focus of this chapter is on the anti-inflam- activity of the corticosteroids used is the pri-
matory properties of synthetic and endoge- mary rationale in their utilization.

Adrenocortical Adrenogential syndrome Hypercalemia Myasthenia gravis


insufficiency
Organ transplants Nephrotic Syndrome Hematologic disorders Neoplastic disease
Liver disease Acute spinal cord injury Rheumatic disorders and Dermatologic
collagen diseases (*) deseases (*)
Allergic conditions and GI dieseases; ulcerative colitis Opthalmic, otic and Respiratory
conjunctivitis (*) and anorectal disorders (*) nasal disorders (*) diseases (*)
Cerebral edema (*) Bacterial meningitis (*)
(Modified from (9))

2.1 Topical Corticosteroids Currently 3 ADVERSE EFFECTS ASSOCIATED WITH


Available THE USE O F ANTI-INFLAMMATORY
STEROIDS
Tables 15.1 and 15.2 are lists of compounds
found in Facts and Comparisons (FC) (9) and The use of anti-inflammatory steroids has led
AHFS Drug Index (DI) (10). The compounds many to relief from discomfort caused by their
listed by FC as topical anti-inflammatories medical condition. However, corticosteroid
were placed within a range of Low to Very use is not without costs, and these are clearly
High Potency (see Table 15.1). The DI list in- seen in the adverse side effects these com-
dicates a range from I (most active) to VI (least pounds can lead to, especially in high doses or
active), and these values are listed next to the prolonged administration. These side effects
drug name in the table. Some compounds have are also associated with Cushing's syndrome
more than one activity class, depending on the (excessive glucocorticoid formation resulting
method of administration and concentration; from, for example, tumors of the adrenal cor-
in such cases only the higher activity class is tex) and are thus sometimes described as
"Cushingoid." Side effects are also dependent
listed.
on the method of corticosteroid administra-
tion and target tissue (11).
[Activity] . . . may vary considerably depend- Dermatologic side effects noted with glu-
ing on the vehicle, site of application, disease, the cocorticoid use include skin atrophy and re-
individual patient, and whether or not an occlu- duced skin thickness (12). Collagen, an essen-
sive dressing is used. The approximate relative tial protein in skin, is not synthesized as
activity is based principally on vasoconstrictor adequately in patients who use glucocorti-
assay andlor clinical effectiveness in psoriasis coids, especially in high doses (12), and this
(10). results in the degradation of skin character.
Anti-Inflammatory Steroids

(2) Hydrocortisone (5a) Betamethasone (6) Ciobetasol propionate


R1=R2=H R1=R2=H
(2a) Hydrocortisone butyrate (5b) Betamethasone dipropionate 0COCH3
R1 = H, R2= COC3H7 R1= R2 = COCH2CH3 /
(2b) Hydrocortisone valerate (5c) Betamethasone valerate
R1 = H, R2 = COC4H9 R1 = H. R2 = COC4HS
(2c) Hydrocortisone acetate (5d) Betamethasone benzoate
R1= COCH3, R2= H R1 = H, R2 = COCBHB
(2d) Hydrocortisone cypionate (50) Betamethasone sodium phosphate
R1= COcyclopentyl, R2= H Rl = P03Na, R2= H
(20) Hydrocortisone sodium phosphate (50) Betamethasone acetate
R1= P03Na, R2= H R1= COCH3, R2 = H
(2f) Hydrocortisone sodium succinate F
R1 = COCH2CH2C02Na,R2= H (7) Diflorasone diacetate

F
(8) Halobetasol propionate (9) Amcinonide (10) Desoximetasone

(1l a ) Flucinolone (12) Halcinonide


(11b) Fluocinoione acetonide R=H
( l l c ) Fluocinonide R=COCH3

F F
(13) Triamcinolone acetonide (14) Clocortolone pivalate (15) Flurandrenolide
(13a) Triamcinolone 16, 17-diol
(13b) Triamcinolone hexacetonide
(21-pivalate ester)

Figure 15.1. Structures of common glucocorticoids.


3 Adverse Effects Associated with the Use of Anti-Inflammatory Steroids 753

F
(16)Fluticasone propionate (17)Mometasone furoate (18)Aclometasone diprorionate

(19)Desonide (20) Dexamethasone R=H (21)Prednisolone R=H


(20a)Dexamethasone
sodium phostphate R=OP03Na

0 &IH/

(22)Fluorometholone (23)Rimexolone (24)Medrysone

(°C0C2H5

F
(25)Beclomethasone dipropionate (26)Flunisolide (27)Budesonide

(28)Methylprednisolone R=H
0
(28a) Methylprednisolone Acetate
R = COCH3
(28b)Methylprednisolone sodium succinate
R = COCH2CH2C02Na
0

(29)Prednisone R=H

Figure 15.2. Structures of common glucocorticoids.


Tablg 15.1 Drug Facts and Comparisons vs. AHFS Drug Information
Very high Potency High Potency Medium Potency Low Potency
Betamethasone dipropionate, Amcinonide, II,21-(Acety10xy)- Betamethasone benzoate, 111, Aclometasone dipropionate,
I,9-Fluoro-llp-hydroxy-16p- 16,17a-[cyclopentylidene- 17-(Benzoy1oxy)-9-fluoro-llp,21- VI,7a-Chloro-llp-hydroxy-
methyl-17a,21-bis(1- bis(oxy)]-9-fluoro-llp- dihydroxy-16P-methylpregna- 16a-methyl-17a,21-bis(1-
oxopropanoxy)-pregna-1,4- hydroxypregna-1,4-diene-3,20- 1,4-diene-3,20-dione(16) oxopropanoxy)-pregna-1,4-
diene-3,20-dione (5) dione(9) Clocortolone pivalate, 9-Chloro- diene-3,20-&one(23)
Clobetasol propionate,I,21- Betamethasone valerate, 111, 21-(2,2-dimethyl-1-oxopropoxy)- Desonide, VI, llp,21-
Chloro-9-fluoro-llp-hydroxy- 9-Fluoro-llfi,17a,2l-trihydroxy- 6a-fluoro-llp-hydroxy-16a- Dihydroxy-16a,17-[(I-
16gmethyl-17-(1-oxopropoxy)- 16p-methyl-17-[(l-oxopenty1)- methylpregna-1,4-diene-3,20- methylethylidene)bis(oxy)l-
pregna-1,4-diene-3,2O-dione(6) oxyl-pregna-1,4-diene-3,20-dione dione(17) pregna-1,4-diene-3,2O-dione
Diflorasone diacetate, I, (10) Flurandrenolide, IV,6a-Fluoro- (24)
17,21-bis(Acety1oxy)-6a,9- Desoximetasone, II,9-Fluoro- llp,21-dihydr0xy-l6a,17-[(1- Dexamethasone,9-Fluoro-
difluoro-llp-hydroxy-16p- 11~,2l-dihydr0xy-l6a- methylethylidene)bis(oxy)l- llp,17,21-trihydroxy-16a-
methylpregna-1,4-diene-3,20- methylpregna-1,4-diene-3,20- pregn-4-ene-3,20-dione (18) methylpregna-1,4-diene-3,20-
dione(7) dione(11) Fluticasone propionate, 6a,9- dione(25)
Halobetasol propionate, 21- Fluocinonide, II,21-(Acety1oxy)- Difluoro-llp-hydroxy-16a- Dexamethasone sodium
Chloro-6a,9-difluoro-llp- 6a,9-difluoro-110-hydroxy-16,17- methyl-3-0x0-17a-(1- phosphate, 9-Fluoro-llp,2l-
hydroxy-l6p-methyl-17-(1- [(I-methylethylidene)bis(oxy)]- oxopropoxy)-androsta-1,4-diene- dihydroxy-16a-methyl-21-
oxopropoxy)-pregna-l,4-diene- pregna-1,4diene-3,20-&one (12) 17p-earbothioicacid,S- (phosphonooxy)-pregna-1,4-
3,20-dione(8) Fluocinolone acetonide, IV, fluoromethyl ester(19) diene-3,20-dione,disodium salt
6a,9-Difluoro-llp,2l-dihydroxy- Hydrocortisone butyrate, V, (26)
16a,17-[(l-methylethy1idene)- 1lp,21-Dihydroxy-17-(1- Hydrocortisone, llp,17,21-
bis(oxy)]-pregna-1,4-diene-3,20- oxobutoxy)-pregn-4-ene-3,20- Trihydroxy-pregn-4-ene-3,20-
dione(13) dione(20) dione (27)
Halcinonide, II,21-Chloro-9- Hydrocortisone valerate, V, Hydrocortisone acetate, 21-
fluoro-l1~-hydroxy-16a,17-[(1- llp,Zl-Dihydr0~-17-[(1- (Acety10xy)-llp,17-dihydroxy-
methylethylidene)bis(oxy)l- oxopenty1)oxyl-pregn-4-ene-3,20- pregn-4-ene-3,20-&one (28)
pregn-4-ene-3,20-dione (14) dione(21)
Triamcinolone acetonide, 111, Mometasone furoate, a,21-
9-Fluoro-11~,2l-dihydroxy-16a,17- Dichloro-17-[(2-furanyl-
[(I-methylethylidene)bis(oxy)l- carbony1)oxyl-lip-hydroxy-16a-
pregna-1,4-diene-3,2O-&one (15) methyl-pregna-1,4-diene-3,20-
dione(22)
Group III: Mometasone acetonide (29) Group V: Prednicarbate (30)
Table 15.2 Steroids Frequently Used in Common Medical Circumstances
Medical Use Drugs Common to This Use
-

Ophthalmic Dexamethasone (20a) Fluorometholone, 9-Fluoro-llp,l7- Medrysone, llp-Hydroxy-6a-


dihydroxy-6a-methyl-pregna-1,4- methyl-pregn-4-ene-3,20-dione
diene-3,20-dione(22) (24)
Prednisolone, llp,l7,21-Trihydroxy- Rimexolone, llp-Hydroxy-16a,17-
pregna-1,4-diene-3,20-dione(21) dimethyl-17-(1-oxopropy1)-androsta-
1,4-dien-3-one(23)
Respiratory Beclomethasone dipropionate, 9- Dexamethasone sodium phosphate Flunisolide, 6a-Fluoro- llp,21-
inhalant Chloro-llp-hydroxy-l6p-methyl- (20b) dihydroxy-16a,l7-[(I-
17,21-bis(1-oxopropan0xy)-pregna- methylethylidene)bis(oxy)]-
1,4-diene-3,20-dione(26) pregna-1,4-diene-3,2O-dione (26)
Triamcinolone acetonide (13)
Intranasal Beclomethasone dipropionate (25) Dexamethasone sodium phosphate Flunisolide, (26)
(20b)
Budesonide, 16a,17- Triamcinolone acetonide (13) Fluticasone propionate (16)
[Butylidinebis(oxy)]-llp,21-
dihydroxy-pregna-1,4-diene-3,20-
dione (27)
Intrarectal or Hydrocortisone (2) Hydrocortisone acetate (2c)
anorectal
Systemic Betamethasone (5a) Hydrocortisone cypionate (2d) Prednisone (29)
Betarnethasone sodium phosphate Hydrocortisone sodium phosphate Prednisolone (21)
(5e) (2e) Prednisolone acetate (2la)
Betamethasone acetate (50 Hydrocortisone sodium succinate (20 Prednisolone tebutate (21b)
Cortisone (1) Hydrocortisone acetate (24 Prednisolone sodium phosphate
Dexamethasone (20a) Methylprednisolone (28) (21~)
Dexamethasone acetate ( 2 0 ~ ) Methylprdnisolone sodium succinate Triamcinolone (13a)
Dexamethasone sodium phosphate (28a) Triamcinolone acetonide (13)
(20b) Methylprednisolone acetate (28b) Triamcinolone diacetate (13b)
Hydrocortisone (2) Triamcinolone hexacetonide (13d
756 Anti-Inflammatory Steroids

Changes in bone metabolism, especially in addition to the aforemetioned, including


leading to osteoporosis and reduced bone for- "Moon face," truncal obesity, hyperglycemia,
mation (13), are another drawback of glu- and ketoacidosis. Tables 15.3 and 15.4
cocorticoid use. It has been found that the rate present, respectively, generalized adverse ef-
of bone mineral density reduction is greatest fects noted with glucocorticoid use and com-
during the initial months of treatment. There parative clinical adverse effects of anti-inflam-
is some debate whether daily dose decreases or matory steroids.
increases bone loss rates. One study suggests
low dose glucocorticoids (predominantly pred- 4 EFFECTS ON ABSORPTION
nisone) lead to increased bone loss (14); mini-
4.1 Intestinal Absorption
mal or no effect on bone mineral density is
noted with low (micro, <10 mg) daily doses Schedl and Clifton (27) showed that the ab-
(15) in another study. In addition, calcium, vi- sorption of steroids by the perfused rat small
tamin D, and estrogen supplementation all intestine is strongly correlated with the polar-
have indicated bone restorative action when ity of the compound. The least polar com-
implemented in conjunction with glucocorti- pound in the study, progesterone (41), was ab-
coid therapy (15). Glucocorticoid-induced sorbed almost completely under the condition
osteoporosis is dependent on the individual of the study, whereas the introduction of polar
steroid used; dexamethasone and methylpred- atoms, and particularly hydroxyl groups, re-
nisone show more effect than prednisolone or duced the extent of absorption (Table 15.5).
deflazacort on human osteoblast-like cell me- Acetylation of the hydroxyl groups invariably
tabolism (16, 17). increased the extent of absorption. Although
Glucocorticoids are frequently used as im- absorption correlates with a physical property
munosuppressants, but this is also an unde- of the steroid, and therefore appears to be a
sired effect when only anti-inflammatory ac- diffusion-controlled process rather than active
tion is required. As with other side effects, the transport, the values in Table 15.5 had no re-
increase in infectious complications is accen- lationship to relative oral clinical potencies.
tuated with large doses and prolonged use Thus, it is likely that the rate of intestinal
(181, whereas reduced effects are noted with absorption is not a controlling factor in the
low daily (<lo mg prednisone) doses (19). activity of the steroids.
The psychological actions of glucocorti- A colon-specific drug-delivery system was
coids have been well documented, although tested with two steroid prodrugs, dexametha-
the cause has been attributed to both direct sone 21-P-D-glucoside(20c) and prednisolone
and indirect effects of steroids on brain func- 21-P-D-glucoside(21a). Despite high levels of
tions (20). Dexamethasone downregulates the P-D-glucosidase producing bacteroides and
expresssion of endothelin B receptors in rat bafidobacteria in the rat upper intestine, these
brain, but produces an increase in ET-1 and glucosides were remarkably selective for the
other neuronal mRNA (21, 22). The mecha- rat lower intestine. These prodrugs may
nisms by which glucocorticoids modulate neu- therefore be useful in humans for the treat-
ronal activity is currently under investigation ment of inflammatory bowel disease. Studies
(23). A general euphoria is commonly associ- showed that upon oral administration of these
ated with initial glucocorticoid use, but pro- drugs to rats, the glucosides reached the ce-
longed use can lead to mania or depression cum in 4 to 5 h, whereupon they were rapidly
(24). Termination of glucocorticoid therapies enzymatically hydrolyzed to the free steroids,
has also led to delirium in patients (25). dexamethasone (20a) or prednisolone (21)
Gastrointestinal effects are a common, (28). Of these, nearly 60% of an oral dose of
though perhaps less serious, side effect associ- (20c) reached the cecum of the rat, whereas
ated with glucocorticosteroid use. only 15% of (21a)was delivered. Interestingly,
Long-term corticosteroid therapy is often when either dexamethasone (20a) or pred-
associated with side effects resembling those nisolone (21) were administered orally, less
of Gushing's syndrome. Carbohydrate and than 1% of the steroid was delivered to the
lipid metabolic malfunction lead to conditions cecum.
Table 15.3 Generalized Adverse Effects Noted with Glucocorticoid Use, Especially Prolonged and High Dosagea
Adverse Condition Some Effects Preventive Action
Adrenocortical insufficiency Suppression of the pituitary-adrenal axis and Mineralocorticoid administration; variation of
inhibition of ACTH production dose, frequency, and time of drug
administration.
Musculoskeletal effects Muscle wasting, muscle pain and weakness, High protein diet
delayed wound healing, atrophy of protein
matrix of bone
Immune system effects Increased susceptibility to, and masked symptoms Vaccinations; antiviral, antifungal, and antibiotic
of infection, especially with high dose and therapy
systemic glucocorticoids
Fluid and electrolyte disturbances Mineralocorticoid-likeaction by glucocorticoids; Dietary salt restriction, potassium supplements
sodium retention. Less frequent with most
V
01 synthetic glucocorticoids.
V
Ocular effects Increases in intraocular pressure (IOP), and the Close monitoring of patient
development of posterior subcapsular cataracts
Endocrine effects Varying effects including hypercorticism, Close monitoring of patient
amenoriea, hyperglycemia
GI effects Varying effects including nausea, anorexia, Antacids and antiulcer agents; oral
increased appetite, and the development and glucocorticoids taken near meal time
increased severity of ulcers
Nervous system and psychological Headache, vertigo, insomnia, increased motor Close monitoring of patient
effects activity, euphoria, mood swings, depression,
anxiety
Dermatologic effects Skin atrophy and skin thinning from a decrease Close monitoring of patient
in colagen synthesis, impaired wound healing,
acne, increased perspiration
"Modified from ref. 9.
4 Effects on Absorption 759

Table 15.5 Steroid Absorption by Perfused Rat Small Intestinea

(41) (31) (33)


Steroid % Absorbed Acetate Derivative
Progesterone (41)
Deoxycorticosterone (33)
Corticosterone (31)
Cortisol (2)
Triamcinolone (13a)
"Ref. 27.

In an application of Lee's "antedrug" ap-


proach, Kimura et al. developed colonic mu-
cosa-specific "pro-antedrugs" for oral treat-
ment of ulcerative colitis (29). The highly
polar P-D-glucopyranosylderivatives (42) and
(43) were found to be stable in the small intes-
tine, but the glucoside bonds were cleaved by
the action of bacteria in the large intestine to
release the antedrug (44). The active cortico-
steroid ester (44) was found to undergo rapid
hydrolysis in the plasma to give the inactive
carboxylic acid (45) (see Sections 10.16 and
10.19).

(20c) Dexamethasone 21-glucoside


4.2 Percutaneous Absorption
Through the use of 14C-labeledsteroids, it has
been shown that cortisol (2) and triamcino-
lone (13a)are absorbed from a topical site of
application and that the radioactivity appears
in the urine (30). The more potent analogs of
cortisol are all effective when applied topi-
cally, although large quantities can produce
systemic effects (31).
In the 1960s it was found that triamcino-
lone acetonide (13)was 10 times as active top-
ically as triarncinolone (13a)itself, but only
equiactive systemically (32).As pointed out by
Popper and Watnick (33), to be topically effec-
0 tive the steroid must penetrate the keratin
layer of the stratum corneum of the skin be-
(21a) Prednisolone 21-glucoside fore it can exert its effect on the squamous cell
Anti-Inflammatory Steroids

(42) pro-antedrug

colonic bacteria

OH

esterases

0
(44) active antedrug (45) inactive

(13) Triamcinolone acetonide

layer of the epidermis. However, to reach the


general circulation and produce systemic side
effects, the steroid must later penetrate the
barrier between the epidermis and the dermis.
Thus for a useful topical agent it is desirable
for the compound to remain in the epidermis
and to migrate only slowly into the dermis.
This property is fostered by the presence of
lipophilic groups and by the absence of hy-
droxy groups in the steroid (4). The conversion
(13a) Triamcinolone of one or two hydroxyl groups in anti-inflam-
4 Effects on Absorption

matory steroids to more lipophilic derivatives, and colleagues examined the influence of a va-
such as esters or ketals, is an effective way to riety of substituted D-ring acetals that had
produce locally active anti-inflammatory their alkyl group (Table 15.6, R,) in the p-ste-
agents, as in the case of triamcinolone already reochemical orientation (36). Introduction of
cited. fluorine at C-6 and/or 9a positions was exam-
ined as was esterification of the 21-alcohol
4.2.1 16a,l7a-Ketals. 16a,l7a-Ketals are group. Selected findings from this work are
formed by the reaction of 16a,l7a-dihydroxy shown in Table 15.6.
steroids and the appropriate ketones or alde- With the 21-alcohol free, introduction of
hydes in the presence of an acid catalyst, to 9a-fluoro in (47) or 9a,6a-difluoro groups in
give a new pentacyclic ring. Unlike other diox-
(48) does not have a dramatic effect on po-
olanes that are readily hydrolyzed by dilute
tency in the rat ear. Although some 50% im-
acid to afford the parent constituents, these
provement in potency was observed for (48),
steroidal dioxolanes are unusually stable. This
led Fried et al. (34) to conclude that the com- its therapeutic index dropped from nearly 1 in
pounds are activeper se, and not as a result of (47) to about 114 in (48). Thus, the additional
hydrolysis in vivo to the parent compound. As F group enhanced systemic activity presum-
mentioned earlier, triamcinolone acetonide ably by curtailing metabolism. Simple acetyla-
displays a dissociation between topical and tion of budenoside led to (49), in which po-
systemic activity. The 6a-fluoro derivative, tency was doubled and the therapeutic index
fluocinolone acetonide ( l l b ) , is also used ex- was substantially improved to about 4.7. Fur-
tensively as a topical agent. ther potency enhancements were gleaned
These compounds can be made even more upon F-substitution in the 21-ester series, but
lipophilic by the preparation of 21-esters. acetate esters were better than longer chain
Thus, fluocinonide (lla), the 21-acetate of esters such as (51).
Human potencies were gauged in skin
blanching studies for the 21-alcohols, as
shown in Table 15.7. An interesting pattern
evolves from this work, where irrespective of
F-substitution pattern, a peak in activity is
observed three times (C-9a = H, C-9a = F,
C-6a and C-9a = F) when n = 2.

4.2.2 Esters. The evaluation of a number of


17-esters, 21-esters, and 17,21-diesters of be-
tamethasone (5)was carried out by McKenzie
and Atkinson (37) (Table 15.8). The least ac-
tive compounds are the unesterified parent (5)
(lla) R = Ac and the highly polar monophosphate ester
(llb) R = H (5g). In all three series shown, activity seems
to have a parabolic dependency on the rr value
of the esterifying acid, as would be expected on
fluocinolone acetonide, is about five times as the basis of the absorption process already dis-
active as the latter compound (35). Other ket- cussed. Betamethasone 17-valerate (5d) is a
als that have anti-inflammatory activity are widely used topical anti-inflammatory agent
the 17,21-acetonides,but these have not found and the related 17-benzoate (5e)has also been
clinical application. used clinically. Betamethasone 17,21-dipropi-
Although many steroidal dimethylketals onate (5L)is in current clinical use as a topical
derived from acetone (i.e., 16a,17a-acetonides) anti-inflammatory steroid. Even the weakly
have been prepared, 16,17-acetals fabricated active 21-deoxyprednisolone (541, which has
from aldehydes are less well studied. Thalbn 25% of the systemic activity of cortisol (38),
762 Anti-Inflammatory Steroids

Table 15.6 Relative Activities of 16,17-AcetalDerivatives Related to Budenoside,


Triamcinolone, or Fluocinolone 16,17-Acetonidesin the Rat Ear Edema Assay"

Compound RI
HO Propyl H H 1
HO Propyl F H 1
HO Propyl F F 1.5
CH,COO Propyl H H 2.3
CH,COO Propyl F H 4.3
CH,(CH,),COO Propyl F H 2.7
CH,COO Propyl F F 4.1
1
1.7
"Ref. 36.
bCroton oil ear edema assay in rats.
"Topical vs. systemic activity in the ear edema assay.
dBudesonide 16,ll-acetonide.
'Triamcinolone 16,ll-acetonide.
fFluocinolone 16,ll-acetonide.

gives a clinically useful compound upon con- sone valerate in the McKenzie assay in human
version to the 17-propionate (33). volunteers. These compounds did have 6a-
A series of 17a-thiomethyl and l7a-meth- methyl groups that would be expected to en-
oxyacetates (55;X = SMe, OMe) were found to hance activity somewhat (see section on C-6
possess activity similar to that of betametha- substitution) (39).
Ueno et al. (40) showed that a variety of
X-substituted l7a-esters have excellent sepa-
ration of topical and systemic activities, as
shown in Table 15.9. Where n = 2 or 3 and X =
polar group such as OMe or OAc, therapeutic
indices were far better than controls such as
clobetasol propionate or betamethasone
dipropionate, whereas anti-inflammatory po-
tency was somewhat less than control. The
least systemically absorbed, or systemically
active, in this table, was the acetate (60),with
a TI of over 18. Although (60)was only a third
as active as CP, on the other hand, it was three
times more active than BMDP.
Clearly, more complicated l7a-esters offer
4 Effects on Absorption 763

Table 15.7 Topical Anti-inflammatory Activity in Blanching of Human Skin, Expressed as


EC,, and Relative Potency in Comparison with Fluocinolone Acetonide (FA)a
OH

Y
Number of CH, EC5,b Relative Potency
Compound Groups (n) X Y (CLg/mL) Compared with FAc
(52a) 1 H H 2.8 (1.6-5.3) 1.1
(46) 2 H H 1.1 (0.7-1.7) 1.9
(52b) 4 H H 3.4 (1.8-8.9) 0.4
(52~) 6 H H > 10
(52d) 0 F H 3.5 (2.0-6.3) 0.6
(52e) 1 F H 0.8 (0.5-1.3) 4.0
(47) 2 F H 0.6 (0.4-0.8) 4.3
(52f) 3 F H 1.1 (0.7-1.9) 1.9
(52g) 4 F H 3.1 (1.8-5.6) 0.7
(52h) 8 F H > 10
(52i) 0 F F 1.7 (1.1-2.8)d 0.9
(48) 2 F F 0.4 (0.2-0.6) 4.0
(52j) 4 F F 1.5 (0.8-3.1) 1.1
(52k) 6 F F 4.6 (2.5-12.8) 0.3
~- -

"Five doses of each compound were tested with 10 parallels/dose.


bThe EC5, values are followed by 95% confidence limits in parentheses.
"Compared with the reference compound (FA) within the same test. The EC50 t SEM value for FA in the five tests was
2.3 t 0.4 p.g/mL.
dThis value was obtained with approximately 100% of the most polar C-22 epimer.

means of separating topical from systemic ef-


fects. It might be argued that metabolism of
(60) renders the analog less active, but this
has not been studied. It was suggested that
improved metabolic stability might account
for the loss in TI for the cyclopropyl esters (63)
and (64).
Given that l7a-benzoate esters were pos-
sessed of excellent topical efficacy, additional
related but heteroaromatic compounds have
been examined. Two classes of compounds
were targeted having furoyl or thienoyl esters 0
at the 17-position, and were further modified z
by oxygenation or halogenation at 6,9,11 and
21 and have the general structure (65): (65)
0
&
Table 15.8 Relative Potencies of Betamethasone and Some of Its Esters in VasoconstrictionAssays against Fluocinolone Acetonidea

/
OCOEt

CH3

(5L) Betamethasone Dipropionate


Betamethasone alcohol
Betamethasone 21-&sodium phosphate
Betamethasone 21-acetate
Betamethasone 21-butyrate
Betamethasone 21-valerate
Betamethasone 21-hexanoate
Betamethasone 21-palmitate
Betamethasone 17-acetate
Betamethasone 17-butyrate
Betamethasone 17-valerate
Betamethasone 17,21-ethylorthoformate
Betamethasone 17,21-ethylorthopropionate
Betamethasone 17,21-ethylorthovalerate
"Ref. 37.
*Fluocinoloneacetonide (llb)= 100.
766 Anti-Inflammatory Steroids

Table 15.9 Activity of 17a-Esters of 21-Desoxy-21-Chlorobetamethasone

Compound X n EEAb Thymusc TI d


(56) CH3 1 0.11 - -
(57) CH3 2 0.38 0.05 7.6
(58) C1 1 0.25 - -
(59) C1 3 0.66 0.12 5.5
(60) OAc 2 0.28 0.015 18.6
(61) SCH, 1 0.35 0.35 1
(62) CN 2 0.07 - -
(63) C-C3H6 0 0.44 1.3 0.34
(64) C-C3H5 1 0.51 1.1 0.46
CP 1 1 1
BMDP~ 0.1 0.03 3.3
"Ref. 40.
*Crotonoil ear edema assay.
"Thymic involution in rat granuloma pouch assay.
dTherapeuticindex determined by EENThyrnus.
'Clobetasol propionate.
Betamethasone dipropionate.

In one series where X and Y = C1, the aro- rination at 21 resulted in a sensitivity to the
matic group was varied from 2-fury1 or thienyl position of attachment to the heterocyclic
to 3-fury1 or thienyl while also varying the 21- ring. Thus, 2-fury1 was significantly better
position, as shown in Table 15.10 (41). As can than 3-fury1 (75 was much more potent than
be seen, not much difference in topical efficacy 76). Halogenation at the 6a-position of the
is evident on changing from an 0 heteroatom better compound in this series (75), providing
(furanyl) to a S heteroatom (thienyl) (i.e., 66 (771, was not beneficial.
versus 68). Similarly, the position of substitu- In a closely related series with an llp-alco-
tion (2- vs. 3-position) on the aromatic ring is hol instead of a halogen, the same investiga-
not highly critical to potency (i.e., 66 versus tors studied the effect of ester substitution at
67). On modifying the 21-oxygen atom from the 17-position (42). As illustrated in Table
alcohol (70) to ester, an expected enhance- 15.11, the effect of heteroatom (S or 0) or
ment is evident but continued increases in heteroatom position (2- or 3-furyl) in this
bulk at 21 (e.g., 72, 73, 74) were without sig- llp-alcohol series is generally similar to the
nificant effect. Upon replacement of the 21 ox- dichlorisone-like series in Table 15.7 and 21-
ygen by C1a sizable potency enhancement was halogenation had a likewise positive effect on
seen (75). Not only was the response rapid, it potency.
appeared to be cumulative, given that the po- These sets of compounds illustrate the
tency after 5 days had risen to over eightfold complex relationships that govern topical po-
better than that of the control, betamethasone tency upon esterification at C-17. Although
valerate. Finally, unlike the 21-01 series, chlo- quite reasonable structure-activity relation-
4 Effects on Absorption 767

Table 15.10 9,ll-Dichloro Corticosteroid l7a-Heterocyclic Esters a


v

(66-77)
Compound X Y R EEA~
(66) H OAc 2-fury1 1.4 (1.2)
(67) H OAC 3-fury1 1.3 (1.4)
(68) H OAc 2-thienyl 1.3 (0.8)
(69) H OAc 3-thienyl 0.9 (3.2)
(70) H OH 2-fury1 0.8
(71) H OH 3-fury1 0.7
(72) H OCOEt 2-fury1 1.1 (0.9)
(73) H OCOPr 2-fury1 1
(74) H OCOCH,OCH, 2-fury1 1(3)
(75) H C1 2-fury1 1.9 (8.2)
(76) H C1 3-fury1 l(1.7)
(77) F C1 2-fury1 2.5 (3)
BVc l(1)
"Ref. 41.
bCroton oil ear edema assay, reported at 5 hand (5 days).
'Betamethasone 17-valerate as control.

ships arise out of homologous esters where the


steroid nucleus is held constant (Table 15.8),
those same relationships do not always trans-
late simplistically into systems where multiple
changes to the steroid nucleus have taken
place.
From the compounds in Tables 15.10 and
15.11, a clinically useful compound was mar-
keted as Elocon or mometasone furoate (17).

4.3 lntra-Articular Administration (17) Mometasone Furoate


Arthritic and inflammatory conditions afflict-
ing skeletal joints may be treated by intra-ar- have a very short duration of action under
ticular injections, in which a hypodermic nee- these circumstances. Figure 15.3 schemati-
dle is passed through the synovial membrane, cally represents the metabolism of corticoste-
and a dose of corticosteroid is injected into the roids. Conversely, aqueous microcrystalline
joint cavity. The specific local anti-inflamma- suspensions of ketals and esters of anti-in-
tory effect lasts from 5 to 21 days or longer. flammatory steroids dissolve suffficient quan-
Water-soluble esters of anti-inflammatory ste- tities to permit local action in the joint but do
roids are rapidly cleared from the joint and not enter the systemic circulation in amounts
768 Anti-Inflammatory Steroids

Table 15.11 9a-Halo-11B-HydrowCorticosteroid l7a-Heterocyclic Estersa

OAc
OAc
OCOEt
OCOPr

CI
OAc
CI
C1
F
C1

"Ref. 42.
bCroton oil ear edema assay at 5 h and (5days).
"Betamethasonevalerate, control.

needed to exert substantial systemic effect. In 25 pg of (13b).The advantage of rimexolone


addition to triamcinolone acetonide, branched (23) over (13b) was its prolonged retention
esters that hydrolyze slowly, such as triamcin- (21 days).
olone hexacetonide (13b)(43) and dexameth-
asone 21-trimethylacetate (20c) (441, are use-
ful for this purpose. 6a-Methylprednisolone
acetate (45) and betamethasone 17,21-dipro-
pionate (46) are also active long enough to be
useful. Figure 15.4 shows a schematic repre-
sentation of the formation of C-21 carboxylic
acid metabolites of cortisol.
Triamcinolone hexacetonide (13b) has
been compared to rimexolone (23) for intraar-
ticular treatment of arthritis (47).Rimexolone
was of interest in this regard for its clinical
availability and reported lack of systemic ef-
fects. In mice, a single injection of 450 kg of
4.4 Water-Soluble Esters
(23) was sufficient to provide a prolonged anti-
inflammatory response, but to generate a sim- The incorporation of ionic groups into anti-
ilar anti-inflammatory response required only inflammatory steroids at the 21 position re-
4 Effects on Absorption

Cortisone Cortisol
\ 6p-Hydroxycortisol

0
H

& $:
Dihydrocortisone Dihydrocortisol Allodihydrocortisol

HO' HO' HO'


H H H

&&::&&::
Tetrahydrocortisone Tetrahydrocortisol (13%) Allotetrahydrocortisol (7%)

HO'
H
HOP
H
HO - H
HO'
H
2Op-Cortolone 20a-Cortolone 20&Cortol 2Oa-Cortol
\ \ 1

Figure 15.3. Metabolism of corticosteroids (figures in parentheses denote percentage of adminis-


tered dose of cortisol recovered as urinary metabolites).
Anti-Inflammatory Steroids

COOH
b OH

HO' .@-o~
H
-
HO' -dPo H
20g-Cortolonic acid

HO' ,&: H HO'


.
H
20a-Cortolonicacid

.&
COOH
b OH

HO'
H
-
HO' .dPoH H
20p-Cortolicacid

COOH
\--OH

HO'
,
HO' *aY H

20a-Cortolicacid
Figure 15.4. Formation of C-21 carboxylic acid metabolites of cortisol.

sults in water-soluble steroids that have rapid phate, Selestoject) and prednisolone sodium
onset of action but are of fleeting duration. phosphate (e.g., Pedi-pred), or 21-hemisucci-
The esters employed include the 21-phosphate nate sodium salts such as hydrocortisone so-
disodium salts, such as betarnethasone so- dium succinate (2f;e.g., Solu-Cortef) and
dium phosphate (5g; e.g., Celestone Phos- methylprednisolone sodium succinate (e.g.,
5 Effects on Drug Distribution

0COC(CH3)3 ined (52,53) and it was concluded that liposo-


/ ma1 preparations were not useful, although
Tween 80 accelerated absorption.

5 EFFECTS O N DRUG DISTRIBUTION

Only about 5% of cortisol in plasma is circu-


lated in the unbound state, whereas the re-
mainder is bound to two major moieties in se-
rum: serum albumin and corticosteroid
binding globulin (CBG) (54). Serum albumin
is present in higher concentration (5500 X
Solu-Medrol). There is extensive evidence in-
lop7M in human serum), whereas the concen-
tration of CBG in human serum is 8 X lop7M.
dicating that such esters rapidly hydrolyze to However, the equilibrium association con-
the active 21-alcohols (48) and therefore rep- stant (K,) for cortisol-CBG is 3 X lop7M-' ,
resent only solubilizing groups. Soluble com- about lo3 greater than that for cortisol serum
pounds such as prednisolone sodium phos- albumin. There is good evidence to support
phate or dexamethasone sodium phosphate the notion that the steroids are complexed to
can be used for ophthalmic purposes as well as these proteins primarily by hydrophobic
for intravenous administration. Moreover, bonds (55, 56). This implies that the steroid
they may be incorporated in long-acting depot interacts primarily with nonpolar portions of
preparations to add a rapid-acting component. the protein. The affinity of steroids to serum
Along these lines, Celestone Soluspan, a mix- albumin decreases with an increasing number
ture of betamethasone sodium phosphate and of hydrophilic or polar groups in the molecule
betamethasone acetate, is useful in providing (57). The hydrophobic effect depends on the
relief from, for example, bursitis, arthritis, displacement of ordered water from two sur-
and dermatological conditions. faces, and it is clear that the presence of a
hydrophilic group on one of the surfaces will
4.5 Corneal Penetration
interfere with this process. The type of sub-
The availability of ocular drugs, administered stituent group is obviously important because,
in solution as drops, is very low because of on this basis, a hydroxyl group should inter-
rapid clearance of the solution from the pre- fere more with binding than would a more li-
corneal area. Highly water soluble drugs are pophilic fluorine congener. The stereochemis-
also not well absorbed from the cornea (49) try of the derivative is also significant; llp-
but decadron phosphate, a 21-sodium phos- hydroxyprogesterone (89) has higher affinity
phate ester of dexamethasone (20b),finds for serum albumin than does the l l a epimer.
clinical utility when formulated as an ophthal- This has led to the suggestion (58) that the
mic ointment in mineral oil or even as a sterile steroids are bound to albumin on their a face.
aqueous solution (50). Schoenwald and Ward Binding of steroids to human and rat CBG
(51) determined the permeability rates across is also diminished by the presence of polar
excised rabbit cornea for 11-steroids. They de- groups (59). Interestingly, the order of binding
rived a parabolic relationship between log per- for rabbit CBG is cortisol (2) > corticosterone
meability and the octanoVwater partition co- (31) > progesterone (41) and thus opposite to
efficient with an optimum at log Po of 2.9 the order one would predict from the polarity
(close to that of dexamethasone acetate). Ste- of substituents (59).This is evidence for struc-
roid combinations containing prednisolone ac- tural specificity in steroid-CBG binding, and it
etate (0.2%in oil base) have found ophthalmic suggests the presence of a "binding site" on
application in humans. the protein, with specific structural require-
Corneal permeability of dexamethasone ments for optimum binding. As discussed in
and selected esters incorporated into lipo- section 9, CBG binding affinity is highly corre-
somes or as solutions in Tween 80 was exam- lated to the 3D structure in corticosteroids.
772 Anti-Inflammatory Steroids

Table 15.12 Comparison of Relative Potencies for Some Systemically Used


Glucocorticosteroidsa
PWPD Clinical
Compound RRBA CL ( l l h ) fu F Potencyb Potency"
Cortisol 9 18 0.20 0.96 1 1
Prednisolone 16 10 0.25 0.81 3.4 4
Methylprednisolone 42 21 0.23 0.99 4.7 5
Betamethasone 58 9 0.36 0.72 17.4 25
Fluocortolone 82 30 0.10 0.84 2.4 5
Dexamethasone 100 17 0.32 0.83 16.3 25
Triamcinolone acetonide 233 37 0.29 0.23 4.4 6
"RRBA, relative receptor binding affinity; CL, total body clearance; Fu,fraction unbound in serum; F, oral bioavailability.
'PWPD potency calculated based on equation (1)and standardized to cortisol = 1.
'Clinical potency based on empiric therapeutic equivalence.

This concept of a specific binding site is also


in harmony with the reduction of binding to
CBG on introduction of numerous substitu-
ents into the cortisol structure, irrespective of
the polarity of the group (60).A study (61)that
illustrates this difference compared the bind-
ing to CBG of cortisol, dexamethasone (20a),
and its 16P-methyl epimer (betamethasone,
5). It was found that all the steroids are bound
extensively to serum albumin, whereas only
cortisol is bound to CBG.
The effect of protein binding on biologic ac-
tivity is not simple to assess. On the one hand,
CBG-bound cortisol is biologically inactive PWPD modeling of corticosteroids. A suitable
(621, suggesting decreased protein binding indirect-response PWPD model was devel-
would lead to enhanced activity. On the other oped that allowed description of the receptor-
hand, it has been shown (63) that CBG pro- mediated drug effects such as endogenous cor-
tects cortisol against metabolic degradation by tisol suppression as a function of time.
rat liver homogenate. Because the two phe- Furthermore, the model allowed for predic-
nomena tend to cancel each other, it is impos- tion of the systemic activity of newly devel-
sible to state a priori the magnitude of oped corticosteroids on the basis of their phar-
changes in pharmacological activity arising macokinetics and their respective receptor-
from effects on plasma binding. binding affinity. The model could also be
Despite these complications, in a clinical applied to systemic steroid effects after topical
setting, it has been possible to carry out administration, or to investigate the effect of
the time of dosing on cortisol suppression.
Comparison of predictions based on this model
and results from large clinical studies were in
excellent agreement (Table 15.12). Corticoste-
roids may represent an ideal class of drugs for
the successful use of P W D modeling during
drug development (64).
Measured EC,, values showed outstanding
correlation with receptor-binding affinities
and allow for the evaluation of new corticoste-
roids in early development. On the basis of
EC,,, the fraction of unbound steroid (protein
6 Clucocorticoid Biosynthesis and Metabolism

bound vs. free) or f,, clearance (CL), and oral A-Ring Reduction (Double Bonds and C-3
bioavailability (F), a parameter for compari- , This is an irreversible reaction that
~arbonvfi.
'

son was found, DR,,, represented by the fol- is a foremost determinant of the secretion rate
lowing relationship: of cortisol. Catalyzed predominantly by corti-
sone p-reductase and 3a-hydroxysteroid dehy-
drogenases, 5P sterols result, although 5a
sterols are more prevalent with other glu-
cocorticoids. Urocortisol and urocortisone
result from the metabolism of cortisol and cor-
DR,, is the dosing rate that produces and tisone, respectively. Compounds can be com-
maintains 50% of the maximum effect. plexed to glucuronic acid at this point.
Evaluation of topical corticosteroid effects C-20 Reduction. Two stereoisomers can re-
through the use of PK/PD modeling is more sult from this transformation, although corti-
challenging, given that markers for acute top- sol is thought to act primarily with (R)20/3-
ical activity are limited. In the case of cortico- hydroxysteroid dehydrogenase. This is a first
steroid inhalation therapy, PK/PD modeling step in the metabolism of corticosterone.
identified the importance of prolonged pulmo- Cleavage of C-17 Acyl/Alkyl Substitu-
nary residence time as an important parame- ents. ~ e s u l t i nprimarily
~ in cholan-17-ones,
ter to improve pulmonary targeting. this is a relatively minor metabolic pathway.
Corticosterone is not known to undergo this
tranformation before excretion.
6 C L U C O C O R T I C O I D BIOSYNTHESIS C-6 Hvdroxvlation. This biotranformation
A N D METABOLISM is more predominant in infants than adults,
and can prevent other metabolic transforma-
Glucocorticoids are biosynthesized from cho- tions.
lesterol and released as needed by the adrenal Glucuronidation. Com~lexationof the ste-
A

cortex; they are not stored. Cholesterol under- roid to glucuronic acid, most predominantly
goes a series of irreversible oxidations during through the C-3 hydroxyl, leads to a consider-
which carbons 22 through 27 are cleaved, re- able portion of the excreted metabolites of all
sulting in pregnenolone. Reversible isomer- glucocorticoids. In infants, sulfurylation (for-
ization of A5 to A4 (progesterone) followed by mation of a sulfate ester) is also predominant
1 1 , 17a-, and 21-oxidations (flavoprotein, (67).
cytochrome P450-mediated) results in corti- Other Reactions. Most of the metabolites of
sol. The major metabolic transformations of cortisol are neutral (alcohol or glucuronide
the adrenal cortical hormones generally follow complex) compounds. However, oxidation at
the metabolism of cortisol, which undergoes C-21 to C-21 carboxylic acids (68) accounts for
the following conversions in vitro (Fig. 15.5). some of the identifiable metabolites of glu-
Cortisol-Cortisone Conversion. Under nor- cocorticoids (69).
mal conditions, this equilibrium slightly fa- Compounds with the 16,17-ketal (e.g.,
vors the oxidized compound. Similarly, the budesonide, amcinonide, fluocinonide, halci-
conversion of corticosterone to ll-deoxycorti- nonide, triamcinolone acetonide, and fluran-
costerone is also mediated by the llp-hydroxy- drenolide) also undergo metabolism by routes
steroid dehydrogenase enzyme system and re- that parallel that of cortisol metabolism.
quires NAD(P)+/NAD(P)H.This conversion is Unsymmetrical acetals such as budesonide
especially important both in the protection of (Fig. 15.6) are also metabolized by routes not
the human fetus from excessive glucocorticoid available to the more metabolically stable
exposure and in the protection of distal symmetrical 16a,l7a-isopropylidiene-dioxy
nephron mineralocorticoid receptors from substituted compounds (desonide, flunisolide,
-

glucocorticoid exposure (65). The impairment triamcinolone acetonide). Isozymes within the
of this conversion is thought to result in hy- cytochrome P450 3A subfamily are thought to
pertension associated with renal insufficiency catalyze the metabolism of budesonide, result-
(66). ing in formation of 16a-hydroxyprednisolone
Anti-Inflammatory Steroids

Pregnenolone

NADP*

-t
1
NADPH + H*

Stema t l p m o n w g e n a s s and
3@Hydroxy-~+stemid
dehydrogsnase
Ferredoxin lo$ Hfl 02 Fenedoxin (red)
0 0
Progesterone
(35)
NADPH + H*

Steroa

I I-Deoxymrti~ol
(cortexolone)
(39)

Figure 15.5. Biosynthetic pathways for formation of cortisol from cholesterol.


6 Clucocorticoid Biosynthesis and Metabolism

Budesonide, (44)
16a-Hydroxyprednisolone,
16-butyrate ester, (45)

Figure 15.6. Metabolism of 22R-budesonide in human liver microsomes.

and 6P-hydroxybudesonide (70, 71) (Fig. effects on metabolism include 2p-methyl,


15.6), in addition to the more common meta- which stabilizes the resulting molecule to the
bolic steps (oxidation through A6, reduction of action of the 4,5-reductase (72) and to the ac-
A3, etc.). tion of 20-keto reductases (73). Similarly, 6a-
Steroids with the greatest number of sub- methyl protects the A-ring against metabolic
stituents generally have the slowest rate of destruction (73). Again, the introduction of
metabolism. Groups that appear to activate by 16a-hydroxyl,as in triamcinolone (13a),pro-
776 Anti-Inflammatory Steroids

Table 15.13 Half-Lives of Corticosteroids in Dog Plasma

CH3

(90)
Steroid Half-Life (min) Reference
Cortisol (2)

Prednisone (29)
9a-Fluorocortisol(3)
Dexamethasone (20a)
Prednisolone (21)

6a-Methylprednisolone (28)
6a-Methyl-9a-fluoroprednisolone (90)
Triamcinolone (13a)

longs the half-life (74). The l6a-methyl and A), indicating a slightly higher molecular
16P-methyl groups have similar action. Tri- mass (94 kDa) than that of the "B" form (81,
amcinolone is unusual in that it is metabolized 82). The functionally active GR has been pu-
principally to the 6P-hydroxy derivative (74). rified (83) and, although an X-ray structure
Table 15.13 presents data on the half-lives of is not available, a significant amount of 3D
corticosteroids in dog plasma (75-80). structural information on the receptor has
been gathered. The GR is a member of the
7 MECHANISM OF ACTION O F nuclear receptor superfamily, which in-
ANTI-INFLAMMATORY STEROIDS cludes receptors for steroids, vitamin D, and
thyroid hormones, and some other proteins
7.1 Clucocorticoid Receptor Structure (84). A high degree of homology is found
The effects of glucocorticoids are thought to within receptors in this class, each contain-
be a consequence of their interaction with a n ing a similar domain organization. These do-
intracellular receptor, and great strides mains (sections of primary structure) have
have been taken in the task of determining a functional duty, and generally describe
the structure and function of the glucocorti- regions of hormone binding, nuclear trans-
coid receptor (GR). Two isoforms of the GR location, dimerization, DNA binding, and
have been identified, with an "A" form (GR- transactivation (85).
7 Mechanism of Action of Anti-Inflammatory Steroids

Within the carboxyl-terminus portion of a-helical substructure interacts with the nu-
the protein (residues 518-795) is the ligand cleotide. This DNA-receptor complex struc-
(hormone) binding domain (HBD) (85). It is ture is supported by the crystal structure of a
here that much of the interaction of the recep- similar DNA-receptor fragment (DBD) com-
tor with hsp90 occurs (86),and mutation stud- plex. Clear dimeric interaction can be seen,
ies have found that three of five cysteine resi- along with the general shape of the zinc finger
dues in this area, spaced close together in the region (Fig. 15.7).
binding pocket (871, are critical to the recep- Two domains (tl and t2) exist that affect
tor's ability to bind specifically glucocorticoids the GR post-DNA binding transcription activ-
(88). Mutation of other amino acid residues ity (96). The major (tl) transactivation do-
within the HBD may or may not have an effect main is 185 amino acid residues in length with
on ligand binding or receptor activity. How- a 58 residue a-helical functional core (97). The
ever, a key amino acid sequence in the rat GR t l domain is located at the N-terminus of the
(residues 547-553) has been shown to be both protein; the minor (t2) transactivation do-
critical for ligand binding and essential for re- main resides on the carboxy-terminal side of
ceptor-hsp90 complexation. It has been sug- the DNA-binding domain. These domains help
gested that hsp90 hellis the GR fold to its ste- control the transcription of target genes by
roid binding conformation by interacting with providing a surface to interact with general
these hydrophobic residues. This sequence transcription factors, and are thought to be
contains an LXXLL motif, often called an NR- bridged by a heteromeric protein complex in-
box, commonly found in other nuclear recep- cluding Vitamin D receptor activating proteins
tor (NR)-interacting proteins. Antiglucocorti- (DRIPS)(98).
coids and certain other modulators (as well as
7.2 Mechanism of Action
sodium molybdate) will interact with the HBD
domain and inhibit activation. The process by which anti-inflammatory ste-
The DNA-binding domain is highly con- roids impart their action is based on the action
served among species, and changes to the of the steroid on a receptor (GR).One result of
amino acid sequence in this region result in this process is the lag between optimum phar-
changes in receptor function (89).A strucutral macologic activity and peak blood concentra-
feature that characterizes the GR-DNA bind- tions. The stages the GR goes through in be-
ing domain are the two "zinc fingers," each in coming active can be divided into five general
which two zinc (+2) ions are held in place by steps (99, loo), and each step is mediated by
tetrahedral coordination to neighboring cys- the glucocorticoid receptor (GR):
teine residues (90). These zinc fingers, com- 1. Subcellular Localization. Some debate
mon to the nuclear receptor superfamily, are still exists as to whether GRs are cytoplasmic
not exactly the same as those found in Xeno- or nuclear in nature (101, 102), although it is
pus TFIIIA, in which histidine residues are believed that the interaction of hormone and
also associated with the metal (91, 92). The receptor occurs in the cytoplasm. The binding
zinc in the GR is thought to stabilize the a-he- of glucocorticoid agonists or antagonists to the
lices at the carboxy ends of the "fingers" as GR results in complete translocation of these
well as aid in carboxy-terminal module fold- receptors into the nucleus (103). Phosphoryla-
ing, needed in the dimerization of the proteins tion sites on the GR do not seem to play a role
(93). Although the entire glucocorticoid recep- in hormone-inducible nuclear translocation.
tor tertiary structure has yet to be elucidated, Sequences controlling nuclear localization
some of the components of the structure, in- have been identified within steroid receptors
cluding the DNA-binding domain, have been in the hinge region. The glucocorticoid recep-
depicted through molecular modeling studies tor has a second nuclear localization signal in
(94), NMR investigations (95), and the crystal the hormone-binding domain (104).
structure data from GR protein fragments 2. Association with Heat Shock Proteins
(93). Solution-state analysis of the DNA-bind- (hsp). Unactivated GRs are complexed with a
ing domain complexed to a nucleotide se- number of protein factors that play various
quence (double helix) reveals areas where an roles in the binding of ligand to the receptor,
778 Anti-Inflammatory Steroids

Figure 15.7. DNA-receptor complex structure. Znf ions can be seen as spheres within the GR DNA
binding domain (ribbon). See color insert.

as well as the localization, DNA binding, and minus of these steroid hormone receptors.
transactivation of the GR. The proteins, in- This domain is also credited with having the
cluding hsp90, hsp70, hsp56, CyP40, and p23 functions of hormone-dependent dimerization
(an acidic protein), have been implicated to be and transactivation. The ligand-binding do-
part of an assembled complex sufficient to ac- main of the glucocorticoid receptor appears to
tivate GR to the ligand-binding state, and this repress transcription in the absence of hor-
complex is termed a foldsome (105, 106). mone and this transrepression is reversed by
Hsp9O has been shown to be associated with hormone. Active glucocorticoid receptor ago-
the ligand-binding portion (C-terminus) of the nists bind tightly to the hormone receptor to
receptor (86), perhaps even blocking this site, elicit their action. Binding of glucocorticoids
although it is needed for the GR ligand-bind- to the GR hormone-binding domain trans-
ing domain to be in the proper conformation forms the receptor into an activated complex
for ligand binding (107). Hsp7O assists the able to interact with DNA sequences, called
binding of hsp90 to the receptor. Upon ligand the glucocorticoid response elements (GREs)
binding, the heat shock proteins dissociate of target genes.
and the receptors become active in dimeriza- 4. Dimerization and DNA Binding. The ac-
tion, DNA binding, and transcriptional en- tive regulatory form of the GR is thought to
hancement (108). Although these hsp proteins be dimeric (109). The GR binds to a palin-
do block certain DNA-binding sites on the re- dromic DNA sequence (GRE), either as a GR
ceptor, protein-free receptor studies have in- dimer (110) or perhaps as a GR monomer,
dicated that the free receptor still needs to be followed by binding of a second GR. Crystal-
bound to hormone to bind to DNA. lographic studies with GR fragments (con-
3. Hormone Binding. The ligand-binding taining the DNA-binding domain) have indi-
domain encompasses almost the entire C-ter- cated this dimerization occurs upon binding
7 Mechanism of Action of Anti-Inflammatory Steroids

to the half-site of the GRE (93). Members of CSF (126), TNFa (119)l. Steroids have been
the steroid hormone receptor superfamily also shown to suppress the formation of cyto-
contain a highly conserved DNA-binding kine receptors (8); dexamethasone, in particu-
domain of about 70 residues, which are com- lar, downregulates gene transcription of an-
plexed around two tetrahedrally coordi- giotensin I1 type 2 receptors (127).
nated zinc atoms. Activated cytoplasmic GRs can be involved
5. Transactivation. Protein synthesis is ini- in the regulation of transcription of genes ex-
tiated or inhibited by the action of the acti- pressing inducible enzymes. Dexamethasone
vated GR on DNA. The use of glucocorticoids can reduce prostaglandin production by inhib-
leads to anti-inflammatory effects by first con- iting cyclooxygenase (COX-2) gene expression
trolling gene expression, which subsequently (128), although this is thought to be through
leads to the synthesis andlor suppression of MAP kinase (p38) interaction (129). Dexa-
inflammation regulatory proteins. methasone inhibits release of prolactin by LC-
One such regulatory protein, inducible by a 1-dependent and LC-1-independent routes
number of glucocorticoids, is the 37-kDa pro- (122). Moreover, glucocorticoid excess down-
tein lipocortin 1 (LC-1) (111,112). Dexameth- regulates the expression of the GR (130).
asone directly effects de novo LC-1 synthesis, The mechanisms by which the glucocorti-
leading to direct increases in intra- and extra- coid receptor is able to inhibit signaling path-
cellular LC-1 concentrations, most notably oc- ways controlled by the transcription nuclear
curring at the cell surface (113). Prednisolone factors AP-1 and NFKBare beginning to be
increases extracellular and decreases intracel- elucidated (131). NFKBplays a very important
lular concentrations (114) of LC-1. Studies role in the transcription of many proinflam-
with LC-1 and with an active LC-1 segment matory genes. The role of the GR in the in-
show direct involvement of this protein in in- flammatory response can be attributed, at
hibiting neutrophil activation through inhibi- least in part, to the inhibition of NFKBfunc-
tion of the release of elastase, PAF, leukotri- tions (132). In addition to COX-2, NFKBup-
ene B4, and arachidonic acid (115), as well an regulates a number of genes that include
inhibition of neutrophil adhesion to endothe- many cytokines (TNFa, IL-1, IL-2,3,6,8,12)
l i d monolayers (116, 117). LC-1 inhibits vari- (133,134), and adhesion molecules that are an
ous prostanoid (inflammation mediator) pro- integral part of inflammatory processes (135).
duction, it suppresses thromboxane A, release Activated glucocorticoid receptors directly in-
from perfused lungs (118), and has thus been teract with and inhibit NFKBsubunits. In ad-
shown to inhibit inflammation in the rat paw dition, glucocorticoids transcriptionally acti-
(carrageenan-induced edema) inflammation vate the I K B ~gene and block nuclear
model (119). This is attributed to LC-1 inhibi- translocation of NFKBand DNA binding.
tion of phospholipase &, which converts Another inducible enzyme, nitric oxide
membrane phospholipids to arachidonic acid, synthase (iNOS) produces NO, which in-
along with its effect on other cellular compo- creases airway blood flow and plasma exuda-
nents of certain inflammatory responses tion, and may amplify T-2 lymphocytes, which
(119). Antibodies raised against LC-1 are able orchestrate eosinophilic inflammation in the
to reverse the anti-inflammatory action of airways. Glucocorticoids probably inhibit
LC-1 (120-122). Corticosteroids reduce phos- iNOS by inactivating NFKB,which regulates
pholipase A, activity, which results in the di- iNOS gene transcription (1361, or by other
minished release of arachidonic acid, and this pre- and posttranscriptional regulation (137).
subsequently leads to limiting the formation A number of cytokines induce iNOS, and glu-
of prostaglandins, thromboxane, and the leu- cocorticoids can also inhibit iNOS activation
kotrienes (123). by inhibiting cytokine formation. LC-1 is also
Glucocorticoids have been shown to inhibit a mediator in iNOS induction inhibition by
gene transcription of other proteins involved dexamethasone (138).
in the inflammatory process, including the key A model has been developed that describes
inflammation mediators called cytokines [in- the relationship between exogenous and en-
cluding IL-1 @), IL3-6 (1241, IL8 (1251, GM- dogenous corticosteroid action in inflamma-
780 Anti-inflammatory Steroids

tion (139). In general, topical glucocorticoste-


roids are found to suppress plasma cortisol
concentrations [adrenal suppression (140)],
especially with prolonged administration and
in high doses.
Certain inflammatory disease states in-
volve an inherent defect in the production
andlor regulation of inflammatory mediators.
In rheumatoid arthritis, an inflammatory dis-
ease targeting mainly joints, glucocorticoid
(hydrocortisone)-induced LC-1 production is
(91a) R = F
impaired (141). In some disease states (ulcer- (9lb) R = C1
ative colitis. Crohn's disease) anti-LC-1 anti- (91c) R = Br
body levels are raised, and this may partly ex- (9ld) R = I
plain why corticosteroid drugs are not always (9le) R = OH
as effective in these circumstances (142). In (910 R = CH3
steroid-resistant asthma, an abnormal recep- (9lg) R = 0CH3
tor-activator protein 1 interaction is observed (91h) R = 0C2H3
(143). (91i) R = SCN
Glucocorticoids also influence other physi-
ological and biochemical pathways, and a gen- A similar situation can be seen from the
eral overview of this action is listed in Table data of Smith et al. (56) (Table 15.16) regard-
15.14. ing the progesterone receptor. Whereas 6-sub-
stituents and the l7a-acetoxy group actually
8 EFFECTS ON D R U G RECEPTOR decrease the receptor binding of progesterone,
AFFINITY Clauberg activity is markedly enhanced (foot-
note, Table 15.16). Decreased metabolic inac-
The effect of structural alteration in steroids tivation may be responsible for this, although
on receptor affinity, which could only be more data are needed. Even the 19-nor modi-
guessed at before 1960, has received increas- fication, which substantially increases recep-
ing study as the fascinating story of steroid tor binding, increases biologic activity by a fac-
receptors has unfolded (144).However, the re- tor of only 6, whereas the most powerful
ceptor affinity of a given steroid is not the sole enhancing groups (Table 15.16) raise activity
or even the major determinant of its pharma- by a factor of 60.
cological potency. This can be appreciated Nevertheless, the relationship between
readily from the data of Wolff et al. (145) (Ta- chemical constitution and receptor binding is
ble 15.15) relating to the glucocorticoid recep- of great interest, given that receptor binding is
tor of rat hepatoma cells. Whereas 9a-F (91a) a sine qua non for biological activity. In a sys-
and 9a-C1 cortisol (91b) have essentially the tematic study of the thermodynamics of bind-
same receptor affinity, their pharmacological ing of 29 different corticoids to the glucocorti-
activity differs by a factor of 2. coid receptor of rat hepatoma cells, Wolff et al.
The enhanced pharmacological potency of (145)formulated a concept of the nature of the
the 9a-F derivative is thus only partially ac- steroid receptor interaction that rationalizes
counted for at the receptor affinity level, and the thermodynamic properties of the steroid
one or a combination of other major processes receptor-binding process and affords a basis
(intrinsic activity, drug distribution, and ef- for predicting the binding affinity of any glu-
fects on metabolism) must also be affected. cocorticoid derivative.
The data in Table 15.15 indicate that intrinsic The temperature dependency of binding of
activity is in fact also affected by the 9a-sub- these glucocorticoids to the rat HTC receptor
stituent, given that TAT induction is en- was determined and a second-degree polyno-
hanced by the 9a- F substituent relative to the mial equation was fitted to the data points ob-
9a-C1group. tained (Fig. 15.8). The enthalpic and entropic
Table 15.14 Glucocorticoids Act by Affecting Many Biochemical Systemsa
Decrease inflammation Stabilizing leukocyte Reducing leukocyte Antagonizing Inhibiting macrophage
by: lysosomal adhesion to capillary histamine activity accumulation in
membranes endothelium and release of kinin inflamed areas
from substrates
Preventing release of Reducing capillary wall Reducing fibroblast Decreasing
destructive acid permeability and proliferation, complement
hydrolases from edema formation collagen deposition, components
leukocytes and subsequent scar
tissue formation
Suppress immune Reducing activity Decreasing Possible depression of
response by: and volume of the immunoglobulin and tissue reactivity to
lymphatic system complement antigen-antibody
concentrations interactions
Producing Decreasing passage of
lymphocytopenia immune complexes
through basement
membranes
Other actions: Simulate erythroid Prolong survival time Produce neutrophilia
cells of bone of erythrocytes and and eosinopenia
marrow platelets
Promote protein Reduce intestinal Promote
catabolism and absorption and redistribution of fat
gluconeogenesis increase renal from peripheral to
excreton of calcium central areas of the
body
aModified from Ref. 9.
782 Anti-Inflammatory Steroids

Table 15.15 Comparison of Receptor Affinity and Intrinsic Activity of 9a-Substituted


Cortisol Derivatives in Hepatoma Tissue Culture Cellsa
-Log MI,, TAT Relative Potency
Compound Log K s Induction (glycogen deposition, rats)

"Ref. 145.

Table 15.16 Receptor Binding and Biologic (Clauberg) Activity in Progestational Steroids a
Compound Receptor Binding (%) Clauberg Activity (%)
6a-Methylprogesterone (92)
Chlormadinone (93)
(94)
6a-Methyl-17a-acetoxy-progesterone
6a-Fluoroprogesterone(95)
19-Norprogesterone (96)
Progesterone (41)
"Ref. 56.
*Claubergassay, in vivo progestation activity determined in rabbit uteri as a f h d i o n of endometrial growth.
8 Effects on Drug Receptor Affinity

Figure 15.8. Plot of in Ka (Kais the association


constant).
Figure 15.9. Plot of the log of the association rate
constant vs. 1/T ("K) for dexamethasone binding to
GR. [Adapted from Wolff et al. (1451.1

cause these phenomena are characteristic of


the hydrophobic effect. From the temperature
dependency of the rate constant (Fig. 15.9),
the enthalpy of activation was found to be 12.8
kcallmol and the entropy of activation to be
17.2 eu, indicating that the driving force for
the formation of the transition state is also the
hydrophobic effect. It is noteworthy that if the
formation of ligand-protein hydrogen bonds
and other oriented structures were of para-
terms of binding were calculated. As was the mount importance in the transition state, the
case for other steroid-protein interactions entropy of activation would be negative,
(591, both enthalpy and entropy decreased as rather than positive. Hydrogen bonding pre-
the temperature was increased (Table 15.17). sumably contributes very little to the overall
It was concluded that the steroid receptor driving force for ligand-protein interactions,
binding forces are mainly hydrophobic in given that the net differences in free energy
character. Both the steroid and receptor are between hydrogen bonding for ligand-protein
extensively hydrated and the displacement of in the bound state and hydrogen bonding be-
water molecules upon binding is a principal tween ligand, protein, and water in the un-
driving force. This is reflected by the positive bound state are probably small.
entropy (AS), negative enthalpy (AH), and From a consideration of the relationship
negative heat capacity (AC,) of association be- between the surface area of proteins and

Table 15.17 Calculated Values of the Enthalpic and Entropic Terms for Corticosterone
Binding to the Rat HTC Glucocorticoid Receptofib
Temperature PC)
Term 0 5 10 15 20
AH, d m 0 1 1200 0 - 1000 -2200 -3200
AS,eu 29 24 20 15 12
"Ref. 145.
b ~ hpolynomial
e function employed was 1.62 X + 118,000(1/T)- 195 = In K,.
lo7 (1/p)
784 Anti-Inflammatory Steroids

Table 15.18 Free Energy Contribution to Binding to the Rat J3T.C Glucocorticoid
Receptor per Substituent of Progesteronea
Fried Glycogen Deposition
Substituent Free Energy (kcal/mol) Enhancement Factor (Rat)
A1
6a-F
6a-CH,
9a-F
9a-C1
9a-Br
9a-OCH,
11p-OH
11p-OH + 11-keto
11-Keto
16a-CH,
16P-CH,
16a-OH + acetonide
17a-OH
21-OH
"Ref. 145.

its contribution to hydrophobic bonding (146- comparison of the binding of 22 corticoids, the
148),it could be shown (145)that the energy of approximate free energy increments of each
binding could best be accounted for if the en- substituent group were calculated (Table
tire steroid were enveloped on both sides by 15.18) (145). These free energy group incre-
the receptor. The steroid appears as a "ham- ments can be added to approximate the bind-
burger patty" enveloped on both sides by the ing constants of steroids whose binding con-
"hamburger bun" of the receptor. stants are unknown. Thus, the free energy of
Interestingly, Anderson et al. (149) pro- binding of fluocinolone (lla)to the rat HTC
posed a similar picture of the binding of an- receptor relative to progesterone is calculated
other nonprotein ligand to a protein. This is as follows for substituents in fluocinolone in
the case of the complex between glucose and excess of the progesterone skeleton.
hexokinase, an enzyme possessing a deep cleft This calculation predicts that fluocinolone
between two lobes. They concluded that a dra- would bind to the receptor with a free energy
matic conformational change occurs in hexo- of -1.32 kcallmol more negative than proges-
kinase as glucose binds to the bottom of the terone, in fair agreement with the experimen-
cleft: the two lobes of hexokinase come to- tal value of -1.65 kcallmol. These free energy
gether, engulfing the sugar. These workers increments may be compared with the phar-
proposed that glucose is sufficiently sur- macological enhancement factors of Fried
rounded by the enzyme in this closed confor- (153) (Table 15.18; also see Section 9). It is
mation that it cannot leave its binding site seen that there is little correlation between
(150), which provides an explanation for the the two parameters, indicating again that
observation (151) that the off-rate of glucose other variables such as the inhibition of met-
from its hexokinase complex is slow (58 s-l). abolic destruction, are of major importance.
It is noteworthy that far slower off-rates are The conformation of the A-ring (154) has a
characteristic of steroid-receptor complexes. pronounced effect on the binding of the steroid
Another interesting point relates to the ques- to the receptor. The difference in the C-3 to
tion of whether the steroid interacts with the C-17 distance from progesterone in the ste-
receptor only on its p face, as suggested by roids was employed as a measure of the A-ring
Bush (152). The thermodynamic data indicate conformation, given that this distance is
that this is not the case and that all of the strongly influenced by such conformational
steroid is in contact with receptor. From a changes. It appeared that binding was greater
8 Effects on Drug Receptor Affinity

(114

Substituent AG Binding Contribution (kcal/mol)


A1
6a-Fluoro
9a-Fluoro
11p-Hydroxy
16a-Hydroxy
17a-Hydroxy
21-Hydroxy

Total

as the distance decreased. The inclusion of a to account for the specific favorable interac-
fluoro group at C-9 or a double bond at C-2 had tion with the hydrogen bond acceptor in the
the greatest effects on the A-ring conformation. receptor. If no group resides in these positions,
Other substituents had varying effects on a value of 0 is assigned. A value of -1 is as-
the direction and magnitude of changes in the signed for the presence of each C-17 or C-16
A-ring conformation. The effects of 9a- polar group, to account for the consequences
methoxy and 9a-bromo substituents stand of placing a polar group in a nonpolar region,
apart in these binding studies. They result in and a value of -2 is given for the presence of
derivatives with low binding affinities (Table an 11-keto functionality, to express the con-
15.18),yet the surface area increases because formational change associated with an sp3 to
of the respective 9a-substituent. Evidently, sp2 transformation and the undesirable di-
the size of these substituents prevents the pole-dipole interaction of the ll-keto group
proper engagement of the steroid within the with the hydrogen bond acceptor of the recep-
receptor site or induces a conformational tor apparently in that position. A total of these
change in the receptor such that binding is values is used for the second parameter, de-
significantly altered. noted as the polar interaction term. The third
A multiple regression analysis relating the parameter (tilt) expresses the conformation of
above-noted four parameters with the loga- the A-ring through the C-3 to (2-17 distance in
rithm of the dissocation constant was made. angstroms. The fourth parameter (XI ex-
The surface area (SA) employed for each de- presses the size limitation at the 9a-position.
rivative was the summation of the Bondi sur- The value employed is the maximum of the
face of each substituent present over that of a function (0, R, - R,,) where R, is the distance
progesterone skeleton. The second parameter in angstroms that a substituent radially ex-
(P)is a de novo variable representing the in- tends from the pregnane ring system. An ex-
teraction of polar groups with the receptor. cellent correlation was found relating these
For each hydroxyl group present in the C-11 four parameters to the logarithm of the equi-
andlor C-21 position, a value of +1 is assigned, librium dissociation constant:
786 Anti-Inflammatory Steroids

callA2, based on the work of Chothia (146).


The absolute value of 0.76 kcal per P unit
agrees well, with the values ranging from
-0.89 kcal (attractive) to +0.86 kcal (repul-
sive) for hydroxyl groups (Table 15.18) and
with the figure of -600 cal per hydrogen bond
in the binding of trisaccharides to lysosome
(155). The high value of the X term indicates
the disruptive effect on binding because of the
9.4 8.6 8.0 7.4 6.6 5.6 introduction of a group larger than the corre-
sponding "pocket" in the receptor. A study by
Calculated log KD
Ahmad and Mellors (156) indicated that the
Figure 15.10. Plot of observed logarithms of the
binding of steroid analogs to specific cytosol
equilibrium dissociation constant versus the values receptor proteins is correlated with the steroi-
calculated from the QSAR equation. [Adapted from dal parachors, although a quantitative rela-
Wolff et al. (145).1 tionship was not derived. Parachor is a molar
parameter defined as the product of the molar
volume and the fourth root of surface tension
(157). Thus, parachor and surface area are
strongly cross-correlated.

9 EFFECT OF STRUCTURAL CHANCE


ON PHARMACOLOGICAL ACTION
9.1 Pharmacological Tests
For this equation, n, the number of data
points, is 29; r, the multiple correlation coeffi- In Table 15.19 are listed various pharmacolog-
cient, is 0.97; s, the standard deviation of the ical tests for anti-inflammatory steroids.
regression, is 0.26; and F, a measure of the Granuloma tests measure the ability of the rat
significance of the regression, is 106. Each pa- to encapsulate a cotton pellet and are a mea-
rameter is significant at better than the 0.999 sure of the anti-inflammatory effect. The liver
level. Shown in Fig. 15.10 is a plot of the cal- glycogen deposition test is an index of glu-
culated vs. the observed logarithm of the equi- cocorticoid action that usually is well corre-
librium dissociation constant. lated with the anti-inflammatory effect. The
An examination of the physical significance other tests in Table 15.20 reflect the diverse
of this equation is of interest, given that it action of these steroids. Nonsteroidal anti-in-
should reflect the thermodynamic contribu- flammatory agents are poorly active in the
tions of the substituents. The equation repre- granuloma tests, indicating the difference in
sents the effects of substituents on the K, their mode of action.
value of progesterone, given by the intercept Animal data do not always predict potency
(-6.52). By multiplyingby 2.303RT, the equa- in humans with accuracy. A number of exam-
tion is transformed to ples are shown in Table 15.20 (26). At least a
part of the problem lies in the fact that the rat
AG,,,,, (cal) = -27(+2)SA (cal/A2) secretes corticosterone (31),which is inactive
- 734(? 62)P (cal/P) as an anti-inflammatory in humans, rather
+ 1865(+435)tilt (cal/A2) than cortisol. The important drugs triamcino-
+ 7585(+609)X (cal/A2) lone acetonide (13)and dexamethasone (20a)
-8143 (cal), are much more potent in rats than in humans.
9.2 QSAR Analyses of Pharmacological
giving the thermodynamic equivalent of each
Action
parameter. The surface area term shows a
contribution of 27 cal/A2, in good agreement In Section 8 we considered the application of
with the temperature-corrected value of 22.5 QSAR techniques to a single process underly-
9 Effect of Structural Change on Pharmacological Action

Table 15.19 Biologic Evaluation of Anti-inflammatory Steroids


Method Species Reference
Granuloma (pellet) Rat
Granuloma (pouch) Rat
Thymus involution Rat
Adrenal suppression Rat
Adrenal steroid concentration Rat
Body weight depression Rat
Eosinopenia Mouse
Dog
Liver glycogen deposition Rat
Mouse
Ulcerogenesis Rat
Sodium retention Rat

ing steroid action, that is, drug receptor bind- certain positions of the steroid molecule.
ing. In this section we examine the attempts These workers discovered the remarkable fact
that have been made to express the gross that each substituent affects the activity of the
pharmacological activity of anti-inflammatory molecule almost independently of the pres-
steroids through QSAR techniques. ence of other activity-modifying groups. The
As already noted, pharmacological activity effect of each substituent was assigned a nu-
represents the summation of a number of pro- merical value, a de novo constant termed an
cesses including absorption, metabolism, drug enhancement factor (Table 15.21). Multiplica-
receptor affinity, intrinsic activity, and drug tion of the biologic activity of a parent com-
distribution. The effect of a given substituent, pound by the enhancement factors for the sub-
such as a 9a-substituent, on these combined stituent groups gives the activity of the final
processes is difficult to parameterize; a single
analog. For example, Table 15.22 (153) illus-
parameter for a substituent can represent
only its average effect. Therefore, QSAR anal- trates the calculation of the potencies of a se-
yses of gross pharmacological activity are nec- quence of steroids, starting with llp-hy-
essarily less accurate than those relating to a droxyprogesterone and culminating in
single process such as drug receptor binding. triamcinolone. The ranges obtained are in
On the other hand, because pharmacological good agreement with the bioassay figures and
activity is the goal of drug design, the attempts their 95% confidence limits. Fried and Bor-
described in this section have considerable in- man were unable to derive similar quantita-
terest. Such studies represent approaches tive expressions for salt-retaining activity, al-
only to the correlation of activity with struc- though the action of the various substituents
ture and have not given final answers. How- on salt retention could be expressed in semi-
ever, because they are relatively rigid mole- quantitative terms.
cules in which the effects of structural change Other investigators have added additional
are easily understood in steric and electronic enhancement factors to those listed in Table
terms, and because we know something of 15.21. In Table 15.23 additional values are
their mechanism of action, steroids represent given, including those for activity in humans.
a fruitful area in QSAR. Although activities in humans and the rat are
similar for many substituents, a major species
9.2.1 Use of De Novo Constants. The ear- difference is seen for the 2a-methyl group and
liest QSAR analysis of anti-inflammatory ste- the 6a-fluoro group. In Table 15.24 are listed
roids, and indeed one of the first QSAR analy- enhancement factors from another laboratory
ses of any kind, was carried out by Fried and (166), in which the 9a-fluoro substituent has a
Borman (153) in an examination of com- value of 3-4 rather than 7-10. A Fujita-Ban
pounds obtained by introducing halogen, hy- analysis on 44 corticoids was carried out by
droxyl or alkyl groups, or unsaturation into Justice (167).
788 Anti-Inflammatory Steroids

Table 15.20 Some Anti-inflammatory Steroids with Atypically Poor Correlation


Between Human and Animal Dataa

(100)
(99)
Anti-Inflammatory Potency
Compound Animals Human
Cortisol (2) 1.0
Corticosterone (31) Inactive
A6-Cortisone (97) Inactive
16a-Methylcortisol(98) 3
2a-Methylcortisol(99) -1
Triamcinolone 16,17-acetonide (13) 4
(100)
21-Deoxy-16a-methyl-9a-fluoroprednisolone 475
Betamethasone (5a) 30-35
Dexamethasone (20a) 30
"Ref. 26.

9.2.2 Hansch-Type Analyses. Wolff and compounds. It was found that activity was cor-
Hansch (168) carried out the first multipa- related with the inductive effect (a,),the size
rameter regression analysis for steroids in an of substituents (molar reactivity, P,), and T,
analysis of the anti-inflammatory activity of giving the equation
9a-substituted cortisol derivatives. A series of
seven active compounds, the 9a- F (91a),
9a-C1 (91b), 9a-Br (91c), 9a-I (91d),9a-OH
(91e),and 9a-CH, (91f) cortisol derivatives as For this equation n, the number of data points,
well as cortisol itself, were analyzed. The re- is 7; s, the standard deviation, is 0.33; and r,
sults were applied to the inactive 9a-methoxy the coefficient of correlation, is 0.96. The
(91g), 9a-ethoxy (91h), and 9a-SCN (91i) equation suggests that the activity of the com-
9 Effect of Structural Change on Pharmacological Action 789

Table 15.21 Fried-Borman Enhancement Factors for Various Functional Groups a


--

Anti-Inflammatory, Effect on
Functional Group Glycogen Deposition, Rat Rat (granuloma) Urinary Sodiumb

"Ref. 153.
'+ retention; -, excretion.
"In 1-dehydrosteroidsthis value is 4.
the presence of a l7a-hydroxyl group this value is < 0.01.

Table 15.22 Fried-Borman Calculation of Activities of Triamcinolone (13a) by Use of


Enhancement Factors

Glycogen Anti-Inflammatory Effect on Urinary


Deposition (granuloma) Sodiumb
Functional
Group ResultingCompound Calculated Found Calculated Found Calculated Found
llp-Hydroxy 0.1 <0.01
progesterone (89)
9a-Fluoro 9a-Fluoro-llp- 1 0.85 <0.1 <0.1 +++ +++
hydroxyprogesterone
(101)
21-Hydroxy 901- 4-7 4.6 <2.5 2.7 ++++++++++
Fluorocorticosterone
l7a-Hydroxy 9a-Fluorocortisol(3) 4-14 11 11 13 ++++ +++
1-Dehydro 9a-Fluoroprednisolone 12-56 28 3344 20 +++ ++++
(101a)
16a-Hydroxy Triamcinolone (13a) 4.8-28 13 3.3-8.8 4 -- --

"Ref. 153.
+, retention; -, excretion.
790 Anti-Inflammatory Steroids

Table 15.23 Enhancement Factors for Important Corticoid Substituentsa


Group Anti-Inflammatory (Rat) Anti-Inflammatory (Human)
1-Dehydro 3 4
2a-CH, 4.5 0.7
6-Dehydro 0.5 Variableb
6a-CH3 2 1.3
6a-F 8 2.5
9a-F 8 10
1601-CH3 1.5 2
16p-C3 1.8 2.5
16-Methylene 1.5 -
16a-OH 0.2 0.5
16,17-Acetonide 8 1, (oral)"
21-F 2 -
21-Deoxy 0.1 <0.1
"Ref. 153.
bUsually<l.
'Topical effect is high.

pound rises with increasing electron with- of the electron-withdrawing group at C-9 was
drawal, decreasing size, and increasing hy- to increase the acidity of the neighboring l l p -
drophobic bonding of the 9a-substituent. hydroxy group and that "the corticoid activity
Moreover, it correctly predicts that the of an llp-hydroxysteroid increases with in-
methoxy, ethoxy, and thiocyano compounds creasing acidity of the llp-hydroxy group."
have little or no activity. The inverse relation- They further speculated that "protein steroid
ship between the radius of the 9a-halogen binding at the site of action could be a function
atom and the magnitude of adrenocorticoid ac- of the acidity of the llp-hydroxy group."
tivity had already been noted by Fried (1691, Newer developments concerning this possibil-
but Fried and Borman argued that the low ity are described in Section 9.3.2.
activity of the 9a-hydroxy and 9a-methoxy The significance of the m- parameter is more
compounds, which also have relatively small difficult to delineate. The increase in activity
substituents, indicates that "it is the electro- with increasing hydrophobicity could be at-
negativity of the substituent rather than its tributable to better transport to the site of ac-
size that determines its enhancement proper- tion for the more lipophilic compounds andlor
ties." It is noteworthy that the quantitative to hydrophobic interactions at the active site.
multiparameter regression technique shows it In a reexamination of this problem Coburn
is perfectly possible for the inverse relation- and Solo (170) found that the activity of 9a-
ship with increasing size to exist, even though substituted cortisols is well correlated with IJI
a qualitative examination of the data led the and a simple steric factor such as molar refrac-
earlier workers to conclude otherwise. Fried tivity (MR), provided that the 9a-hydroxy-
and Borman (153) suggested that the function lated compound is excluded from the series.
Table 15.24 Adrenocorticoid Activity
They suggested that compounds containing
Enhancement Factorsa strongly hydrated 9a-substituents would have
a larger effective bulk than is found in the
Glycogen unhydrated species and hence a lower pre-
Group Deposition Thymolytic dicted activity. In another reexamination, Ah-
9a-F 3.3 3.9 mad and Mellors (156) found that molar para-
1-Dehydro 2.3 2.8 chor gives a better fit than T in single-
6a-Methyl 2.4 2.9 parameter equations but not in a three-
16a-Hydroxy 0.4 0.3 parameter equation.
16,17-Acetonide -2 -2.5 Pattern recognition approaches have been
"Ref. 166. applied to the analysis of glucocorticoid test
9 Effect of Structural Change on Pharmacological Action 791

data. Bodor applied the linear learning ma- set as potent or nonpotent. Finally, three dif-
chine (LLM) approach of Nilsson (171) and ferent methods were used for prediction of the
Jurs (172, 173) to human vasoconstrictor remaining 37 corticoids: LDF, KNN, and PC
(McKenzie)data as well as rat granuloma data plots. Within the set of 37 test compounds, 14
for 122 corticoids (174). Of these compounds, were unambiguously classified as P or NP.
over 70 were considered potent, whereas the KNN and PC methods correctly predicted 12
remainder were considered nonpotent relative of this unambiguous test set of 14. The au-
to hydrocortisone butyrate (2a).Of 33 start- thors suggest that with certain prescreening
ing, Free-Wilson-like indicator variables, 22 criteria, this approach may be useful in pre-
were used in the final analysis, in which an dicting potent vs. nonpotent corticosteroids.
LLM generated a nonparametric linear dis-
criminant function that correctly classified all 9.2.3 Use of Neural Networks to Predict
122 of the corticoids. Descriptor values were Corticoid Properties. Artificial neural net-
determined for separate substituents as well works such as a Kohonen self-organizing neu-
as for certain combinations of substituents. It ral network can be developed by conventional
was concluded that these values would allow means to describe various corticosteroid data
one to predict the contribution to potency of sets (176). New approaches to coding molecu-
various substituents. Stouch and Jurs (175) lar structures for QSAR that also employ neu-
later argued that these results (i.e., 122 data ral networks have been described recently.
points divided 48/74 between two classes, a Their relevance here is that they use a bench-
22-dimensional data space) can support cor- mark CBG-corticosteroid data set originally
rect classifications attributed to chance of be- described by Crarner et al. in 1988 (177), and
tween 85 and 90%. These authors, by use of later corrected in 1998 (178) in the seminal
the same vasoconstrictor test data as those paper on comparative molecular field analysis
used by Bodor, describe the compounds as po- (CoMFA).
tent (PI or nonpotent (NP) and coded the com- One reported method for reducing the di-
pounds instead with 10 descriptors: three mensionality of the 3D input data (molecular
indicator descriptors (A1*'; 16-methylation; data) was to produce self-organizing neural
andlor 16,17-acetonide)and seven descriptors maps (SOMs) (179). A transformation called
coding for the log P at the sites of varying sub- "hypermolecule" is carried out for each mole-
stitution (positions 6, 9, 17, 21) or esterifica- cule in the steroid data set, reducing the data
tion (positions 17 and 21). The data set was to a 2D-coded SOM. The SOM map for the
separated into a training set of 88 compounds most active compound was used for training. A
and a test set of 37, and then probed by linear series of comparative SOMs from each analog
discriminant analysis (LDF). Statistical anal- are then used in the unsupervised neural net-
ysis by use of Mahalanobis distances revealed work, to furnish a single trained neuron. In
that the two classes were well separated in the this way, different properties were studied
10-dimensional space. such as shape (r2 = 0.89, s = 0.49) and
Additional evidence for data structure was electrostatics.
provided by K nearest-neighbor (KNN) and In another approach called comparative
principal-component (PC) analysis. Based on molecular surface analysis (COMSA), the
initial analysis in which esters were misclassi- molecular surface electrostatic potentials
fied, presumably because of the nonlinear re- (MEPS) of this corticoid data set were ana-
lationship between chain length and activity, lyzed by a Kohonen self-organizing neural
the descriptors that coded for the log P of the network (180). Neighboring MEP points were
side chains were transformed. For example, placed into adjacent neurons in the Kohonen
the C-17 position was described by subtracting map. The most active corticoid in the data set
the log P of the side chain of propanoic acid was used as a template, and PLS analysis pro-
(1.55) and squaring the result for each value in vided statistical comparisons of models. The
the descriptor. By use of this modified set of 10 best model used D = 40, MD = 0.2 A, and had
descriptors, a linear discriminant resulted a cross-validated r2 value of 0.88 (s = 0.424),
that correctly classified all 88 of the training which outperformed CoMFA, with a cur2 value
Anti-Inflammatory Steroids

of 0.73 (s = 0.657). In this method, D was the increase its flexibility. In the case of many
density of the points sampled from the molec- anti-inflammatory steroids the presence of un-
ular surface and MD was the maximal dis- saturation at C-4 and C-1 produces com-
tance of the points allowed in a single neuron. pounds that may exist as a range of conform-
A unique approach combining neural net- ers oscillating over a broad energy minimum,
works with data reduction involves the or as several conformers of nearly equal en-
method called 3D-MORSE (Molecule Repre- ergy separated by a significant barrier. This
sentation of Structures based on Electron Dif- flexibility complicates the question of inter-
fraction) (181).A 3D-MORSEcode is a set of 32 atomic distances between such key atoms as
evenly distributed values of s in the following 0-3 and 0-20 and the effect of structural
equation: change on these distances. An obvious point of
interest in this connection is the conformation
N i-1
sin srij of the side chain of glucocorticoids, given that
I(s) = C 244
i = 2 j=1
srij S = O ,.... , 31.0A this portion of the molecule includes the key
0-20 and 0-21 oxygen functions. In principle,
a 360" rotation of the side chain is possible, but
in which I is the intensity of the scattered ra-
it is obvious that steric factors will tend to
diation; and A can be set to atomic number,
favor only selected conformations. Numerous
atomic mass, partial atomic charge, residual
investigators have studied this matter by
atomic electronegativities, or atomic polariz-
abilities. The values of s were obtained from chemical (182), physicochemical (183, 1841,
this function and the atomic coordinates cal- quantum chemical (1851, crystallographic
culated in the 3D structure generator (186, 187), and energy minimization (188)
CORINA. These sets of 32 values could then methods. Most of these analyses have indi-
be studied by PC analysis or counterpropaga- cated that 0-20 approximately eclipses C-16.
tion neural network (CPG). As applied to the In the energy minimization work of Schmit
above corrected CBG data set for 31 cortico- and Rousseau (188) significant differences
steroids, the network correctly classified low were found relative to the crystallographic
to medium to high affinity ligands. studies cited. They suggested that one reason
for the discrepancy between energy optimized
9.3 Some Steric and Electronic Factors and X-ray diffraction data could be the influ-
Affecting Anti-Inflammatory Activity ence of intermolecular hydrogen bonds and
packing forces in the crystal. Moreover, as
In the following we discuss a number of stud- they pointed out, in certain cases two indepen-
ies that use the techniques of extended Huckel dent crystallographic structures are observed,
theory (EHT) and geometry optimization indicating that the crystallographic data do
through energy minimization to determine not necessarily give an unambiguous picture
the preferred conformation of steroid mole- of side chain conformation.
cules. In some cases it is possible to compare The most important factor influencing the
these theoretical results with the findings of position of the C-20 carbonyl is the presence or
X-ray crystallography. Also discussed are elec- absence of a l7a-hydroxyl group, according to
tronic structure examinations through the use the study of Schmit and Rousseau (188).Duax
of CND012 molecular orbital calculations. Al- et al. (189) studied crystallographic data on
though none of these examinations has provided over 80 pregnanes, including numerous corti-
a comprehensive understanding of the effect of costeroids, and showed that the C(16)-C(17)-
structural change on biologic activity, a few un- C(20)-O(20)dihedral angle is consistently be-
derlying principles are beginning to emerge. tween 0" and -47". With the exception of 16-
substitution, this dihedral angle was usually
9.3.1 Effect of Structural Change on Steroid in the range of -20" for steroids having either
Conformation. Some steroids have greater 17a or 21-hydroxy groups, or their corre-
flexibility than others. The introduction of un- sponding esters. Hydroxylation at both the
saturation into the steroid molecule tends to 17a and 21 positions tended to shift this angle
9 Effect of Structural Change on Pharmacological Action 793

an average of -15", and into the range of -35". 9.3.2 Effects of Structural Change on Elec-
These studies did not demonstrate a correla- tronic Characteristics of the Steroid. As noted,
tion between side-chain hydoxylation and the electronic effect of the 9a-substituent was
side-chain orientation (intramolecular hydro- already invoked by Fried in attempting to ex-
gen bonding) as suggested by Schmit and plain how substitution at C-9 could modify bi-
Rousseau. Intermolecular complexation did ologic activity. More recently, the electron
not result in a change in the preferred dihe- densities in portions of cortisol, 6a-fluoro cor-
dral angle. These and other findings led Duax tisol, and 9a-fluorocortisol were calculated by
to suggest that force field calculations cannot use of X-ray structures and the CNDOA
provide accurate side-chain orientations in method by Kollman et al. (195). They con-
cluded that hydrogen bonding differences at-
pregnanes and corticosteroids. Underestima-
tributed to electron density changes engen-
tion of eclipsing interactions between the
dered by the 9a-substituent could not fully
C-16lC-17 bond and the C-20 carbonyl bond,
account for the observed activity differences.
and repulsive interactions between C-21 and Moreover, Divine and Lack (196) showed that
the 17-substituent were cited as shortcomings the hydrogen bonding ability of the hydroxyl
of the force field calculations. proton of 9a-substituted 11P-hydroxyprogest-
Weeks et al. (154) examined the structures erone derivatives decreases in the order F = C1
of six corticosteroids and noted a correlation > Br > H. If the cortisols behave similarly,
between the degree of bowing of the fused-ring there must be additional factors explaining
system toward the a face and the anti-inflam- the greater glucocorticoid activity of 9a-fluoro
matory activity. The presence of a C-1 double cortisol relative to 9a-chlorocortisol, which
bond and a 9a-fluoro group brought about has only four times the activity of cortisol it-
these changes. It is clear that bowing could be self.
only one of several factors affecting biologic
activity, given that 9a-chlorocortisol (190) is 9.3.3 3D-Quantitative Structure-Activity
about four times as active as cortisol, but is not Relationships of Steroids. Comparative molec-
bowed more than cortisol, whereas 9a- ular field analysis, or CoMFA, is a "3D-QSAR"
methoxy cortisol (191) is inactive but is bowed methodology whose development began some
more than cortisol. Schmit and Rousseau 12 years before the seminal work was pub-
(188)undertook a Free-Wilson (192) analysis lished by Cramer, Patterson, and Bunce in
of the effect of various substituents on the con- 1988 (177). CoMFA is described in some detail
formation of the steroid on the basis of the in volume 1 of this series. In this study a train-
ing set of 21 corticosteroids whose corticoste-
predicted energy minimization structure (not
roid-binding globulin (CBG) affinities were
the X-ray structure). In accord with the X-ray
known was selected for analysis. Ten other
results, they found that the curvature of the corticosteroids were excluded from the analy-
entire steroid molecule (AID angle) is in- sis and were later submitted for prediction in
creased 30% by a C-1 double bond and 14% by the pharmacophoric model.
an lip-hydroxy substituent. It is decreased In the CoMFA approach, a critically impor-
14% by a l7a-hydroxy substitution. These re- tant input variable is the representation of the
sults parallel the biologic data in rats. A geom- molecules and their mutual alignment or
etry minimization study by Marsh et al. (193) alignment rule. For relatively rigid molecules
indicated that the conformational effect of the such as the corticoid ring system, structures
9a-fluorogroup is attributed to its effect on B-, are reasonably represented by molecular me-
C-, and D-rings which then induces a confor- chanics. For the training set, Cramer first im-
mational change in the A-ring. Dideberg et al. ported crytallographic coordinates before con-
(194)analyzed the crystal structure data of 20 ducting molecular mechanics calculations
corticoids. On the basis of their results they with the Tripos force field. At this point, min-
suggested that the distance between the at- imization resulted in alteration of the C(16)-
oms on the receptor capable of binding the C(17)-C(20)-O(20) dihedral angle. The dis-
steroid at 0-3 and 0-20 is 16.5 k crepancy between this important tortional
794 Anti-Inflammatory Steroids

Figure 15.11. CoMFA steric contour plot of the 3D-QSAR of corticosteroid binding to the human
corticosteroid-binding globulin. Light-shaded contours correspond to regions where steric bulk from
the steroid is predicted to decrease binding, whereas black-shaded contours correspond to regions
where steric bulk is predicted to improve binding (177).

angle in the crystal state and from force field atom at each corner of the lattice. Finally, the
calculations has been pointed out by Bodor. target property of CBG affinities were ana-
Thus, although some question exists with re- lyzed relative to these field values by the pro-
gard to the reality ofthe D-ring side-chain con- cess of partial least squares (PLS) analysis
formation chosen for this study (e.g., does the with cross-validation. Matrices of lattice size
energy minimized structure mimic the recep- and so on were examined, as was the robust-
tor bound conformation of the steroid?), the ness of the QSAR, with q2 values over 0.75.
results are nevertheless meaningful for dem- The resulting QSAR equations could be dis-
onstrating the existence or nonexistence of a played for each analysis in three-dimensional
QSAR. The "alignment rule" for these mole- space as a contour plot. In this manner steric
cules was then defined by least-squares fit of contour plots provide visual information in
C-3, C-5, C-6, C-13, C-14, and C-17 to the cor- the region surrounding the data set regarding
responding atoms of the template molecule, the importance for binding of steric bulk (Fig.
deoxycortisol. The molecules were stored in a 15.11). Likewise, an electrostatic contour plot
molecular database and linked to a molecular provides visual clues regarding the effect of
spreadsheet within Sybyl. The database was polar groups on activity (Fig. 15.12). Ulti-
then enclosed in a box sufficient to encompass mately, structures can have their activity pre-
all of the molecules, with further subdivision dicted within the fields. Inspection of the con-
into lattices of variable size, ranging typically tour plots in Fig. 15.12 reveals some expected
from 1 to 2 A. A number of steric and electro- relationships on the basis of simple SAR (e.g.,
static field energy values were then deter- that the &position and the 20-position benefit
mined for each molecule by placing a probe qualitatively from polar groups such as ke-

Figure 15.12. CoMFA electrostatic con-


tour plot of the 3D-QSAR of corticosteroid-
binding globulin. Light-shaded contours
correspond to regions where partial nega-
tive charge from the steroid is predicted to
improve binding, whereas black-shaded
contours correspond to regions where par-
tial positive charge is predicted to improve
binding (177).
9 Effect of Structural Change on Pharmacological Action 795

tones). Of course, the magnitude of the effect


that position and orientation of these polar
groups have on activity is not revealed visu-
ally; for this, the QSAR equations underlying
the contour plots must be accessed. A set of 10
steroids for which binding data were available,
but that were excluded from the analysis, was
predicted in this model, as shown in Table
15.24.
Six of the 10 randomly chosen steroids had
activity predicted very accurately, with one
other still quite closely predicted. In the set of
steroids that were not predicted well were
structures that were not represented well by
the original data set of 21 compounds. For ex-
ample, members of the set of 10 steroids that
were excluded from the original CoMFA model
that were not predicted well were an A-ring Figure 15.13. Steric contribution of the 3D-QSAR
dienone, a C-2 substituted steroid, and a of pregnane and corticosteroid binding to the hu-
9-fluoro steroid. man corticosteroid-binding globulin. Black polyhe-
Through the use of PC analysis, Norinder dra correspond to positive nonbonded interactions,
examined QSARs for the binding of pregnanes whereas gray polyhedra correspond to negative non-
to human corticosteroid-binding globulin bonded interactions (197).
(197). Affinity data for 37 steroids, consisting
of 26 steroids having hydroxyl groups at both dominated the interactions, whereas MLP
or either of the 17- and 21-positions and 11 contours were not shown because of weak cor-
steroids having no hydroxyl group at either relations. The contour plots from this study
the 17- or 21-position, were collected. In a (Fig. 15.13) were quite similar to those ob-
manner analogous to the CoMFA procedure, tained by Cramer.
the energy-minimized steroids were aligned When this technique was applied to both
and encased in a gridwork of 1 . 5 4 spacings. human and guinea pig CBG binding data, as
Through use of a methyl probe atom, non- described above, the QSAR coefficients could
bonded interactions and charge-charge inter- be subtracted, providing difference contour
actions were calculated. A molecular lipophi- maps that could be useful in examining issues
licity potential (MLP) was calculated with the of species selectivity (198).
atomic lipophilicity factors for each grid point More recent approaches to QSAR of glu-
as well. A PC analysis was performed on the cocorticoids still employ the Cramer CBG
matrix, with each molecule represented by the binding data set, but focus on molecular eig-
three fields (7425 points). Four principal com- envalue descriptors for QSAR (199), as well as
ponents were determined that described over E-state topological descriptors of atoms and
89% of the variance in the binding affinity. A hydrogens (200). A more unique approach has
training set of 16 compounds was chosen that, been the use of C-13 data for corticosteroids to
after PLS analysis, resulted in a predictive r2 develop a QSDAR relationship (quantitative
value of 0.94. Estimation of the remaining 21 spectrometric data-activity relationship) (201).
compounds gave a predictive r2 value of 0.51, Tuppurainen and coworkers evaluated a
or 0.70 when the outlier 5-pregnene-3,20- novel electronic eigenvalue (EEVA) molecular
dione was excluded. Finally, when all 37 of the descriptor for QSARIQSPR studies, in which
original data set were included in the PLS they validated, by use of a benchmark CBG
analysis, a predictive ? value of 0.80 was ob- steroid data set, a novel EEVA descriptor of
tained. Through use of CHEM-X, 3D contour molecular structure for use in the derivation
plots of the grid points were generated in of predictive QSARIQSPR models. Like other
which nonbonded and charge-charge terms spectroscopic QSARIQSPR descriptors, EEVA
Anti-Inflammatory Steroids

was also invariant as to the alignment of the bolic stability. The sum of these effects is man-
structures concerned. Its performance was ifested as the observed increase in biologic ac-
tested with respect to the CBG affinity of 31 tivity. The action of structural changes on
benchmark steroids. It appeared that the elec- only one individual process, receptor binding,
tronic structure of the steroids (i.e., the "spec- can be predicted with accuracy (46). Gross
tra" derived from MO energies) is directly re- pharmacological activity has been predictable
lated to the CBG binding affinities. The for two decades through use of the technique
predictive ability of EEVA was compared to of Fried and Borman (153). QSAR studies on
other QSAR approaches, and its performance metabolic stability, drug distribution, and in-
was discussed in the context of the Hammett trinsic activity remain for the future. Combin-
equation. The good performance of EEVA is ing all these relationships by use of mathemat-
an indication of the essential quantum me- ical modeling techniques may lead to a
chanical nature of QSAR. The EEVA method complete quantitative theory of anti-inflam-
is a supplement to conventional 3D-QSAR matory steroid design in the next two decades.
methods for corticosteroids, which employ
fields or surface properties derived from cou- 10 EFFECT OF INDIVIDUAL STRUCTURAL
lombic and van der Wads interactions. CHANCES ON ANTI-INFLAMMATORY
The E-state modeling of corticosteroids ACTIVITY
binding affinity and validation of the model for
a small data set was recently conducted by In this section we examine the SAR at each
Maw and coworkers. Data for 31 steroids bind- position in the steroid nucleus. First, however,
ing to the CBG were modeled by use of E-state it is useful to make an overview of the more
molecular structure descriptors and a kappa important changes.
shape index. Both E-state and hydrogen E-
10.1 Overview of Major Changes
state descriptors appeared in the model as at-
in Anti-Inflammatory Steroids
om-level and atom-type descriptors. A four-
variable model was obtained that was In Tables 15.25 (202),15.26 (166),15.27 (203),
statistically satisfactory: r2 = 0.81, s = 0.51; and 15.28 (204)are displayed the activities of a
rpress2 = 0.72; spress = 0.62. Structure inter- number of anti-inflammatory steroids in
pretation was given for each variable in the large-scale studies in four major laboratories.
model. A leave-group-out (LGO) approach to These reports are of considerable interest.
model validation was presented, in which each They make it possible to compare the activi-
observation was removed from the data set ties of clinically important steroids and other
three times in random groups of 20% of the steroids from data obtained by a single labora-
whole data set. The average of the resulting tory. It can be seen that the potency of poly-
predicted values constitutes consensus predic- substituted compounds is predicted well by
tions for these data for which rLOo2= 0.70. the enhancement factors of Tables 15.20,
These collective results support the claim that 15.22, and 15.23. The success of the medicinal
the E-state model may be useful for prediction chemist in increasing potency and decreasing
of pK, binding values for new compounds. sodium retention is apparent in Table 15.27.
Flumethasone acetate, the 21-acetate of (40),
9.4 Summary of the Results
is 424 times as active as cortisol in the granu-
of Theoretical Studies
loma assay and has no more sodium retention
Theoretical studies have shown that a given than that of cortisol itself-a remarkable
structural change in an anti-inflammatory achievement. Unfortunately, as mentioned in
steroid affects not one but a multitude of fac- section 3, other side effects have not been re-
tors. The introduction of a 9a-fluoro substitu- duced with respect to the therapeutic effect.
ent, for example, alters the conformation of
10.2 Skeletal Changes in the
the entire steroid molecule and increases the
Cortisol Molecule
acidity and hydrogen bonding capacity of the
llp-hydroxyl group. These changes influence Unlike the situation in the progestational
receptor binding, protein binding, and meta- agents, removal of the 19-angular methyl
Table 15.25 Relative Potencies of Glucocorticoids (I)"

Relative Activity in Adrenalectomized R a t


V
rD
v Compound Granuloma Thymolytic Glycogen Deposition
Cortisol 21-acetate (2c)
Cortisone 21-acetate
Prednisone 21-acetate
Prednisolone 21-acetate (21a)
6a-Methylprednisolone (28)
9a-Fluoroprednisolone (39)
Triamcinolone (13a)
Triamcinolone acetonide (13)
6a,9a-Difluoro-16a-hydroxy cortisol, 16a,l7a-acetonide (102)
Fluocinolone (1la)
Fluocinolone acetonide ( l l b )
6a-Chloro-9a-fluoro-16a-hydroxyprednisolone 16a,17a-
acetonide, 21-acetate (103)
Paramethasone (37)
Dexamethasone (20a)
Betamethasone (5a)
"Ref. 202.
Table 15.26 (Continued)
Steroid Glycogen Deposition Thymolytic
Cortisol(2) 1.0 1.0
16a-Hydroxycortisol(104) 0.4 0.3
16a-Hydroxycortisol16,17-acetonide
(105) 2.0 2.2
Prednisolone (21) 4.1 2.3
16a-Hydroxyprednisolone(106) 0.9 1.3
16a-Hydroxyprednisolone16,17-acetonide(107) 13.0 17.0
6a-Methylcortisol(10S) 9.5 5.8
16a-Hydroxy-6a-methylcortisol(109) 1.1 1.2
16,17-acetonide
16a-Hydroxy-6a-methyl-cortisol (110) 10.0 14.0
9a-Fluorocortisol(3) 6.1 5.6
16a-Hydroxy-9a-fluorocortisol(111) 7.9 2.4
16a-Hydroxy-9a-fluorocortisol16,17-acetonide
(112) 14.0 14.0
6a-Methyl-9a-fluorocortisol(113) 9.0 9.8
16a-Hydroxy-6a-methyl-9duorocortisol (114) 2.8 4.2
(115)
16a-Hydroxy-6a-methyl-9a-fluorocortisol16,17-acetonide 15.0 29.0
6a-Methylprednisolone(28) 8.4 10.0
16a-Hydroxy-6a-methylprednisolone(116) 2.0 2.2
16a-Hydroxy-6a-methylprednisolone16,17-acetonide
(117) 24.0 28.0
9a-Fluoroprednisolone
(39) 13.0 16.0
Triamcinolone (13a) 6.1 3.9
Triamcinoloneacetonide(13) 32.0 33.0
6a-Methyl-9a-fluoroprednisolone
(90) 16.0 25.0
(118)
16a-Hydroxy-6a-methyl-9a-fluoroprednisolone 4.9 5.5
16,17-Acetonide(119)
16a-Hydroxy-6a-methyl-9a-fluoroprednisolone 21.0 70.0
"Ref. 166.
Table 15.27 Effect of 6a-Fluoroand 9a-FluoroSubstitution on Activity of Cortisol Derivativesa
Table 15.27 (Continued)
Relative Activity
Sodium Retention
Compound Granuloma Glycogen Deposition (DOCA = 1.0)
Cortisol (2) <0.02
16a-Methylcortisol(98) <0.02
Prednisolone (21) <0.02
16a-Methylprednisoloneacetate (120)~ <0.02
9a-Fluorocortisol acetate (3Ib 5.0
Dexamethasone (20a) <0.02
2a-Methylcortisolacetate (99aIb 2.7
9a-Fluoroprednisoloneacetate (39aIb 20.0
6a-Fluorocortisol acetate (32aIb Slight
6a-Fluoro-16a-methylcortisol(37) <0.02
6a-Fluoroprednisoloneacetate (121Ib Slight
Paramethasone acetate (37a)b <0.02
6a,9a-Difluorocortisol acetate (122)b 4.0
Flumethasone acetate (40a)b <0.02
6a-Fluoro-2a-methylcortisolacetate (124)b 3.0
6a,9a-Difluoroprednisoloneacetate (125)~ 2.5
"Ref. 203.
*21-Acetate.
10 Effect of Individual Structural Changes on Anti-Inflammatory Activity

Table 15.28 Human Topical


Anti-inflammatory Potency"
Compound Activity
Cortisol (2)
Cortisol 17-butyrate (2a)
Cortisol 17-valerate (2b)
Dexamethasone (20a)
Dexamethasone 21-acetate (20c)
Dexamethasone 21-phosphate (20b)
Prednisolone (21)
Prednisolone 21-acetate (21a)
Prednisolone 17-valerate (21b)
Flurandrenolide (15)
Triamcinolone (Ma)
Triamcinolone acetonide (13)
Betamethasone (5)
Betamethasone 21-acetate (5e)
Betamethasone 17-valerate (5c)
Fluorometholone (22)
Fluoroprednisolone (121)
Fluocinolone ( l l a )
Fluocinolone acetonide (1lb)
Fluocinonide (1lc)
Flumethasone pivalate (40bIb
Halcinonide (12)
Desonide (19)
"Ref. 204.
bPivalateester.

tem a 16a-ester grouping but lacking the con-


ventional side-chain hydroxylation pattern, is
group reduces anti-inflammatory activity. 19- beneficial. In clinical trials, domoprednate was
Norcortisol (126) has only 3/10 the activity of comparable to betamethasone valerate as a
cortisol in the granuloma test (205,206).How- 0.1% ointment (209).
ever, if the removal of the 19-angular methyl Contraction of the A-ring to A-norcortisol
group is accompanied by aromatization of the (130)gives an inactive compound (210). The
A-ring, as in (127), the compound retains ac- A-ring can be modified by isosteric replace-
tivity equal to that of cortisol in the granuloma ment to afford a heterocycle like 2-oxacortisol
test (207). D-Homocortisone acetate (128) is acetate (1311,which has one quarter the activ-
less active than cortisol but retains some cor- ity of cortisol. The introduction of a 9a-chloro
tical hormone activity (208). Domoprednate group and a 16a-methyl group gives (132),
(129), incorporating into the D-homo ring sys- which is equal to cortisol in activity (211).
Anti-Inflammatory Steroids

loma activity in rats about fourfold, but this


enhancement is not observed in humans
(213). The 2a-methyl group also produces a
marked increase in sodium retention. The 2a-
methyl group exerts its effect through a re-
duced rate of reduction of the A4,3-ketone sys-
tem (73). It is likely that metabolism in other
positions, such as at C-11 and C-20, is also
retarded.
Introduction of a 2a-fluoro group reduces
the biologic activity in several tests (214,215)
but, by contrast, 2-halogenation in A1r4-3-keto
corticosteroids can provide highly potent com-
pounds. In the latter case, overall substitution
of the ring system is important for activity, as
is shown in Table 15.29.
The effect of simultaneous substitution at
both C-2 and C-6 is profound. Fluorination at
C-6a is well known to improve potency, but
generally 6p-substitution is thought to be
detrimental toward good anti-inflammatory
properties (see Section 10.7). In this series,
however, 6p- F is better than 6a-F when there
is halogenation at C-2. Of additional signifi-
cance is the absence of apparent systemic ef-
fects as gauged by the lack of thymic involu-
tion. The most potent within this group,
2-bromo (134) is virtually without systemic
effect. No explanation for these unusual find-
ings has been reported, but it was speculated
that the steroids were rapidly metabolized in
the systemic circulation (215).
10.5 Alterations at C-3
0
Almost all active anti-inflammatory steroids
(132) have a carbonyl group at C-3, but some excep-
tions exist. One is the ring A phenol (127) dis-
cussed in Section 10.2. 2'-Phenylpregn-4-
10.3 Alterations at C-1 ene[3,2-clpyrazoles of corticoids are more
potent anti-inflammatory agents than their
The introduction of a double bond at C-1leads parent A4,3-keto steroids.
to enhanced anti-inflammatory activity, as Activity can be enhanced still further by
has already been discussed, but a hydroxyl p-fluorophenyl substitution of the pyrazole
group in this position leads to an inactive ring, as in (139), which has 2000 times the
product. Thus la-hydroxy-9a-fluorocortisol, potency of cortisol (216-218). The pyrazole
obtained by microbial hydroxylation, is virtu- derivatives do not exhibit mineralocorticoid
ally inactive in anti-inflammatory assays activity. They have high affinity for cytoplas-
(211). mic glucocorticoid receptors (219).
A simplified class of pyrazoles, lacking the
10.4 Alterations at C-2
16-methyl, 9-fluoro, and A6,7 p u p s present in
The introduction of a 2a-methyl group into (139),was found in some cases to provide ac-
cortisol and its analogs (212) increases granu- tivity comparable to that of betamethasone
10 Effect of Individual Structural Changes on Anti-Inflammatory Activity 805

Table 15.29 6cy,9-and 6~,9-Difluoro-ll~-hydroxypregna-1,4-diene-3,2O-dione


and Their
a
2-Halogenated Derivatives and Acetyl Esters

Compound X C-6 CPG Assayb Thymusc


(133) H 6P 40 >40
(134) Br 6P 0.01 >lo00
(135) C1 6P 0.45 >40
(136) H 6a 0.10 5
(137) Br 6a >40 >40
(138) C1 6a >40 40
Flucinolone acetonide 1.02 5
"Ref. 215.
*Cotton pouch granuloma weight inhibition determined at the ED,, (pglpellet).
"Thymus weight inhibition determined at the ED,, for the same group.

In this approach, the heterocyclic derivatives


are themselves biologically inactive, but slow
valerate in the human vasoconstriction assay. hydrolysis results in opening of the thiazoli-
In this series, R was varied from OH to C1 to a dine ring and liberation of the anti-inflarnma-
variety of ester (OCOR') groups. Surprisingly, tory steroid (Fig. 15.14).
the most topically potent steroid, (140), had R Significant anti-inflammatory potency has
= OH and R, = ethyl (220). been observed for many of these derivatives,
A unique class of 3-spirofused heterocyclic as shown in Table 15.30. Potency was en-
corticosteroids was developed as "soft drugs" hanced in general by a factor of about 3 to 4
through use of the chemical delivery system relative to that of hydrocortisone, with only
(CDS)approach of Bodor and coworkers (221). slight changes in potency being noted upon
Anti-Inflammatory Steroids

Inactive

Active

Figure 15.14. Corticosteroidal 3-spirothiazolidine derivatives as chemical delivery systems for


hydrocortisone.

substitution of the ester R group. Various duced for the soft drug (142). Triamcinolone
methods for assessing the degree of systemic acetonide, a more potent topical anti-inflam-
absorption were applied to the prototypical matory steroid in this group, had higher per-
ethyl ester, (142).The percentage reduction in centages of skin thinning.
thymus weight or thymus involution for
(1421, an indirect measure of systemically cir- 10.6 Alterations at C-4
culating corticosteroid, was 21%. In contrast,
The A4 double bond is important but not es-
hydrocortisone and its 21-acetate were 35%,
sential for anti-inflammatory activity. Thus,
but hydrocortisone 17-butyrate was less well
the 5a-pregnan-3-one (147) derived from tri-
absorbed, as evidenced by a reduction of 20%
in thymus weight. Another interesting ap-
proach to assessing the degree of absorption
was accomplished in the mouse skin model.
After 24 h, 8 mol % of hydrocortisone had
passed through isolated, hairless mouse skin,
whereas 14 mol % was observed for hydrocor-
tisone 21-acetate, and only 3 mol % for the soft
drug (142).
The decrease in systemic absorption noted
for analogs such as (142) was presumably at-
tributable in part to binding to skin tissue
through formation of disulfide bonds between
partially hydrolyzed prodrug (142) and -SH-
containing amino acid residues in skin pro-
teins, as shown in Fig. 15.14.
A clinically significant side effect of topi- amcinolone 16a,17a-acetonide is more active
cally applied corticosteroids is thinning of the than cortisol in glycogen deposition (222). Be-
skin or atrophogenicity. This effect has been cause 5a-steroids have approximately the
estimated in animal models by measuring same shape as the corresponding A4 com-
thinning of mouse ear skin. As shown in Table pounds, it appears that reduction of the A4
15.31, substantial thinning of the skin was ob- double bond in this manner still allows recep-
served for hydrocortisone and its 21- and 17- tor binding but results in a 100-fold decrease
ester derivatives, but was substantially re- in the activity of the compound.
10 Effect of Individual Structural Changes on Anti-Inflammatory Activity 807

Table 15.30 Relative Anti-Inflammatory Activity of Selected Cysteine-Based


3-Spirothiazoldine Derivatives of Hydrocortisonea

~ood
Compound R R1 ED50 (Wb Relative Potencyb
(141)
(142)
(143)
(144)
(145)
(146)
(2), Hydrocortisone
(2c),Hydrocortisone 21-acetate
(2a),Hydrocortisone l7a-butyrate
"The test compounds were applied in acetone solution containingZ%croton oil on the anterior and posterior surface ofthe
right ear. Three hours later, the mice (male DDY)were euthanized and both ears were removed. Circular sections were
punched out and the drug effect expressed as percentage inhibition of inflammation compared to the control. 100% potency
is defined as that of hydrocortisone (33).
bLinear regression analysis of data obtained a t 3 x lo-', 3 X 3X and 3 X 10-'M.

4-Methylcortisol acetate is inactive in the (226) and 6a-hydroxycortisone is less active


granuloma and glycogen deposition tests than cortisone in the granuloma test (227).
(223). The reported anti-inflammatory activ- 6-Oxocortisone 21-acetate is only weakly ac-
ity of 4-fluorocortisol is very low (224).Allen et tive (228). The enhancing activity of the 6a-
al. (225) prepared Cmethyl and 4-ethyl deriv- methyl group has been mentioned in a number
atives of 9a-fluorocortisol and the Cmethyl of preceding sections of this chapter. The
derivative of 9a-fluoroprednisolone. In all closely related 6-methylene group, as in
cases the 4-methyl analog was less potent than 6-methylene 16a-methylprednisolone, impairs
the parent compound and the 4-ethyl deriva- activity (229). 6a-Chloro groups (230) markedly
tive was even weaker. enhance activity in 6a-chlorocortisol acetate,
but have no effect on cortisone or prednisone
10.7 Alterations at C-6
(231),perhaps because the group interferes with
In general, polar substituents such as hydroxy reduction of the 11-keto moiety.
or 0x0 in the 6a-position decrease biologic ac- The 6a-fluoro group is very similar to the
tivity, whereas 6a-substituted hydrophobic 9a-fluoro group in enhancing the activity of
substituents, such as alkyl groups or halogens, corticoids (203). 6a-Chloro and 6a-fluoro (73)
tend to increase activity. 6P-Substituents of groups give weakly active or inactive products
any type impair biologic activity, except as when substituted in cortisone. Introduction of
noted in Section 10.9. Thus, 6P-hydroxycorti- 6a-fluoromethyl groups into prednisolone
sol-6,21-diacetatehas less than one-third the and 9a-fluoroprednisoloneaffords compounds
thymolytic potency of its parent compound that are still active as anti-inflammatory ste-
808 Anti-Inflammatory Steroids

Table 15.31 Ear Thinning in Mice after Topical Application of Steroidsa


Compound Concentration (M) % Reduction in Ear Thickness
Hydrocortisone

Hydrocortisone 21-acetate

Thiazolidine (142)

Hydrocortisone 17-butyrate

Hydrocortisone 17-valerate

Triamcinolone acetonide

Vehicle
"Left ears treated for 4 days with 10 pL of the steroid solution.

roids (232).The more polar 6a-methoxy group methyl cortisol derivatives are less active than
drastically reduces thymolytic activity when the parent compound (236). Introduction of a
introduced into 9a-fluoroprednisolone (233). methylthio, ethylthio, acetylthio, or thiocyano
Although the introduction of a 6,7 double group into the 7a-position of cortisol or corti-
bond has little effect on the activity of cortisol sone reduces the activity of the resulting com-
(213), A6-6-chloroprednisolone is about twice pound (237).
as active as prednisolone in arthritis (234). 7a-Halogenation of non-9a-halogenated
When the 6-azido group was introduced into steroids, derivatives that are conceptually ob-
9a-unsubstituted A6-corticosteroids, to give tained by moving the halogen from the 9a- to
A6-6-azidocortisol, systemic anti-inflamma- the 7a-position, has led in some cases to potent
tory activity was increased five to eight times,
synthetic corticosteroids having activity com-
whereas the corresponding change in 9a-fluo-
parable to that of 9a-fluorinated steroids (e.g.,
rocorticoids left potency unaffected (235). A dexamethasone dipropionate) (238). Diaste-
6-formylpregnadiene derivative (148), upon
reomeric 16-methylated corticoids (a and p)
oral administration, had a relative potency of with various halogenation patterns were com-
680 compared with that of cortisol acetate in pared to controls, as shown in Table 15.32.
the granuloma pouch assay (235). This inter- When the 16-position is not methylated, activ-
esting result could imply that a 3-keto group is ity appears to be independent of the 7a-halo-
not a prerequisite for anti-inflammatory activ-
gen, in that relatively similar activity is ob-
ity, although (148) is an enol-ether and thus
tained for both 7a-chloro or 7a-bromosteroids
might be expected to undergo acid-catalyzed
(149) and (150),respectively.
hydrolysis in stomach acid to provide, initially, For the 16a-methylated series, activity is
enone-al (148a). Equilibration to (148b), fol-
generally enhanced as expected (see section
lowed by deformylation, might then give the 10.13) but, surprisingly, 7a-F (152) is equipo-
ester of triamcinolone acetonide (13b). tent with its corresponding nonhalogenated
derivative (151). This observation might - im-
10.8 Alterations at C-7
ply that other bulkier, less electronegative
Both 7a and 7p substituents reduce anti-in- halogens placed at the 7a-position would lead
flammatory activity. 7a-Methyl and 7p- to less active compounds. This is particularly
10 Effect of individual Structural Changes on Anti-Inflammatory Activity 809

CHO CHO

(13b) Triamcinolone acetonide, acetate ester

true, given that activity follows the order of F 10.14). On the other hand, if an axial interac-
> C1 > Br > I for 9a-halogenation (section tion at a 1,3-diaxial distance to the 9-position
9.2). However, as is seen for (152), (1531, were more important, then 7a-F steroids
(1541, and (155) (7a- F versus C1 versus Br might be expected to have activities compara-
versus I), the trend appears to be Br > C1> I > ble to those of their 9a-F counterparts. In-
F. The noteworthy analogs in this series, 7a- deed, (151)and/or (157) do have activity in the
chloro analog (153) and 7a-bromo analog range of that of betarnethasone 17-valerate, a
(1541, are both as potent as betarnethasone highly potent 9a- F steroid. Given that receptor
17,21-dipropionatein this assay system. binding data for all of the axially halogenated
This is a remarkable finding, which may C-7,9 and 12-fluoro derivatives are not pub-
not support the contention of Fried (169). If lished, it is difficult to assess whether these dif-
the enhancement produced by a 9a-halogen is ferences are truly attributable to a sigma-bond
attributed to an inductive effect, similar en- electronegativity effect, a through-space inter-
hancement would be expected from a 12a- action, a conformational effect, or an effect on
halogen because the 9a- and 12a-positions are the metabolism of the steroid. It is interesting
equivalent with respect to their electronic ef- that 6a-halogenation (preceding section) of-
fect on C-11. Fried found that 12ar-F substitu- ten leads to significant potency enhance-
tion led to steroids with potency comparable to ments, even though 6a derivatives are pseudo-
that of 9a-F steroids (see Section 9.2,9.3, and equatorial rather than axial, like the 7,9- and
Table 15.32 Topical Anti-inflammatoryPotencies of 7a-HalogenoCorticosteroids and Their 7a-Hydrogenand 6,7-DehydroDerivativesa

(245-246)
Compound X Topical Potencyb
OCOEt C1
OCOEt C1
OCOEt OCOEt
OCOEt OCOEt
OCOEt OCOEt
OCOEt OCOEt
OCOEt OCOEt
OCOEt OCOEt
OCOEt OCOEt
OCOEt OCOEt
OCOEt OCOEt
OCOEt OCOEt
OCOEt OCOEt
OCOEt OCOEt

--

"Refs. 238,239.
bPotencyis a percentage reduction in inflammation relative to that of betamethasone 17-valerate ( 5 4 in the croton oil ear assay
"Betamethasone17-valerate (5c)as control.
dBetamethasone 17,21-dipropionate(5b).
10 Effect of Individual Structural Changes on Anti-Inflammatory Activity 811

l2a-positions. Perhaps the latter effect is re- 10.1 0 Alterations at C-8


lated to a reduction in steroid metabolism, and
The introduction of an 8(9) double bond into
thereby an increase in activity.
prednisolone derivatives gives products of
The lea-methylated series is similar to the
somewhat lower anti-inflammatory potency
16P-series, in that potency is maximal at 7a-
than that of the parent compound (241). Thus,
bromo, although the series is ranked Br > C1
- F > I, which is at odds with the 16pmethyl
6a-fluoro-S(9)-dehydroprednisolone acetate
(164) has 2.3 times the thymolytic activity of
set of analogs. The 6,7-dehydro derivatives
cortisol.
(161) and (162) were surprisingly active in the
croton oil ear assay, the 16P-methyl analog
(161) being a third as active as its 9a-fluori-
nated relative, betamethasone 17,21-dipropi-
onate (BDP).

10.9 6,7-Disubstituted Compounds


6a,7a-Dihydroxycortisone21-acetate is much
less active than cortisone acetate in the gran-
uloma and thymolytic tests (237). The epoxide
6a,7a-epoxycortisoneis about 1/10 as active as
cortisol in the glycogen deposition test (229).
However, the introduction of a 6,7-difluorom-
ethylene substituent can give rise to highly
active compounds (240). Both the 6a,7a-diflu-
oromethylene derivatives and the 6p,7p-de- 10.11 Alterations at C-9
rivatives are active. Compound (163)has 1400
Modifications at C-9 are discussed in Sections
9.2 and 9.3.

10.1 2 Alterations at C-10


Modifications at C-10 are discussed in Section
---OH 10.2.
-.---
10.1 3 Alterations at C-11
N The C-11 oxygen group is not essential for
\
anti-inflammatory activity if enough other en-
hancing groups are present in the molecule.
Thus the 16,17-acetonide (166;) is an active

<OH

(163)

times the systemic anti-inflammatory activity


of cortisol on subcutaneous administration.
The high potency of both a and P isomers was
0
taken as evidence that the 6,7 region of the
steroid is not in contact with the receptor, al- CH~
though the opposite case could be argued
equally well. (165)
812 Anti-Inflammatory Steroids

anti-inflammatory steroid, in spite of the ab-


sence of a C-11 oxygen group (242). However,
changes at C-11 profoundly affect biologic ac-
tivity. The lla-hydroxy epimer of cortisol is
inactive in the glycogen deposition test (243).
12P-Hydroxyprednisolone llp,l2p-acetonide
is much less active than cortisol in the granu-
loma and thymolytic assays (244). Converting
the llp-hydroxy group to a tertiary alcohol
through the formation of lla-methylcortisol
21-acetate gives an inactive compound (245).
The introduction of a 9a-fluoro atom into
deoxycorticosterone acetate (DOCA) gives a
9a-fluorinated steroid lacking an 1lp-hydroxy
group (166) that has 12 times the mineralo-

corticoid action of DOCA. However, (166) has tate (the 21-acetate of 168)is a useful topical
only 5-10% the anti-inflammatory action of anti-inflammatory steroid (248). However,
cortisol (246). This is an indication that some 9,ll-dichloro steroids undergo solvolysis, as
of the enhancing effect of the 9a-fluoro group shown in the formation of (169) from (168)
is the result of its action on the l l p - OH group. (249).
An especially interesting series of compounds Thus, the activity of 11p-chlorosteroids
is the 9a,llp-dihalocorticoids. These deriva- may be attributable to their conversion to the
tives lack the 11-oxygen atom and yet have llp-hydroxy compounds in vivo. In support of
useful anti-inflammatory activity. The llp- this is the poor activity of (167), which would
fluoro-9a-bromo derivative (167) has less than not be expected to undergo such a solvolysis,
one-quarter the activity of cortisol in the gran- given that fluorine is a poor leaving group.
uloma assay (247), although dichlorisone ace- 16a-Methylation causes a major increase in
potency of dichlorosone, which is unexpected
on the basis of the enhancement factor of this
group. However, the 16a-methyl compound
undergoes the solvolysis to the corresponding
llp-hydroxy derivative much more readily
than the parent (168). This also provides
evidence that some of the activity of the 9,ll-
dichlorosteroids is attributable to the solvoly-
sis reaction shown (250). Thus, these com-
pounds do not unequivocally represent highly
active 11-deoxyanti-inflammatory steroids, as
has sometimes been claimed.
10 Effect of Individual Structural Changes on Anti-Inflammatory Activity 813

10.1 4 Alterations at C-12


12a-Hydroxycorticosteronehas 1/10 the activ-
ity of cortisol in the glycogen deposition test
(251). 12a-Methyl-11-oxodeoxycorticosterone
is less active in the glycogen deposition test
than the corresponding compound lacking the
12a-methyl group (252). The effect of the 12a-
halo substituent in corticoids was used by
Fried (161) in an effort to understand the ac-
tion of 9a halogens. He reasoned that if the
enhancement produced by a 9a-halogen is the
result of an inductive effect, similar enhance-
ment would be expected from a 12a-halogen,
that the acetonides (171a) and (171b) have
given that the 9a- and 12a-positionsare equiv-
equivalent biologic activity (10 times that of
alent with respect to their electronic effect on
hydrocortisone) in the glycogen deposition
C-11. It was shown that the liver glycogen and
test. On the other hand, the parent steroid
sodium-retention activities of l2a-halo-llp-
(170b) has only 10 times the activity of
hydroxyprogesterones paralleled those of the
(170a). Thus, masking the 17a-hydroxyl
corresponding 9a-halo-llP-hydroxyprogest-
erones (253, 254). Again, Taub et al. (251) group restores the 12a-halogen to parity with
showed that 9a- and 12a-fluoro groups pro- the 9a-halogen as an enhancing group.
A variety of 12p-hydroxy (173-175) and
duced equivalent activity enhancement in the
12P-acyloxy (176-179) analogs of betametha-
glycogen deposition test when substituted in
sone 17,21-dipropionate (5b) were synthe-
corticosterone and 1-dehydrocorticosterone,
sized and tested for topical efficacy in a modi-
and that 9a- and 12a-hydroxy groups cause
fied croton oil ear assay (mouse), by use of both
similar reduction in activity in corticosterone.
Surprisingly, l2a-chlorocortisone is inac-
(5b) and beclomethasone 17,21-dipropionate
(172) as standards, as shown in Table 15.33
tive in the liver glycogen and sodium-reten-
tion assays (255), whereas the 9a-chloro ana- (256).
Topical potency is not greatly affected on
log has a glycogen deposition activity of 3.5
introduction of a 12P-hydroxylgroup into ei-
times that of cortisone acetate (3). Fried and
ther (5b) i . . 173) or (172) i . . , 174). Al-
Borman hypothesized that hydrogen bonding
though the topical potency of the ga-bromo-
from the l7a-hydroxyl group to the 12a-halo-
12P-hydroxy analog (175) is slightly reduced
gen is responsible for the loss of activity (153).
(by about 1/21 relative to that of the analogous
To test this hypothesis they synthesized the
C1 or F analogs, it is still more potent topically
9a- and 12a-fluoro-16a-hydroxycortisol deriv-
than would be expected on the basis of the
atives, (170a) and (170b) and their respective
Fried-Bormann enhancement factors (sys-
acetonides, (171a) and (171b). It was found
temic) for 9a-halogens. On the other hand, the
result for (175) is perhaps not unusual in light
of studies by Green et al., (235, 238) who ex-
amined the effect of 7a-halogen substitution
on topical potency in betamethasone-like base
structures (e.g., alclometasone), and showed
that potencies range in the order of 7a-Br > C1
> F > (5b).
The degree of systemic absorption of these
analogs was measured in three ways. First,
contralateral (distal ear) topical application,
in the modified croton oil ear assay, allowed
for an indirect determination of the degree of
systemic absorption. As shown in Table 15.34,
814 Anti-Inflammatory Steroids

Table 15.33 Estimated Topical Potencies of 12-Substituted Betamethasone Analogsa


0

Compound R X ED1oob Relative Potency


(5b) H F 110 1
(172) H C1 110 1
(173) OH F 140 0.78
(174) OH C1 160 0.69
(175) OH Br 325 0.34
(176) OCOC,H, F 400 0.28
(177) OCOC,H, C1 400 0.28
(178) OCOC,H, Br 325 0.34
"Ref. 256.
bMicrogram per mouse.

both BMDP (5b)and BCDP (172) exhibited strating a high degree of systemic absorption.
marked systemic absorption, at the topical For the fluoro alcohol (173), both thymus
EDloo values. When applied at a site distant weight and adrenal suppression were influ-
from the inflammation, (5b)was 75% as effec- enced by increasing dose in a regular manner,
tive as when applied directly, whereas (172) whereas the bromo (175) and chloro (174) al-
was 50% as potent. cohols were not. This effect is presumably re-
The 12P-hydroxyanalogs (173), (174), and lated to the lower relative intrinsic systemic
(175) all showed similar, if slightly attenu- activities of (1741175) compared to that of
ated, signs of systemic absorption. As can be (173). In any event, all three 12-hydroxy ana-
seen in this series (12P-OH), varying the halo- logs are clearly absorbed through the skin.
gen substituent at C-9 did not greatly influ-
In stark contrast are the corresponding
ence the degree of systemic absorption, in that
propionate esters (176), (177), and (178),
all three analogs displayed about 15-20% dis-
tal topical potency. none of which demonstrates any evidence of
The other method used to assess the de- systemic absorption, even after multiple high
gree of systemic absorption was to examine dose applications. It is interesting to note that,
hypothalamic-pituitary-adrenal axis function, upon esterifcation of the 12P-hydroxy group,
based on thymic involution (thymus weight) topical potency becomes relatively indepen-
and adrenal suppression (plasma cortisol), af- dent of the 9a-halogen (Table 15.34).
ter multiple topical applications of the corti- Topical potency was sensitive to the spe-
coid. The results shown in Table 15.20, for the cific 12-ester. Thus, potency follows the order
controls BMDP (5b)and BCDP (172),are con- propionyl > butyryl > isovalyryl for aliphatic
sistent with those obtained for the single dis- esters. On the other hand, bulky aromatic es-
tal topical application. Thymus weights were ters such as benzoyl (179) or furoyl are still
dramatically reduced (by 70-90%) as were quite potent. Furthermore, none of these es-
plasma cortisol levels (36%), clearly demon- ters was systemically absorbed.
10 Effect of Individual Structural Changes on Anti-Inflammatory Activity 815

1 Table 15.34 Anti-Inflammatory Activities of 12-Substituted Betamethasone Analogs


i
i 0

No. R X Dosea Topicalb Systemicc Thymolyticd Adrenale


(5b) H F 2.5 59 27 30 12
25 76 30 74 24
75 93 72 87 37
(172) H C1 2.5 60 12 12 1
25 78 40 52 32
75 93 52 67 36
(173) OH F 10 46 7 9 1
(174) OH C1 10 41 0' - -
40 67 5 0 13
100 81 21 5 14
(175) OH Br 50 57 16 7 12
100 86 17 0 14
200 91 17 7 15
(176) OCOC,H, F 10 21 0' 0 0
40 50 0 1 6
100 69 0 4 0
(177) 0COC2H5 C1 50 47 0" 4 5
100 53 0 5 5
200 71 0 0 6
(178) 0COC2H, Br 50 36 Oe 2 2
100 45 0 6 6
200 70 0 2 0
(179) OCOC,H, Br 300 84 0' - -
"Dose of drug in pg/mouse ear, after TPA challenge of 8.8 nMImouse.
bTopicalpotency, % reduction in inflammation of mouse ear.
'Systemic potency, % reduction of inflammation in mouse contralateral ear.
dEffect was measured by weight loss of mouse thymus.
'Effect was determined by measuring the inhibition of increased plasma cortisol induced by stress.

In addition to systemic toxicity, prolonged the analogs (174) and (177), vs. (5b) and
topical corticosteroid therapy can result in (1721, on skin thickness is shown in Table
clinically significant atrophy of the skin, 15.35.
which is thought to arise primarily by an inhi- Both beta- and beclomethasone dipropi-
bition of DNA synthesis. The 9a-chloro-12P- onate (5b)and (172), respectively) are quite
hydroxycorticoid (174) and its corresponding atrophogenic, whereas the 12P-alcohol (174)
propionate (177) were examined for atropho- is moderately toxic. In contrast, the corre-
genicity, as previously described in the mouse. sponding tripropionate (177) is completely in-
The results of repeated topical applications of ert. These results would seem to indicate that
816 Anti-Inflammatory Steroids

Table 15.35 Atrophogenicity of Betamethasone Analogs


Structure Dosea Skin Thickness % Potency

"Applied daily for 3 weeks.

12P-hydroxybeclomethasone 12,17,21-tripro- duction of a 16pmethoxy group (260). The


pionate (177)acts purely as an anti-inflamma- 16pacetoxy group abolishes the glucocorti-
tory agent. coid action of 9a-fluorocortisol or 9a-fluoro-
One matter that has not been discussed ad- prednisolone (261). Introduction of the 16a-
equately in this theory is why the hydrogen chloro group into 9a-fluorocorticoids greatly
bond formation between a l7a-hydroxy group, increases anti-inflammatory and glycogenic
which is not even needed for activity in the rat, activity (262). 16a-Chloro 6a,9a-difluoropred-
and the 12a-halogen should impair activity. nisolone 21-acetate has 1100times the activity
One possible explanation would be through a of cortisol (180)(262). 16a-Ethylsubstitution
conformational effect on the steroid molecule.
This would be an interesting area to explore
through X-ray crystallography.

10.1 5 Alterations at C-15


Methyl groups can be substituted in the 15a-
position of anti-inflammatory steroids, with
enhancement factors of approximately 0.5 on
both anti-inflammatory action and sodium re-
tention. A 15p-methyl group has little effect
on glycogen deposition. Parent steroids sub-
stituted with the 15P-fluorogroup include cor-
tisol, prednisolone, 9a-fluorocortisol, and 9a-
fluoroprednisolone (257). Bioassays suggest a
small increase in anti-inflammatory activity
because of the 15P-fluorogroup and a 97-99%
reduction in sodium retention.
is similar to l6a-methyl and 16a-hydroxyl
substitution in eliminating effects on electro-
10.16 Alterations at C-16
lytes (263). Beal and Pike (264) synthesized
The effect of introducing l6a-methyl, 16a-hy- the 16a-fluoromethyl derivative of 9a-fluoro-
droxy, and 16a,l7a-acetonides has already prednisolone 21-acetate. It is highly active as
been discussed. 16a-Fluoro derivatives of an anti-inflammatory agent and produces
prednisolone and 9a-fluoroprednisolone hav- mild electrolyte excretion similar to that of
ing 16 times and 75 times the anti-inflamma- 16a-methyl steroids. The 16a-methoxy deriv-
tory activity of cortisol were reported by Mag- ative of cortisol exhibits twice the thymolytic
erlein et al. (258). This group also enhances activity of the parent compound (265). Iso-
the activity of 6a-fluoroprednisolone deriva- meric 16-methylene and A15,16 methyl ste-
tives (259). By contrast, the 16p-fluoro group roids (266) show interesting differences in ac-
in cortisol, 9a-fluorocortisol, and 9a-fluoro- tivity. The A15,16-methylcompound (181)has
prednisolone produce compounds with de- 156 times the oral activity of cortisol in the
creased anti-inflammatory activity (260). granuloma test and produces sodium reten-
Likewise, loss of activity is seen upon intro- tion. By contrast, the isomeric 16-methylene
10 Effect of Individual Structural Changes on Anti-Inflammatory Activity 817

the systemic circulation, ubiquitous esterases


effect hydrolysis of the ester moiety, thus ren-
dering the drug impotent. There is essentially
no conceptual difference between Lee's ante-
drug and Bodor's inactive metabolite ap-
proach to soft drugs, only a difference in the
position of the carboxylate group. Whereas
Bodor focused on derivatization of corteinic
acid (2251, a C-20 acid), Lee chose 20-dihydro-
prednisolonic acids, or cortolic acid-like struc-
tures (a C-21 acid) such as (2241, for elabora-
tion into active prodrugs. In these cases, it is
the prodrug that is bioactive, not the liberated
steroidal skeleton. In contrast, betametha-
sone dipropionate is itself inactive and de-
pends on esterase cleavage for release of the
active species. Lee expanded this concept be-
yond the C-21 metabolite to encompass car-
boxylic acid esters at unnatural positions of
the steroid nucleus, such as C-16 carboxylic
acid esters and amides (183; R, = 0-alkyl,

compound (182) has only one-third the anti-


inflammatory action of (181) and produces so-
dium excretion.
The 16P-methoxy group reduces the anti-
inflammatory potency of 9a-fluorocortisone
21-acetate (247). 16,16-Dimethylprednisone
21-acetate is inactive in the systemic granu-
loma test (267).
Along the lines of Bodor's "inactive metab-
olite approach" or soft drugs (see Section
10.191, Lee coined the term antedrug for his NH,) (270-272). The carboxyl group can be
concept to topical-selective delivery of cortico- incorporated as part of an otherwise beneficial
steroids (268, 269). In this approach, an in- 16,17-acetonide grouping, as in (189) (273).
active corticosteroid side-chain metabolite More recently, Lee described 16,17-acetonide-
(generally a carboxylic acid) is synthetically like, fused isoxazolidine esters, (190) (274).
converted to an ester, whereupon topical ac- Esters of (183), where R, = Me (184),Et
tivity is obtained. However, on absorption into (1851, i- Pr (1861,or benzyl(187), synthesized

Table 15.36 Anti-Inflammatory Potencies of Ester and Acid Derivatives of 183: Croton Oil
Ear Edema Assay of 184-188
Compound X R RI Relative Potency Log P
Prednisolone 1 1.48
(184) H H CHs 1 1.57
(185) H H Et 1.3 1.58
(186) H H i-Pr 4.0 1.62
(187) H H CH&,H, 4.7 1.66
(188) H H OH inactive
Anti-Inflammatory Steroids

support for separation of topical and systemic


effects was garnered from the rat cotton pellet
granuloma assay (CPG).Body weight, thymus
weight, and adrenal weight were obtained at
the ED,, dose, on the basis of which it was
concluded that (186) and (187) had local to
systemic relative activity ratios of 6.1 and 6.4,
whereas prednisolone showed rather less sep-
aration, with a therapeutic index of 1.6. The
ester (184) and its 17-hydroxylated analog
(191) were compared in the CPG assay and

found to have IC,, values of 4.1 and 0.4 mg/


pellet, whereas the value for prednisolone was
2.2 mg/pellet. Thus, in the CPG assay, (184)
was roughly half as potent as prednisolone,
whereas (191) was about 5.5 times more ac-
tive. Neither compound showed increased sys-
temic anti-inflammatory activity compared to
that of prednisolone; (191) did reduce thymus
weight by 33% in contrast to 47% for pred-
from prednisolone in seven steps, had relative nisolone. Although (192), the putative acidic
potencies ranging from 1 to 4.7 in the croton metabolite of (184), did not have anti-inflam-
oil ear edema assay (Table 15.36). matory activity in the CPG, surprisingly, the
Whereas methyl and ethyl esters (184) and
(186) were roughly equipotent with pred-
nisolone, more lipophilic isopropyl and benzyl
esters had substantially improved potency. It
has been suggested that increases in lipophi-
licity can be correlated with skin permeability,
and this explanation has been invoked by Lee
to explain the improved potency of (186) and
(187) compared to that of (1841186). Perhaps
most noteworthy is the absence of detectable
glucocorticoid activity for the free acid (188),
the putative metabolite of (184-187), thus
supporting the antedrug concept. Further
10 Effect of Individual Structural Changes on Anti-Inflammatory Activity 819

the CPG assay. They were 20-40% as topi-


cally potent as prednisolone and were without
systemic effect.
Studied in some detail, acetonide-ester
(189) presented promising results as a topi-
cally selective anti-inflammatory steroid when
compared side by side with several well known
corticoids. As shown in Table 15.37, (189)had
intrinsic potency at the receptor level compa-
rable to that of prednisolone.
In the croton oil ear assay, doses required
to achieve the ED,, values allowed determina-
acidic metabolite of (191), (193) did produce tion of a topical potency ranking for dexa-
significant local anti-inflammatory activity. methasone of 1; (189), 0.5; prednisolone, 0.17;
Insertion of F at the 9a-position of (184) to and triamcinolone, 0.04. These results corre-
give (194) was accompanied by a twofold en- late well with receptor binding data for dexa-
hancement in topical potency, as determined methasone and (189). It is satisfying to note
in the croton oil ear edema assay. There were that, although (189) is nearly as potent as
no concomitant increases in untoward sys- dexamethasone, it has only a fraction of the
temic effects for (194) compared to those for systemic activity evidenced for dexametha-
prednisolone; thymic weight was minimally sone in the CPG assay.
impacted and HPA suppression was not ob- For the novel isoxazolidine (190) recently
served (275). reported by Lee, when X = H, the resulting
Acetylation or diacetylation of the 21 or compounds were not improved relative to
17,21alcohols of (183)was examined. The 21- prednisolone. However, when X = F and R =
monoacetate (195) was about 50% more active H (197) or Ac (198), useful topical activity was
than prednisolone in the CPG assay, whereas achieved. Based on ID,, doses, the relative po-
the corresponding diacetate (196) was three tency (prednisolone = l) for (197) was 4 and
times more active than prednisolone in the for (198), 5.3.
same system (275). In the croton oil ear edema In the croton oil ear edema assay, (197) did
assay, the mono- and diacetates were substan- show signs of systemic absorption as gauged
tially more active than prednisolone. These ac- on the contralateral ear, but (198)did not. It
etates appeared to be systemically inconse- was also consistently found that plasma corti-
quential, again presumably attributed to sosterone levels were reduced for (197) but
enzymatic hydrolysis to inactive carboxylic ac- not for (198). It is clear that 9a-fluorination is
ids. required to improve potency in this class,
Other interesting derivtives of (183), with whereas the acetate group at (2-21is needed to
R, = NH, and X = H or OH, were examined in help avoid systemic absorption. These results

Table 15.37 Receptor Binding Affinity, Croton Oil Ear Edema Assay, and Cotton Pellet
Granuloma Assay of 114 versus Three Common Control Corticosteroids
Compound ICs,, GCRa ID,,, CEEb CPG, TopicalC CPG, Systemicd
Dexamethasone 17 nM 0.001 m g 81%
Triamcinolone 46 0.026 62
Prednisolone 35 0.006 57
(189) 30 0.002 55
Control - - -
"Glucocorticoid receptor binding affinity.
bCrotonoil ear edema assay.
"Percentage inhibition of inflammation on treated side.
dPercentage inhibition of inflammation on untreated side.
Anti-Inflammatory Steroids

are difficult to reconcile with the antedrug


concept and, interestingly, it was found that 15.39). Furthermore, after semichronic sys-
the putative metabolite (199) did display (ar- temic dosing with 4-5, no effects on weight
guably) systemic activity of 19%. were seen for overall body weight, adrenal
Subsequently, these researchers examined gland, or thymus. However, under these con-
the effect of deleting the carboethoxy group, as ditions, potent weight loss was seen for pred-
shown in Table 15.38. First, potency is re- nisolone (e.g., 53% reduction in thymus
tained in the simple ear edema assay with po- weight).
tencies relative to hydrocortisone of 1.9-6.8
10.1 7 Alterations at C-17
for compounds with or without 9a-fluorina-
tion, or withlwithout an acetyl group at (2-21 The l7a-hydroxy group is not essential for ac-
(276). tivity. The introduction of fluorine, chlorine,
In the contralateral ear assay, although or bromine into the l7a-position of 11-dehy-
these new compounds are less potent than ei- drocorticosterone gives compounds of inferior
ther (197) or (198), there does not appear to activity (277-279). The activity of 16,17-ketals
be any sign of systemic absorption (Table and 17-esters has already been discussed.
Table 15.38 Estimated ED,, and Relative Potencies in the Acute Ear Edema Assapb

Compound R X ED50 (40 Relative Potency


Hydrocortisone - - 1.36 1
Prednisolone - - 0.64 2.2
(200) H H 0.71 1.9
(201) H F 0.33 4.1
(202) Ac H 0.42 3.3
(203) Ac F 0.20 6.8
"Ref. 276.
bCroton oil-induced edema
10 Effect of Individual Structural Changes on Anti-Inflammatory Activity

Table 15.39 Contralateral Ear Edema Assay for Compounds (200-203)"


% Inhibitionb

Compound R X Left Ear Right Ear L/S Ratioc


Prednisolone - - 57.8
(200) H H 45.7
(201) H F 55.2
(202) Ac H 49.1
(203) Ac F 57.0
"Ref. 276.
bCompoundand croton oil applied to right ear, croton oil only to the left ear. "Local effect" measured as ear thickness at
5 h.
'Ratio derived from local/systemic anti-inflammatory activities; local effect measured after 5 h, systemic effect deter-
mined after 5 once-daily applications.

17a-Methylcorticosterone 21-acetate has hydroxylation at C-6 leads to almost indect-


nearly half the activity of cortisol in the glyco- able binding affinity. It was thus proposed
gen deposition assay (280). Rimexolone (23) that the high ratio of topical to systemic activ-
(Fig. 15.2), in which the 16,17- and 21-posi- ity noted in previous pharmacological studies
tions have methyl groups instead of hydroxyl for both rimexolone and flunisolide could be
groups, is a moderately potent topical anti-in- explained by rapid systemic metabolism to in-
flammatory agent. Interestingly, (23) has un- active metabolites.
detectable dermal atrophogenicity, adrenal, or The classical hydroxy-ethanone corticoste-
thymolytic effects (281). Apparently, rimex- roid side chain attached at (2-17 is not a re-
olone is systemically impotent as a conse- quirement for activity, as was seen for 17a-
quence of rapid metabolism to inactive 6-hy- propynylated steroids. The glucocorticoid
droxylated or 5-reduced metabolites. Three receptor is apparently quite tolerant of substi-
rationally proposed but hypothetical metabo- tution at this position. Noteworthy in this re-
lites, 6P-hydroxyrimexolone (204) and both gard are 17-thioketal derivatives, such as ti-
5P- and 5a-dihydrorimexolone(205 and (206), predane (2091, which retain potent receptor
respectively), displayed poor if any binding af- binding affinity and topical anti-inflammatory
finity in human glucocorticoid receptor bind- activity. These androstene-17 thioketals have
ing assays (282). On the other hand, hydroxy- relative potencies in mouse ear edema assay,
lation in the side chain ((3-21) did provide glucocorticoid receptor binding, and inhibi-
potent derivatives (207) and (208), albeit with tion of DNA synthesis, as shown in Table
less activity than that of the control, dexa- 15.41.
methasone (20a), as shown in Table 15.40. It is clear from the significant binding af-
In the same study, binding affinities were finities of these androstenes that they possess
determined for flunisolide (26) and its puta- intrinic activity at the receptor level, which is
tive metabolite, 6p- HO-(26). As can be seen, in many cases competitive with halcinonide or

Table 15.40 Relative Binding Affinities (RBA) of Various Steroids and Rimexolone
to the Human GRa
Compound RBA Compound RBA
Dexamethasone, (20a) 100 (204) <1
T A ,(13)
~ 233 (205) 8
Flunisolide, (26) 191 (206) <1
Rimexolone, (23) 134 21R4207) 41
Hydrocortisone, (2a) 10 215'4207) 107
60-hydroxy-(26) <1 (208) 34
"Human synovial tissue.
bTriamcinoloneacetonide.
Anti-Inflammatory Steroids

(23) Rimexolone

synthesis or reduction of inflammation indi-


cate a reduced activity of (212) versus ti-
predane (209). That these differences might
have a bearing on cellular metabolism seems
verified by the finding that fluorination of the
SEt group (i.e., 213), which has a metaboli-

cally more robust 17p- C,H,F group, now


demonstrates lower receptor binding affinity
but greatly enhanced DNA synthesis inhibi-
tion and anti-inflammatory activities com-
triamcinolone acetonide. Binding affinity is pared to those of its nonfluorinated counter-
enhanced on interposing SMe with SEt groups part, (212).
(209) versus (212), amounting to a preference The more potent of these thiasteroids, ti-
for the more bulky SEt group in the 17p-posi- predane (209) and (213), were the subject of
tion. On the other hand, other measures of more intense pharmacological scrutiny (284).
intrinsic activity such as inhibition of DNA A comparison of a wide range of corticoste-
10 Effect of individual Structural Changes on Anti-Inflammatory Activity 823

Table 15.41 Biological Potencies of Andostene-17-thioketalsversus Reference Steroids"

(209) Rl = SCH3, R2 = SEt


(210) Rl = SEt, Rz = SEt
(211) R1 = SCH3, R2 = SCH3
(212) R1 = SEt, & = SCH3
(213) Rl = SC2H4F,R2 = SEt
(214) R1 = SCH3, Rz = SC2H4F
(215) Rl = SEt, R2 = SC2H4F
Compound
(205)
(206)
(207)
(208)
(209)
(210)
(211)
Halcinonide (12)
TA (13)'
Hydrocortisone (2)
aRef. 283.
bRelativeglucocorticoid receptor (rat liver cytosol) binding affinity.
'Thymidine incorporation into DNA of mouse thymocytes.
dCrotonoil-induced mouse ear edema assay.
eTriamcinoloneacetonide (13).

roids vs. these two thiasteroids is shown in than 1 h to a plethora of metabolites having
Table 15.42. Although clobetasol butyrate greatly reduced anti-inflammatory activity.
demonstrated very high intrinsic activity, as The primary pathways (Fig. 15.15) are oxida-
gauged by receptor binding, it had only weak tion of (209) to sulfoxides, followed by loss of
influence on thymic involution and was a rel- thioketal grouping and formation of andro-
atively weak anti-inflammatory agent against sten-17-01, -one, and -thiols (285).
edema, being more readily compared to hydro- A series of related thioketals, also situated at
cortisone valerate. the 17-position of an androstan-17-one core,
Tipredane and (213) are both bound to the have been described that employ an antedrug
GR and have markers of high anti-inflamma- strategy (see below) (286). In this approach, a
tory potency, as indicated by their inhibition 17P-thiol moiety is attached to a butyroladone
of DNA synthesis and reduction of edema rel- ring in the active drug. Rapid hydrolysis in
ative to controls. Their lack of effect on thymic plasma leads to relatively inactive metabolites.
involution is unexplained, but clearly metabo- This design emulates other topically potent
lism of these steroids is more complicated than corticosteroids such as fluticasone propionate
for a classical corticosteroid having the usual (Flonase), in which hydrolysis of a side-chain
20-keto-21-01 functionality. For example, ti- ester group leads to inactive metabolites. As
predane (209) is avidly metabolized in mouse shown in Table 15.43, (216) and (217) are
liver preparations or in human skin in less stable in buffer solution but are rapidly hydro-
824 Anti-Inflammatory Steroids

Table 15.42 Pharmacological Profiles of a Variety of Corticosteroidsversus (153) and (157)


Compound GRBa DNA~ EEAc Thymusd
Clobetasol butyrate 2 0.23 0.22 0.04
Betamethasone Dipropionate 1.4 0.17 0.84 2
Betamethasone valerate 2.2 0.90 0.48 0.36
TAe 1 0.28 0.44 0.46
Halcinonide 1 1 1 1
FA^ 0.86 0.73 0.83 2.8
Dexamethasone 0.72 - 0.12 -
Budesonide 0.57 0.63 0.68 0.33
Clobetasol propionate 0.57 1.6 0.98 0.70
Fluocinonide 0.43 0.12 0.98 0.93
Hydrocortisone valerate 0.34 0.04 0.11 0.01
Hydrocortisone 0.23 0.008 0.08 0.04
Tipredane, (209) 0.66 0.65 1.2 0.04
(213) 0.31 1 1.1 -
"Relative glucocorticoid receptor (rat liver cytosol) binding affinity.
bThymidineincorporation into DNA of mouse thymocytes.
"Croton oil-induced mouse ear edema assay.
dThymus involution following s.c. administered corticosteroids.
'Triamcinolone acetonide.
fFluocinoloneacetonide.

lyzed in human plasma to the relatively inac- effects typical of inhaled corticosteroids. More
tive hydroxyacid (219) (racemization). Like- remarkable is the fact that these derivatives
wise, the related species (218) is also stable in are relatively stable to human lung S9 fraction
buffer but hydrolyzed rapidly in plasma to the (t,,, = hours), perhaps making them useful for
inactive metabolite (220). All three of these treatment of asthma.
antedrugs have half-lives on the order of min- The thiolactones apparently act as conven-
utes, clearly establishing that they would not tional corticoids in that the intrinsic activity
likely have the usual constellation of systemic at the hGR receptor is on the order of dexa-

Figure 15.15. Metabolic profile of tipredane (209) after 0.5-h exposure to human skin (285).
826 Anti-Inflammatory Steroids

Table 15.44 Inhibition of Rat Ear Edema and Thymus Weights by Table 15.43 Antedrugs
% Decrease % Reduction
Compound Dose ( p d a (ear weightIb (thymus weighty

Fluticasone propionate
Fluticasone propionate
Fluticasone propionate
Budesonide
"Standard croton oil ear assay, compound applied in acetone to both ears.
*Decrease was calculated relative to control animals treated only with croton oil.
"Thymus involution test; thymus weight determined relative to controls after several days of dosing

methasone itself. The in vitro cell studies par- Whether the compound must first be hydro-
allel the receptor assays, again demonstrating lyzed to the 20-ketone is not known, although
excellent potencies for these three leads. it is stable in weak acid and enzymatic hydro-
Compared with fluticasone propionate in lysis in liver brei did not occur. The related
the mouse model of inflammation (croton oil bismethylenedioxy compound (222) is more
ear assay; see Table 15.44), (218) was less ef-
fective than fluticasone propionate, but still
showed significant anti-inflammatory activity
at 100-pg doses.

10.1 8 Alterations at C-20


Ketalization of the C-20 carbonyl group of cor-
ticoids with ethylene glycol gives ketals that
retain biologic activity, although it is lower
than that in the parent compound (287). The
most active compound is the 9a-fluoro-6a-
methylprednisone derivative (221), which is
four times as potent as triamcinolone.

active than cortisol itself, although the activ-


ity is lower than that of the parent steroid
(288).

10.19 Alterations at C-21


Reduction of cortisol to 21-deoxycortisol (new
90) gives a compound having about one-third
the thymolytic activity of its parent (289). If
activating groups are introduced into the mol-
ecule, clinically useful ophthalmic anti-in-
flammatory steroids such as medrysone (24)
result. Medrysone has good anti-inflammatory
activity with relatively little effect on intraoc-
10 Effect of Individual Structural Changes on Anti-Inflammatory Activity

ular pressure (290). Fluorometholone (22) is Chemical synthesis


an efficacious topical agent (291) and also has
little tendency to increase intraocular pres-
sure when used as an anti-inflammatory agent
in ocular diseases (292). / 3
'

Inactive metabolite Antiinflammatory


agent

Biological transformation

Figure 15.16. Bodor's inactive metabolic approach


to soft corticosteroids.

Oxidative metabolic degradation of the di-


hydroxyacetone side chain of cortisol (2) pro-
ceeds in a stepwise fashion to provide the al-
dehyde (2231, the ketoacid (2241, and, finally,
corteinic acid (225). The acidic metabolites
corteinic acid (225) and the pyruvic acid, cor-
tolic acid (224), are inactive and have been
used as leads to design isosteric andtor isoelec-
tronic replacements for potent corticosteroids.
A steroid containing a 21-carboxylic acid
ester, fluocortin butyl(226), shows significant
activity on human skin, but little or no sys-
temic action in rats (295). Related diesters of
17a-hydroxyandrostane-17P-carboxylic acids,
such as (227), are active topically (296).
Building on these leads, but with special
The propionate and butyrate 17-mo- attention to isosteric/isoelectronic principles
noesters of 6a,9cu-difluoro-2l-deoxypred- from molecular modeling, Bodor designed a
nisolone have high topical anti-inflammatory class of corteinic acid esters, (228), having a
activity (293) (refer also to section 4.2.2 and carbonate group at the l7a-position (221).
ref. 4). Oxidation of cortisol to the 21-aldehyde These compounds have demonstrated topi-
gives a product that retains its anti-inflamma- cal efficacy in the cotton pellet granuloma
tory activity (2941, but it is not known (CPG) assay, with apparent diminished sys-
whether this is attributable to reduction back temic action, as gauged by noneffect on thy-
to the primary alcohol. mus weight. Representative examples, shown
In addition to Bodor's chemical delivery in Table 15.45 , have the Y group = H.
systems for soft drugs applied to corticosteroi- It can be seen that the soft steroids are very
dal 3-spiro thiazolidines such as (142), an- potent and that the 9a-F derivative (231)
other approach to soft drugs is the use of inac- shows good activity at a much lower dose than
tive metabolites. The inactive metabolite reference steroids while having no systemic
approach depends on enzymatic cleavage of a effect. In contrast, widely used reference ste-
biologically active synthetic steroid to an inac- roids such as betarnethasone valerate re-
tive structure or metabolic product, and is quired a much higher dose to approach the
shown diagrammatically in Fig. 15.16. degree of topical activity demonstrated by
Anti-Inflammatory Steroids

(228) R1,R2= alkyl, haloalkyl, alkoxyalkyl, etc.


RS= H, OH, CH3
X, Y = H or halogen

(231),at which point signs of systemic absorp-


tion were prominent. Potencies were deter-
mined for (230), (2311, and (232) relative to
that of BMV (=I)in the granuloma assay.
They were determined at the ED,, to be 4,
430, and 474, respectively. Clearly, both (231)
and (232) demonstrated excellent topical effi-
cacy relative to that of betamethasone esters,
being more than 400 times more active than
10 Effect of Individual Structural Changes on Anti-Inflammatory Activity 829

Table 15.45 Effect of Locally Administered Selected "Soft Steroids" and Reference Steroids
on Granulation Tissue Formation Caused by Implantation of Cotton Pellets in Rats

Compound X R R1 Dosea Body Weightb (%I Inhibitionc Thymusb


None 32
(229) H i-Pr H 1000 34 70 -
(230) H, A1 i-Pr H 1000 29 77 -
(231) F, A C,H, a-CH, 10 36 73 -
(232) H, A i-Pr Pa 10 33 63 -
HC-17Bud 1000 26 56 78
BMV 1000 5 53 83
"Dose expressed in microgram per pellet.
bChangesin body weight (gain, in grams) and thymus (% reduction in weight) indicative of systemic effect.
CPercentageinhibition of granulation of cotton pellet implanted under skin along with dose of steroid.
dHydrocortisone17-butyrate (2a).
'Betamethasone valerate (512).

BMV. A therapeutic index for (230) was deter- Many compounds in the (228) series, where,
mined, by the ratio of the relative anti-inflam- for example, X or Y was F, were as potent as
matory potency and the relative systemic po- betamethasone valerate or clobetasol propi-
tency (% thymus weight reduction), to be 48. onate in the human vasoconstrictor assay. Thus,
In another slightly different example, (2331, although it appears that these steroids do not
achieve systemic circulation in substantial con-
centrations on the basis of the foregoing data,
they are exceedingly topically potent. When
these compounds were injected subcutaneously
in mice at relatively high doses, some signs of
systemic toxicity were noted that were nonethe-
less attenuated relative to control.
Compound (231) has been examined for its
effects on cell growth and wound healing. It is
generally accepted that corticosteroids ad-
versely affect healing and inhibit cell growth.
Compared to betamethasone dipropionate,
wounds treated with (231) healed twice as fast
and were comparable to untreated groups.
Studies have also demonstrated the useful-
where R = Me, R, = a-Me, X = F with A1 ness of these soft corticosteroids in corneal
unsaturation, the therapeutic index was over healing (297).
7000. In the case of (233), the absolute relative The introduction of an additional methyl
potency was still over 200 compared to that of group at C-21 to produce a secondary alcohol
HC-17Bu,or over 50 compared to that of BMV. gives compounds with about 112 the activity of
Anti-Inflammatory Steroids

serves in this instance a drop in potency upon


incorporation of a polar ester tail, as in (235).
Esterification of the 21-hydroxyl group, giv-
ing, for example, (238) (a mixed ester very
similar to betamethasone dipropionate), is re-
flected in its good relative potency. However,
in this series, the effect of ester substitution in
the l7a-side chain is to decrease overall activ-
ity. This loss is clearly mitigated in some in-
stances by incorporation of 21-halogen, the ef-
fect of which is seen for the various succinoyl
esters (239-247). When the ester group is
the parent structure (298, 299). The related methyl (239), topical potency is better than
20,21-diketone (234) is also active (300). Re- that of BV. The bulkier esters, ethyl through
placement of the hydroxyl group at C-21 by a cyclohexyl, are relatively topically weak. Cer-
fluorine atom (301) doubles the anti-inflam- tainly, (239) is of interest because it incorpo-
matory activity. The effect of substitution of rates an ester that is capable of enzymatic
the 21-hydroxyl function by chlorine in ste- cleavage to inactive metabolites while main-
roids with a 16a,17a-acetonide group depends taining excellent topical potency in humans.
greatly on other molecular substituents (302). In closely related studies, Ueno et al. (40)
When the 9a-fluoro substituent is present in showed that structures of this type have very
the parent compound a useful compound, hal- low thymolytic involution activity in the gran-
cinonide (12), results. The introduction of uloma pouch assay and, further, that human
diazo and azido groups into prednisolone gives skin metabolites are lacking in activity. In Ta-
compounds that retain moderate amounts of ble 15.47, relative potencies and receptor
binding values are provided for the esters
activity (303,304).
(248-254).
Taken together with the lack of systemic
absorption signaled by undetectable thymic
involution in the granuloma assay, these com-
pounds clearly fulfill the necessary criteria for
antedrug status. Of these compounds, the
methyl ester (248) was most potent. More
bulky esters such as isopropyl (252) or tert-
butyl(253) were much less active than (2481,
perhaps not surprisingly on the basis of the
foregoing discussions.

The combination of 21-chlorination with a


soft drug or antedrug approach has been de-
scribed by formation of functionalized 17-es-
ters. Mitsukuchi and colleagues (305) pre-
pared a series of l7a-succinoyl esters (235-
247) and observed topical potency in the
McKenzie assay. The potencies illustrate the
enhancing effect of 21-chlorination on topical
efficacy. As illustrated in Table 15.46, when R
= OH, the resultant structure is somewhat
reminiscent of betamethasone valerate (BV,
relative topical potency of 1) (305). One ob-
10 Effect of Individual Structural Changes on Anti-Inflammatory Activity 831

Table 15.46 Vasoconstrictive Assay of 17a-SuccinoylEsters o f Betamethasonea

Compound R RI Topical Potencyb


OH
OCOCH,
OCOEt
OCOPr
C1
C1
C1
C1
C1
C1
C1
C1

"Ref. 305.
bMcKenzieassay in human volunteers; BV = control.
'BV = betamethasone valerate (Sc).

An amide at C-20, as in (255),gives steroids


that retain more than half the activity of the
I
parent compound in the liver glycogen test
(306).
( COOH
Sulfur-containing substituents at C-21,
such as 21-mercapto, 21-methylthio, 21-thio-
cyano, and 21-acetylthio, gave corticoids as of
1961 that were not of biologic interest (307).
Thirty years later, an acetyl cyteine derivative
at C-21, (256), was shown to possess about a
tenth of the activity of dexamethasone. On the
other hand, systemic absorption appeared to
be curtailed on the basis of thymus weight,
body weight, and adrenal-suppressive data
(308). heres to the soft drug concept in providing an
Replacement of the 21 carbon by S, on the inactive corteinic acid-like metabolite, (267)
other hand, provided 21-thioesters related to (see page 835).
(2271228)that have found clinical utility. The Although fluticasone propionate (16), mar-
trifluoro-thioester (16) is a leading example in keted by Glaxo as Cutivate, exhibits some
the series (257-266) and (16)that is reminis- HPA suppression when administered subcu-
cent of Bodor's andlor Laurent's esters. Me- taneously, it does not posssess oral antiglu-
tabolism studies have revealed that (16) ad- cocorticoid activity, an important consider-
832 Anti-Inflammatory Steroids

Table 15.47 Activity of 17a-Alkanoate Esters of 21-Desoxy-21-Chlorobetamethasonea

(248-254)
Compound R n EEA~ Thymusc GRBd

"Ref. 40.
'Croton oil ear edema assay.
'Thymolytic involution in the granuloma pouch assay.
dGlucocorticoidreceptor-bindingassay; IC,, values, nM.
"Not detectable at the highest dose.
fclobetasol propionate.
SBetamethasone dipropionate.

ation for inhaled drugs in which much of the Lee's antedrug concept has been applied to
metered dose is actually swallowed (309). Bio- steroidal 21-esters, which on ester hydrolysis
assay data for these thioesters are shown in lead to inactive steroidal acids (see Section
Table 15.48. Human vasoconstriction data re- 4.1). First reported in 1982, methyl pred-
veal, for example, that when the 6a-position is nisolonate (268) and its diastereomeric 20-al-
unsubstituted, Y = H or (259), a very potent cohols, methyl 20R-dihydroprednisolonate
steroid results. Unfortunately, this same ana- (270) and methyl 20s-dihydroprednisolonate
log (259) is substantially more active system- (269), retain significant local anti-inflamma-
ically than (16). Interestingly, replacement of tory activity, but are devoid of prednisolone-
the 9a-position of (259) with C1, resulting in like side effects, such as pituitary adrenal sup-
(258), provides an analog of potency in the pression and thymus involution (268). The
realm of Cutivate but, again, with substantial orientation of the alcohol group at C-20 was
systemic activity; further, the 6a-F group in important for potency, given that (269) was
this series generally appears to be a predictor several times more potent than (270) (269).
of systemic activity. Perhaps not surprisingly, That the acidic metabolites were indeed de-
both Br or I = X in this series were detrimen- void of anti-inflammatory activity was ascer-
tal to activity; the ranking of potency for X is F tained independently (310).
> C1> Br, I. When the prednisolonates (2681, (2691,
In the series (256-266), potency is depen- and (270) were applied locally at equivalent-
dent on the size of the 17-ester group, where potency anti-inflammatory doses compared
the R = Me or CH,CH3 esters are more active with that of other corticosteroids, they mark-
than the esters where R = CH,CH,CH3. edly inhibited granuloma formation but did
10 Effect of Individual Structural Changes on Anti-Inflammatory Activity 833

Table 15.48 Biological Activities of Halomethyl Androstane-17p-carbothioates

Mouse
Compound Z Y X R 16 Human Va AITb HPAc

"Human vasoconstrictor activity relative to fluocinolone acetonide (104).


bTopicalanti-inflammatory activity relative to fluocinolone acetonide (104).
'Systemic corticosteroid activity after topical application relative to fluocinolone acetonide (104).
dStandard.

not inhibit skin collagen synthesis nor cause The aldehyde (276) showed signs of sys-
dermal atrophy in rats (311). temic absorption and activity that were com-
Removal of the l7a-hydroxyl group of the parable to that of prednisolone but the ester
prednisolonates above gave the deoqpred- (273) was virtually devoid of systemic effects,
nisolonate structures (2731, (2741, and (275), on the basis of thymus weight changes and
which led to interesting changes in activity rel- reduction of plasma corticosterone levels (ad-
ative to that of their hydroxy counterparts. Pro- renal suppression) (270).
duced during synthesis, the aldehyde (276) was Interestingly, acetonide formation across
also examined for anti-inflammatory activity. the 17,20-diolarrangement led to more potent
The keto-ester (273) and keto-aldehyde (276) analogs compared to the free diols. In Table
were as potent as prednisolone in the granuloma 15.49 the diols (269) and (270) and their cor-
pouch assay, whereas the alcohols (274) and responding acetonides, (277) and (278)., re-
(275) were almost inactive. It is clear that the spectively, are examined in croton oil ear
l7a-hydroxyl group is needed for activity in the edema assay, cotton pellet granuloma pouch
21-ester series when the 20-position is reduced, assay, and for glucocorticoid receptor affmity.
but is not necessary in 20-keto steroids. Although none of these analogs is more potent
Anti-Inflammatory Steroids

HO

(269) active antedrug


0
k esterases

Liver
___f

0
F (271) inactive

(279) nor (280) had systemic effects on thy-


mus weight, and were promptly metabolized
to (281) (active) within 2 min of i.v. dosing. A
second metabolic event deactivates (281) by
deesterification, to furnish the inactive car-
boxylic acid (282), with a t,,, value of about 2 h
(312).
The effect on anti-inflammatory activity of
replacing the ester group of the diols (269) or
(270) by metabolically tenacious amides has
been examined (275). The carboxamides (283)
(268) active antedrug and (284), where R = Me or bulkier benzyl,
exhibited potent local as well as systemic ac-
than prednisolone (211, the acetonide (278) tivities compared to those of prednisolone (see
more closely approximates the intrinsic bind- Table 15.50). The more potent topically active
ing affinity and topical efficacy of (21). It is compounds were unfortunately also the most
fascinating that the stereochemical depen- systemically absorbed, as gauged by several
dency on activity for the flexible diols, where measures of systemic potency: thymic involu-
20s > 20R, is reversed for the conformation- tion, reduction in plasma corticosterone, and
ally restricted acetonides, where the potency contralateral ear effects. As with the ester-
of 20R > 208. acetonides above, the 20R isomers were more
The succinate ester antedrug (280) was potent than the 20s isomers.
comparable to dexamethasone in an ear It is probable, based on the presence of un-
edema assay for potency, whereas the less li- desirable side effects for these amides, that
pophilic acid was slightly less potent. Neither metabolism has been curtailed. This is per-
10 Effect of Individual Structural Changes on Anti-Inflammatory Activity

Table 15.49 A n t i-M a m m a t o r y Activity of Steroidal 21-oate Esters

Compound ID,, GPAa ID,, EEAb IC,, GRB'


(269) 2.8 5.9 31.6
(270) 13.5 32.8 11.5
(277) 15.4 3.6 1.6
(278) 2.6 1.4 0.36
(md 1.4 0.4 0.18
"Cotton pellet granuloma pouch assay, pmoUpellet.
bCrotonoil ear edema assay, pnol/ear.
'Rat liver cytosol receptor, ptvf inhibition.
dPrednisolone (21).

Table 15.50 Anti-Inflammatory Activity of 20-Carboxamide Derivatives a


HN-R HN-R

Compound EEAa GPA, top.b GPA, S ~ S . ~ Thymusd Plasma colt.'


2022-(2841, R = CH, 33.5 75.5 48.6 45.6 73.1
203-(283),R = CH, 24.5 21.1 -4.5 96.7 66.1
20R-(2841,R = benzyl 42.6 69.3 38.9 65.4 23
208-(283),R = benzyl 21.9 20.9 5.2 103.4 67.8
Prednisolone (21) 68.6 60.6 26.6 43.1 29.6
"Ref. 275.
b%Inhibition following application of 0.75 mg/mouse ear.
'% Inhibition of granuloma formation after insertion of cotton pellet treated with 2 mg compound.
d% Inhibition of granuloma formation on distal (contralateral) ear.
'Thymus weight as % of control in granuloma assay.
'Plasma corticosterone levels as % of control in granuloma assay.
Anti-Inflammatory Steroids

(270) active antedrug

esterases

1
H02C,
OHC,

(272) inactive
ence of amides such as (283) in the systemic
circulation could lead to agonistic glucocorti-
coid behavior because it has been shown that
(283) has receptor affinity comparable to that
of prednisolone. Thus, for (283), where R =
Me, the IC,, value is 275 pM (prednisolone =
196 a); and when R = benzyl, the IC,, value
a.
is 54 It is consistent with earlier discus-
sions that partition coefficients correlate
loosely with topical potency. The P values
have been measured for several of these amide
analogs and those with P values of 30 (log P >
1.48) or better were the most active in the se-
ries (313).

Antiglucocorticoids, or GR antagonists, are


compounds that bind tightly to the GR but do
not illicit a corticosteroidal action. Clinically,
antiglucocorticoidsare used to reduce intraoc-
ular pressure (IOP) and in the treatment of
Gushing's disease (glucocorticoid excess). Re-
cently, it has been proposed that antigluco-
haps not surprising, in that arnides are gener- corticoids might have a use in reducing the
ally more stable than esters in vivo. The pres- GR-mediated side effects associated with glu-
11 Antiglucocorticoids 837

cocorticoid use while not significantly inter- 1. Unsaturation of the A-ring: alteration of
fering with the anti-inflammatory action of the A-ring conformation. Except for the A-
the compound (12). ring diazoles, all the GR antagonist known
It is thought that there is a correlation be- have the 4-ene-3-one structure. Because
tween degree of antagonism and steroid bind- this is shared with the most potent glu-
ing affinity, although strong binding affinity cocorticoids, it is thought that this struc-
does not distinguish between agonistic and an- tural feature increases binding affinity to
tagonistic activity. It is difficult to define exact the steroid receptor (314).
stuctural features that lead to strictly agonist 2. The hydroxy groups at C-11, (3-17, and
or antagonistic activity at the GR. Although C-21, although not necessary for a com-
X-ray structures may reflect certain SAR pat- pound to bind to the GR, seem to be indi-
terns, the X-ray structure of an agonist or an- vidually or collectively responsible for ago-
tagonist bound to the GR is yet unavailable, nistic activity, although some compounds
and the presumption that a certain steroid with hydroxyls at one or more of these po-
conformation is indeed that in the binding site sitions still act as GR antagonists [e.g., RU-
is yet speculative (although the rigidity of a 486 (285) and dexamethasone oxetanone
steroid backbone allows for some predictivity). (29611. The absence of any of these hy-
Some general structural features that lead droxyls may be one factor related to antag-
to both observed differences in steroidal con- onistic activity (314), although not neces-
formation (determined by X-ray structures) sarily a determining factor. The lack of a
and to noticeable effects in bioactivity (RBAs, hydroxyl at C-11 is a noticeable factor with
agonism vs. antagonism, magnitude of activ- some GR antagonists. Whereas all anti-in-
ity, etc.): flammatory glucocorticoid agonists have a
838 Anti-Inflammatory Steroids

The most clinically useful GR antagionist is


RU-486 (315, 3161, which also shows signifi-
cant PR antagonism. This compound has been
more completely characterized as a partial an-
tagonists in certain cell lines (317, 318), and
thus sometimes has been called a type I1 an-
tagonist. Although a type I1 antagonist-occu-
pied receptor can bind to DNA, it cannot inter-
act in a productive manner with the DNA in a
way equivalent to an agonist-occupied recep-
tor (319). RU-486 blocks the transactivation of
the GR and inhibits the production of associ-
(285) RU486 GR antagonist ated proteins, notably dexamethasone-in-
duced extracellular LC-1 (112).
hydroxyl at C-11 (or are readilly solvolized The defining feature of RU-486 is the l l p -
to a C-11 OH), many antagonists (though aryl moiety. Most steroidal GR antagonists in-
not all) have no hydroxy moiety at C-11. clude llp-aryl or other l l p substituents. The
For example, compounds that have shown tight binding affinity of RU-486 to the GR is
antagonistic (ZK 98299, 286) or mixed ag- presumed to be in part attributable to a tight
onist activities (RU-486) have substan- fit of this llp-aryl component in a lipophilic
tially bulky groups at C-11. pocket within the GR hormone-binding re-
gion. ZK98299 is an llp-aryl GR antagonist
very similar in structure to RU-486, although
it is a pure (type I) antagonist. Like RU-486, it
also has antiprogestational activity. The llp-
N-methyldihydroindol-5-yl analog of RU-486
(2871, another compound with GR antagonis-

(286) ZK98,299 GR antagonist

An interesting comparison between ago-


nist and antagonist to the GR is that of dexa-
methasone (20a, agonist) and dexamethasone (287) the N-methyl dihydroindolo-5-yl-analog
of RU486
oxetanone (296, antagonist). The X-ray struc-
tures of these compounds indicate that all four
rings overlap very well and should thus fit into tic properties and a strong GR binding f i n -
the ligand-binding site in a similar manner. As ity, has a rotomer that fits well in the space
with differences in the magnitude of agonist (llp-pocket) occupied by the llp-substituent
activity, the agonistlantagonist distinction of RU-486.
may be attributable to chemical factors, in- Another class of glucocorticoid antagonists,
cluding the hydrogen bond donating or accept- the lop-androstanes (e.g., RU-39305 and RU-
ing ability of the ligand. In this example, both 43044) also bind tightly to the GR (Table
compounds can accept a hydrogen bond at 15.51). Relative binding affinities (RBA)to the
C-20, although only the agonist is able to do- GR (vs. dexamethasone) do not lead to clear
nate hydrogen bonds (314). SARs with regard to unsaturation at A' or A6.
11 Antiglucocorticoids

Table 15.51 Relative Binding Affinities of log-Androstanesa

RU Code and
Structure Thymocytes IC,,
Number Unsaturation R Thymus GR RBAb PR RBA (nM)
39305,(288) H 57 <0.1 100
43044,(289) p-CH3 130 <0.2 200
43065,(290) p-0CH3 17 <0.1 500
46759,(291) A1, A6 p-CH3 33 <0.1 500
44068, (292) A1 P-CH~ 15 <0.1 >lo00
44427, (293) A6 p-CH3 3 <0.1 1000
"Modified from Philibert (320).
bDexamethasone = 100.

However, space-filling models indicate a simi-


larity between the 3D space occupied by the
lop- and llp- compounds (3201, although the
aromatic moiey of RU-39305 (288) is not in
the same orientation as that of RU-486. These
compounds have very little affinity for the pro-
gesterone receptor (PR).
On the other hand, "moving" the aryl
group from the C-18 angular methyl up to the
C-19 methyl provides a similar looking ste-
roid. Two compounds were tested, (294) and

methasone 21-mesylate) that covalently bind


to the GR (3221, has better defined the struc-
ture and function of the GR. Spiro C-17 oxet-
anes have shown potent antiglucocorticoid ac-
tivity in whole-cell systems (323, 324).

(2951, and were found to be about as potent as


RU-486 in contraceptive assessments. Both
(294) and (295) had lower antiglucocortiocoid
activities than those of RU-486 (321).
Research done by Simons by use of antiglu-
cocorticoids, including compounds (e.g., dexa-
840 Anti-Inflammatory Steroids

Although the lop- or llp-group is found


important with many GR antagonists, it is not
a necessity for antiglucocorticoid activity. Cor-
texelone (34) acts as a GR antagonist in the
true definition, blocking GR transactivation
(325-3271, although it does not include an
11p-substituent.

(331). For this reason, a continued effort has


been seen in the design of selective (anti-) glu-
cocorticoids and selective (anti-) progestins.
Certain structural features have been impli-
cated as consequential in the differentiation
between GR and PR binding. As seen previ-
ously, RU-486 is a potent antiprogestin and
antiglucocorticoid. It is apparent that an l l p
group increases RBA for both GR and PR, al-
though structural modification of the l l p
Although 11-deoxycortisolis an in vitro GR group affects GR and PR binding differently
antagonist, it is an agonist in vivo, presumably (323, 332). As shown in Table 15.52, for in-
through the metabolic hydroxylation at C-11. stance, although vinyl andp-tolyl substituents
The reduced "affinity" for llp-hydroxylation both lead to significant binding in both recep-
shown by unsaturation at C-9 (i.e., 299) (11) tor types, some differentiation can be made in
led to the development of ll-oxa-ll-deoxycor- favor of GR binding (m-MeO-phenyl,p-F-phe-
tisol (298), a compound with GR antagonistic nyl, and p-NH,-phenyl substitnents have
activity in vivo (although with a GR RBA 20- GRPR "RBA ratios" of 17, 3.2, and 2.9, re-
to 60-fold less than that of dexamethasone) spectively, vs. 0.6 for RU-486) or PR binding
(328,329). (p-MeS-phenyland Me substituents have 0.09
Most progestins and antiprogestins bind to and 0.25 GRPR "RBA ratios," respectively).
the GR and can elicit some antiglucocorticoid An interesting observation is that the l l p -
activity (330). There is probably a relation be- pocket of the GR receptor seems to be more
tween the high degree of homology between spacious than that of the PR in the area closest
PR and GR and the fact that certain com- to the steroid backbone. This idea is supported
pounds show both GR and PR antagonism by increased GR RBA of steroids with bulky
llp-substituents such as t-butyl and cyclopen-
tyl (GRPR RBA ratios of 60 and 43, respec-
tively).
Addition of an a-CH, at C-6 increases the
GR selective binding, whereas a p-CH, was
equivalent to no substitution. Substituting
the (2-17 1-propynyl group of RU-486 with a
chloro-ethynyl group (Table 15.53) had only a
small effect on GR binding affinity (range
from 10% increase to 3% decrease) and de-
creased PR binding affinity (by up to 30%)
(332). By reversing the C-17 stereocenter, the
GRPR RBA ratio is increased to 30 (vs. 0.58
11 Antiglucocorticoids

Table 15.52 Effect of 11-Substituenton Progesterone and


Glucocorticoid Receptor Binding Af6nitiesa

llp-Substituent (R) GR Bindingb PR Binding"


p-F-phenyl, (300) 285 85
p-NH2-phenyl, (301) 118 40
p-(CH3),N-phenyl, (285) 300 530
m-CH30-phenyl, (302) 245 15
p-CH30-phenyl, (303) 300 505
p-tolyl, (304) 295 295
p-CH3S-phenyl,(305) 180 605
H, (306) 2 15
Vinyl, (307) 340 390
t-Butyl, (308) 50 1
Phenyl, (309) 240 65
"Ref. 323.
bDexamethasone = 100%.
"Progesterone = 100%.

for RU-486), although the GR RBA is reduced PR antagoism altogether from GR effects. The
to about 40% of that of RU-486 (332). SARs from these PR-focused studies are use-
Researchers have examined other alter- ful in understanding GR SARs. The data from
ations at C-16 and C-17 intended to separate Table 15.54 show that RTI-012 (315)and RTI-

Table 15.53 Effect of Substitution at C-17 on Relative Binding Affinities in Progesterone


and Glucocorticoid Receptorsa

"Ref. 332.
bDexamethasone = 100%.
'Progesterone = 100%.
842 Anti-Inflammatory Steroids

Table 15.54 Relative Binding Affinities of Mixed-PRIGR Ligandsa

Compound
RTI-012, (315) OAc
RTI-022, (316) H
Dexamethasone
RU-486,(285)
Progesterone
"Ref. 333.
bProgestin receptor (h-PR) from baculovirus expression system, competitive assay with tritiated progesterone as refer-
ence ligand.
"G1ucocorticoid receptor (h-GR) from MDA-231 expression system, competitive assay with tritiated dexamethasone as
reference ligand.

022 (316)both have greater binding affinity' to translocate GR to the nucleus. On the other
than that of either progesterone or dexameth- hand, they are active antagonists of PR tran-
asone, with little apparent separation of ef- scriptional activity. Thus, a separation be-
fects. However, the issue of antagonism, par- tween GR and PR antagonism occurs by virtue
tial agonism, and pure agonism for these of differences in mode of action of these com-
compounds was studied in some detail. These pounds at the receptor level (333).
compounds function as competitive antago- In a later study (Table 15.55),relative bind-
nists of GR function because they are unable ing affinities of several aryl-substituted a-pro-

Table 15.55 Relative Binding Affinities of Mixed-PRIGR Ligandsa

Compound R r-PRb r-GRc PRIGR


RTI 6413-003,(317) (CH&N- 118 20
RTI 6413-018,(318) CH3CO- 144 5
RTI 6413-033,(319) CH3S- 106 10
RTI-012, (315) See above 134 86
"Ref. 334.
*Progestin receptor from Rabbit, competitive assay with tritiated progesterone as reference and ligand.
"Glucocorticoid receptor from rabbit thymus, competitive assay with tritiated dexamethasoneas reference and standard.
11 Antiglucocorticoids

Table 15.56 Relative Binding Afihities of Mixed-PRIGR Ligandsa

Compound At-I GR (%Ib PR (%)" GRPR

"Ref. 335.
*IM-9 cells, cytosol; relative binding affinity; dexamethasone = 100%.
"MCP-7 cells, cytosol; relative binding affinity; Org 2058 = 100%.

panolyl derivatives of RU-486 were compared. has not yet been found, several general obser-
Compared to RTI-012, the new compounds vations could be made. Apparently, GR-activ-
demonstrated lower GR binding, but unlike ity and selectivity are critically dependent on
RTI-012 (315), the new compounds lacked both the substituent at Ar, and Ar,. The opti-
oral antiprogestational activity in the anti- mum substituent at Ar, seems to be the
Clauberg assay (334). 4-N,N-dimethylamino- or the 3,4-methyl-
A series of l7a-arylalkyl analogs reminis- enedioxo group. The 3-(thio)methoxy- or
cent of RU-486 were tested for their ability to 3-N,N-dimethylamino- group, which have
bind to the human glucocorticoid receptor been reported to induce high selectivity for the
(GR; IM-9 cells, cytosol) and the human pro- GR, led to a significant decrease in affinity for
gesterone receptor (PR: MCF-7 cells, cytosol), the GR upon combination with a substituted
as shown in Table 15.56. Of approximately 80 21-phenyl group (data not shown). As shown
different compounds tested by the authors in Table 15.56, substitution of Ar, by a 4-N,N-
(335), only a limited number of compounds dimethylamino (321), 4-sulfone (322,323), or
combine a high binding to the GR with a low 4 pyrrolidone (324) moiety leads to a dramatic
binding to the PR (thus a high GRPR). Al- increase of the GR/PR ratio compared to that
though a solid structure-activity relationship of the unsubstituted Ar, (320). A 4-carboxa-
844 Anti-Inflammatory Steroids

Table 15.57 Nonsteroidal Glucocorticomimetic lH-Quinolin-2-onesa

Compound
(328)
(329)
(330)
(331) (racemic)
(+)-(332)
(-14333)
(334)
(335)
(336)
Prednisolone
"Ref. 336.

amide or a 6sulfonamide group in Ar, has a In vivo antiglucocorticoid activity of the


similar though slightly less pronounced effect novel compounds is assessed by measuring the
(not shown). Reduction of the triple bond to effect on body weight gain and thymus weight
the E-double bond (as in 325) leads to a signif- of dexamethasone-treated immature male
icant decrease in GR and an increase in PR- rats. In this test, compound (321) clearly an-
binding. Similar results were obtained upon tagonized the dexamethasone effect, with an
reduction of the triple bond to the 2-double (as ED,, value of 10 mgkg after oral administra-
in 326) or the saturated bond (as in 327; re- tion.
sults not shown).
Apparently the GR can accommodate 11-
phenyl substituted steroids with a rigid 17a- 12 NONSTEROIDAL GLUCOCORTICOIDS
substituent as large as a phenylethynyl func-
tion; moreover, specific polar substitution of The synthesis and biological charactarization
this 17a-phenylethyny1 group provides com- of novel nonsteroidal 2-quinolinones, whose
pounds that are highly selective for the GR. It functional activity was equivalent to pred-
has been reported that the GR is more hydro- nisolone, was recently reported (336). A gen-
philic than the PR in the area that interacts eral structure for these compounds (reminis-
with the steroidal D-ring. However, consider- cent of RU-38486) is shown in Table 15.57.
ing the distance between the polar phenyl- First, 9-substituted compounds showed PR ac-
ethynyl substituent and the steroidal D-ring tivity and so the ring system was examined at
(-8.5 A), it seems unlikely that this hydrophilic other positions with a Me0 group at C-8
area in the GR accounts for the high selectivity through C-10. Once C-10 was established as
observed for compounds (321-324). having the best profile, modification of the
References 845

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be separated from unwanted PR, MR, AR, and cists, Bethesda, MD, 1995.
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Index

Terms that begin with numbers Acetazolamide, 64, 66, 70 African homozygous sickle-cell
are indexed as if the number were discovery of, 69 disease, 456
spelled out; e.g., "3D models" is Acker's Symmetry rule, 386 Afterdepolarizations, 159-160
located as if it were spelled Aclomethasone dipropionate, Afterload, 9
"ThreeD models." 750, 753 Aggrastat, 29,287
Activated partial thromboplastin Aggregated band 3
50-125 time, 291 interaction with sickled cells,
endothelin receptor antago- Active transport 458
nist, 206 in kidneys, 57-60 Aggrenox, 287
A-127722 Acyl-CoA cholesterol AcylTrans- AGI-1067,373
endothelin receptor antago- ferase (ACAT) inhibitors, L-Agrobactin, 483,485,490
nist, 206 370 pMvalues, 484
A-192621 Addison's disease, 594, 748 synthesis, 522
endothelin receptor antago- Adenosine, 32 AHC-52
nist, 206 cardiovascular effects, 44 cardioprotective drug, 47
A-216546 multiple inhibitory mecha- AHC-93
endothelin receptor antago- nisms, 33 cardioprotective drug, 47
nist, 206, 207 Adenosine receptor antagonists Albumin
A-308165 xanthines, 121-122 binding of androgens to, 686
endothelin receptor antago- Adenylyl cyclase, 158 for plasma volume expanders,
nist, 206 ADME studies, See Absorption, 429,430,431-432
Abbott 53385,94 distribution, metabolism, thyroid hormone binding, 569
Abciximab, 29, 318 and excretion (ADME) Albumin-heme, 421
ADME, 297 Adrenaline Alcaligin, 486,487
clinical trials, 293, 294 role in clotting, 304 synthesis, 527-528
formula weight, mechanism of Adrenal steroidogenesis, 112- Aldactone, 111, 115, 117
action, and route of admin- 113,615 Aldehydes
istration, 287 sickle hemoglobin modifiers,
Adrenal steroids
for thrombolytic therapy, 167 461
biosynthesis, 612-616
Aldosterone
treatment regimen, 298 a-Adrenergic receptor ACE inhibitor effects on, 28
Absorption, distribution, metab- activation after myocardial angiotensin 11effects on, 197
olism, and excretion infarction, 158 steroidal antagonists, 111-120
(ADME) p,-Adrenergic receptor synthesis, 612, 613-614, 615
anticoagulants, antithrombot- activation after myocardial transport mechanism in renal
ics, and hemostatics, 295- infarction, 158 tubules, 57-58,59
298 0-Adrenergic receptor antago- Aldosterone biosynthesis inhibi-
antihyperlipidemic agents, nists, See p-Blockers tors
355-358 Adrenocorticoids, 598 diuretic agents, 120-121
AC-3056,373 synthesis, 612-616 Alipamide, 82,84
Acebutolol, 28, 32 Adrenocorticotropic hormone Alkylating agents
cardiovascular effects, 39,40 (ACTH),612-613 sickle hemoglobin modifiers,
uses and side effects, 38 Adrenomedullin, 218,219,220 461
ACE inhibitors, 198 therapeutic potential, 221 Allantoin, 138
with aldactone (spironolac- Adrenomedullin receptor, 220- Allantoinase, 138
tone), 111-112 221 Allogeneic bone marrow trans-
effect on endothelin, 207 Advicor, 343, 368 plantation
for prevention of ventricular Aerobactin, 486 for sickle-cell anemia, 465-
remodeling, 180-181 pMvalues, 484 466
Acenocoumarol, 311 synthesis, 524 Allopurinol, 139, 141
855
Index

Allosteric effectors Anabolic steroids Angiomax, 286,316


of hemoglobin, 386,388394 absorption, distribution, and Angioplasty, 156
Alloxanthine, 141 metabolism, 707-715 Angiotensin-convertingenzyme
&-Blockers,28 toxicities, 715-716 (ACE), 195.See also ACE
Alteplase, 166,322 Anandron, 717 inhibitors
clot selectivity, 290 Anastrozole, 632,643 Angiotensin I
treatment regimen, 298 Ancestim, 268 formation, 195,196
Alzheimer's disease Androcur, 717 and renin-angiotensin-aldoste-
role of iron, 504-505 Androderm, 697 rone system, 114
Amanozine, 123 AndroGel, 697 Angiotensin I1
Amcinonide, 750,752 Androgen antagonists, 716-721 binding to receptors, 196-197
Amdlodipine, 21 Androgen binding protein, 686
biological actions, 197
bioavailability and half-life, Androgen insensitivity syn-
biosynthesis and metabolism,
20,23 drome, 695
cardiovascular effects. 20 Androgen receptors, 694-695
195
N-Amidino-3-aminopyrazinecar- Androgen response elements, formation, 195,196
boxamides, 132 694,696 and neutral endopeptidase
N-Amidino-3,5-diamino-6-chlo- Androgens, 598,680-681.See inhibitors, 135
ropyrazine-carboxamide, also Anabolic steroids and renin-angiotensin-aldoste-
130 absorption, distribution, and rone system, 114,197-199
Amiloride, 124,130-132,142 metabolism of endogenous, structure, 195-196
hydrochlorothiazide-amilo- 620,685-692 and vasopressin, 200,201
ride, 78 absorption, distribution, and and ventricular remodeling,
Amino acids metabolism of synthetic, 164
transport mechanism in renal 703-705 Angiotensin 111, 195,196
tubules, 57-58 biosynthesis, 616-618,682- Angiotensin IV, 195
4-Amino-6-chloro-1,3-ben- 685 Angiotensinogen, 195
zenedisulfonamide, 70-71, conversion to estrogens, 690- Angiotensin, receptor, 196-197
73,80 692 Angiotensin, receptor, 197
Aminoglutethimide,643,724 and estrogen biosynthesis,642 Angiotensin, receptor antago-
applications, 631,632 mechanism of action, 692-697 nists, 198-199
2-Aminomethyl-3,4,6-trichloro- occurrence and physiological effect on endothelin, 207
phenol, 108-109 roles, 681-682 Anhydron, 77
Aminometradine, 122-123,124 synthetic, 697-706
Animal models and studies
Aminophylline, 124 temperature-dependent up-
diuretics, 62
Aminouracils, 122-123 take, 686
Amiodarone, 32,41,167 toxicities of synthetic, 705 iron chelators, 534-540
ATP-sensitive potassium Android, 697 for nitrogen retention, 705
channel inhibition, 178 Androisoxazol, 712,715 sickle-cell anemia, 459
cardiovascular effects, 40,42 anabolic activity, 716 Anionic polysaccharides
with implantable cardioverter 5a-Androstane-3a,17dol,696 marketed antithrombotics,
defibrillator, 46 Androstanes, 597,598,749 286
increased use of, 45 Androstenedione,616,621,682 mechanism and sites of action,
multiple inhibitory mecha- absorption,685 310
nisms, 33 and estrogen biosynthesis, 642 Anisoylated plasminogen strep-
synergy with P-blockers,46 metabolism, 620 tokinase-activated complex,
thyroid hormone antagonist, Androsterone, 681 165
576 cholesterol lowering effect, Anordiol, 658
uses and side effects, 40 682 ANP clearance receptor block-
Amisometradine, 124 testosterone metabolite, 687 ers, 134
Amlodipine Anemia, 386,503-504.See also Antianginal agents. See also Va-
vascular-to-cardiac selectivity Sickle-cell anemia sodilators
ratio, 172 Angina pectoris, 3,8,9-11.See factors affecting myocardial
Ammonium chloride also Antianginal agents oxygen supply, 8-9
osmotic diuretic, 64 and blood clotting, 290 factors governing myocardial
Amylin, 218 treatment and prevention, 11, oxygen demand, 9
Amy1 nitrate, 13 298-299 future directions, 46-47
Index

Antiarrhythmic agents corneal penetration, 771 future developments, 331-332


Class I: membranedepressant 3D-QSAR, 793-796 marketed, 286-287
agents, 31,32,34-38,167,168 drug distribution, 771-773 mechanisms and sites of ac-
Class IA,31,32,3436, 167 drug receptor affinity, 780- tion, 308-322
Class IB, 31,32,36-37, 167 786 nitrates as, 12
Class IC, 31,32,37-38, 167 electronic factors affecting, treatment regimen, 298-299
classification, 31-33, 167-168 793 Anturane, 139
Class 11: P-adrenergic blocking intestinal absorption, 756-759 Aorta, 3, 4
agents, 31,32,38-40,167 intra-articular administration, AP-811, 134
Class 111: repolarization prolon- 767-768 Apo E gene, 10-11
gators, 31,32,40-43, 167 mechanism of action, 776-780 Apolipropotein AI, 341
Class IV: calcium channel
neural networks application, Apolipropotein B48, 341
blockers, 31,32,43-44,
791-792 Apolipropotein B100, 341
167-168
future directions, 45-46 percutaneous absorption, Apolipropoteins, 340 3 4 1
miscellaneous agents, 44 759-767 Apoptosis
multiple inhibitory mecha- QSAR analyses, 786-792 necrosis contrasted, after
nisms, 33 skeletal change effects on ac- myocardial infarction, 157
Anticlotting agents, See Antico- tivity, 796-837 AT, receptor effect on, 197
agulants; Antiplatelet steric factors affecting, 792- Aquaphor, 88
agents; Thrombolytic agents 793 Aquatag, 76
Anticoagulants, 166,284. See structure-activity relation- Aquex, 82
also Heparin ships, 786-837 Aranidipine, 21
ADME, 295-298 water-soluble esters, 768-771 bioavailability and half-life, 23
calcium channel blockers, 29 Anti-obesity agents cardiovascular effects, 20,24
clinical use, 284-299 thyroid hormones (potentially) AR-C69931,320,321
next-generation, 291 as, 565,575 Argatroban, 29,285
treatment regimens, 298-299 Antioxidants ADME, 296
Antidiuretic hormone for atherosclerosis treatment, clinical trials, 293
transport mechanism in renal 372-374 development of, 314-315
tubules, 57-58 a2-Antiplasmin inhibitors, 329- formula weight, mechanism of
Antiendothelial receptor anti- 331 action, and route of admin-
bodies, 462 Antiplatelet agents, 285 istration, 286
Antiestrogens, 631 with alternative mechanism of
currently marketed, 632 structure, 288
actions, 326-329
Antiglucocorticoids, 837-845 Arginine vasopressin
calcium channel blockers, 29
Antihyperlipidemic agents, 339- clinical uses, 284-299 biological actions, 200-201
342 mechanisms and sites of ac- biosynthesis, metabolism, and
ADME, 355-358 tion, 308-322 structure, 199-200
clinical applications, 342-352 next-generation, 291 Arginine vasopressin antago-
new treatments for lowering nitrates as, 12 nists, 201
LDL and TG, 368-370 structures of marketed, 289 Arginine vasopressin receptors,
new treatments for raising for thrombolytic therapy, 166 200
HDL and lowering TG, 368- treatment regimens, 298-299 Arimidex, 632
370 Antiprogestins, 632,651-652 Arixtra, 286,310
side effects, adverse effects, binding to glucocorticoid re- Aromasin, 632, 728
drug interactions, and con- ceptors, 841 Aromatase
traindications, 352-355 Antithrombotic agents. See also in androgen metabolism, 692
structure-activity relation- Anticoagulants; Antiplatelet in estrogen metabolism, 642
ships, 363-367 agents Aromatase inhibitors, 604,642-
thyroid hormones (potentially) ADME, 295-298 643
as, 565,571,575 with alternative mechanism of C-20 steroids as, 726-728
Anti-inflammatory steroids, actions. 323-326 currently marketed, 632
747-751. See also Glucocor- bleeding and other adverse nonsteroidal, 668-669
ticoids effects, 291-294 Aromatic aldehydes
adverse effects, 751-756 challenges, 284 hemoglobin allosteric effec-
clinical uses, 751 clinical use, 284-299 tors, 394396
Index

Arrhythmias, See Antiarrhyth- ATP-sensitive potassium chan- Basophilic granulocytes, 252,


mic agents; Cardiac ar- nel agonists, 163-164, 177- 253
rhythmias 178 BAY-139952,368
Arterial thrombosis, 290 ATP-sensitive potassium chan- Baycaron, 82
antithrombotic treatment reg- nel antagonists, 162-164, Baycol, 343
imen, 298-299 175-176 Bay g2821, 108
Arthrobactin, 483,486 Atrial fibrillation, 9,31 Bay K8644,178
synthesis, 524 Atrial flutter, 9, 31 calcium channel agonist, 160,
Artificial neural networks Atrial natiuretic peptide, 133-138 161,175
anti-inflammatory steroids biological action, 217 Bay Y5959
application, 791-792 biosynthesis, metabolism, and calcium channel agonist, 175
structure, 214-216 BE-18257A
Arylalkylamine calcium channel
and endothelin, 205 endothelin receptor antago-
blockers, 16-20
therapeutic potential, 218 nist, 206
Aryloxy acetic acid high ceiling BE-18257B
Atrial natiuretic peptide (ANP)
diuretics, 93-94 clearance receptor blockers, endothelin receptor antago-
Arzoxifene, 668 134 nist, 206
estrogen receptor affinity, 659 Atrial natiuretic peptide recep- Bebulin
Aspirin, 285 tor, 218 hemostatic preparation, 292
ADME, 297-298 Atrial tachycardia, 9 Beclomethasone dipropionate,
clinical trials, 291-292,293 Atrioventricular (AV) node, 3,4 750,753
effects on thromboxane &, and disorders of signal genera- Bemitradine, 87-89
326 tion, 29 Bendroflume thiazide, 76
formula weight, mechanism of and reentry excitation, 30-31, Benefix
action, and route of admin- 161 hemostatic preparation, 292
istration, 287 Atrium, 3 , 4 Benemid, 139
mechanism and sites of action, Augmented-Acute Nonnovolemic Benidipine, 21
321-322 Homodilution A-ANH. 427 bioavailability and half-life, 23
structure, 289 Automaticity, of myocardial cardiovascular effects, 24
for thrombolytic therapy, 166 cells, 5 Benzbromarone, 139,141-142
treatment regimen, 298 Autoplex Benzolamide, 69, 70
Atenolol, 28, 179 hemostatic preparation, 292 Benzothiadiazine diuretics,
Avasimibe, 370 76-77
cardiovascular effects, 39,40
AV nodal reentry, 9 Benzothiazepine calcium chan-
uses and side effects, 38
AV node, See Atrioventricular nel blockers, 16-20
Atherosclerosis Benzthiazide, 76
(AV) node
and myocardial oxygen supply, AV reciprocating tachycardia, 9 Bepridil, 17, 18, 32
9 AZ-242,370,371 cardiovascular effects, 16,44
new antihyperlipidemic treat- 5-Azacytidine, 464 multiple inhibitory mecha-
ments, 372-374 2-Azetidinone, 366 nisms, 33
role of lipids, 340-342 Azimilide, 41 uses and side effects, 43
Atherosclerotic angina, 9 cardiovascular effects, 43 Beraprost, 328,329
Atherosclerotic plaque rupture, potassium channel subtype p2-Antagonists,See p-Blockers
290 selective, 45 p-Blockers
Atorvastatin, 343,345,609 uses and side effects, 40 as antiarrhythmic agents,
ADME, 356 Aziridine, 665 179-180
for angina treatment, 11 Azolimine, 132-133 as class I1 antiarrhythmic
discovery, 362363 Azopt, 67 agents, 31,32,38-40,167
enzyme activity, 364 Azosemide, 88, 104 effect on coronary care unit
side effects, 354 mortality after introduction,
ATP 156
depletion after myocardial in- endothelin-converting enzyme with implantable cardioverter
farction, 156, 159 inhibitor, 208 defibrillator, 46
ATP-sensitive potassium chan- BAPTA-AM, 160,170-171 synergy with amiodarone, 46
nel, 7,162-163 Barnidipine, 21 Betamethasone, 604,750, 752,
and arrhythmias, 163-164 bioavailability and half-life, 23 764-766,770,772,788,797,
and preconditioning, 157 cardiovascular effects, 24 803,813-816,824,830-833
Index

Bezafibrate, 343, 347 formula weight, mechanism of Bradykinin


allosteric effectors of hemoglo- action, and route of admin- biological actions, 225-226
bin, 388,389-394 istration, 286 biosynthesis, metabolism, and
for angina treatment, 11 structure, 288 structure, 224-225
enzyme activity, 365 Bleeding disorders, 291 Bradykinin antagonists, 226
metabolism, 357 Blood cell development, 252-255 Bradykinin receptors, 225
Bezalip, 343 Blood clotting, 284-291, 299- Brain natiuretic peptide, 214,
BIBN4096BS 308 215
cdcitonin gene-related peptide Blood coagulation, 284,299408 biological action, 217-218
antagonist, 222 Blood oxygenation, 8-9 biosynthesis, metabolism, and
BIBP3226 Blood substitutes, 388 structure, 216
neuropeptide Y antagonist, clinical trials, 398-421 Bretylium, 32,41, 167
212
history of, 396-398 cardiovascular effects, 42
Bicalutamide, 717,720
Blood thinners, 29 multiple inhibitory mecha-
Bicarbonate
neutralization in kidneys, 58 Blood transfusion, 396 nisms, 33
Bicyclic polyaza diuretics, 128- erythropoietin use leads to uses and side effects, 40
130 decline in, 272 Brinaldix, 82
Bi-directional conduction block, B-lymphoblasts, 253 Brinzolamide, 67
29 B-lymphocytes, 253,254 Bristuron, 76
Bile acids, 594 BM573,327 BRL-32872,45-46
Bile acid sequestrantslcholes- BMS- 182874 Budesonide, 750, 753, 775,824,
terol absorption inhibitors endothelin receptor antago- 827
ADME, 357 nist, 206 Bumetanide, 88,95103, 142
for angina treatment, 11 BMS-201038,368 Bundle branches, 4
clinical applications, 347-349 BMS-298585,370,371 Bundle of His, 3 , 4
marketed agents, 343 BMY-15037-1,87 Burinex, 88
new treatments for lowering BO-653,373 Burst-forming units, 253,256
LDL and TG, 368-370 Bolasterone, 709 Buthiazide, 76
side effects, adverse effects, Bombesin, 229 3-Butylamino-4-chloro-5-sulfa-
drug interactions, and con- BOMT, 719 moylbenzoic acid, 95
traindications, 355 Bone marrow transplantation Butyrate
structure-activity relation- for sickle-cell anemia, 465- for sickle-cell anemia, 465
ships, 364-365,365-366 466
Birth control agents, See Con- Bones
traceptives thyroid hormones effect on, allosteric effector of hemoglo-
Bis[3,5-dibromo salicyllfumarate 572-573 bin, 388, 394,395
sickle hemoglobin modifier, 461 Bosentan Caffeine
N-,N'-Bis(2-hydroxybenzy1)eth- endothelin receptor antago- diuretic action, 121
ylenediamine-N-,N'-diacetic nist, 206, 207 Calcitonin, 218-219
acid (HBED) Bowman's capsule, 57 Calcitonin gene-related peptide,
animal studies of iron chela- BQ-123 218-220
tion, 515520 endothelin receptor antago- and substance P, 223
formation constants with iron nist, 206 therapeutic potential, 221
and other metals, 506 BQ-153 Calcitonin gene-related peptide
pM values, 484 endothelin receptor antago- family
preclinical toxicity trials, 520- nist, 206 biological actions, 221
522 BQ-485 biosynthesis, metabolism, and
use with animal models, 537- endothelin receptor antago- structure, 218-220
540 nist, 206 in spinal cord neurons, 214
18,19-Bisnortestosterone,707 BQ-610 therapeutic potential and an-
Bisoprolol, 28 endothelin receptor antago- tagonists, 221
Bisucaberin, 483,486 nist, 206 Calcitonin gene-related peptide
synthesis, 527-528 BQ-788 receptors, 220-221
Bivalirudin, 29, 285, 316-317 endothelin receptor antago- Calcitriol
ADME, 296 nist, 206 and calcium reabsorption in
clinical trials, 293 Bradycardia, 9,31 kidneys, 59
Index

Calcium Carbonic anhydrase, 68 Celestone Phosphate, 770


and arrhythmias, 159-162 action in kidneys, 58,59,61 Celestone Soluspan, 771
reabsorption in kidneys, 59 Carbonic anhydrase inhibitors Centchroman, 662,663
role in heartbeat, 4-5 diuretic agents, 66-70 Centocor, 29
role of cytosolic after myocar- Carbon monoxide Central hypothyroidism, 574
dial infarction, 157-159 binding to hemoglobin, 386 Cerebral occlusion, 289
Calcium channel agonists, 175- p-Carboxybenzenesulfonamide, Cerebral vasodilators, 12
176 70,71 Cerebrovascular disease, 340
Calcium channel antagonists, Carboxypeptidase A Cerivastatin, 343,345,353
168 effect on urotensin-11,212 ADME, 356
Calcium channel blockers, 6, Cardiac arrhythmias, 3.See also for angina treatment, 11
15-16.See also ACE inhibi- Antiarrhythmic agents; enzyme activity, 364
tors; @ Blockers Long QT syndrome cGMP
anticlotting agents, 29 and calcium, 159-162 after myocardial infarction,
arylalkylamines and benzo- disorders in electrical signal 158-159
thiazepines, 16-20 generation and conduction, and endothelin, 203
cardiac glycosides, 28 29 role in clotting, 306
as class IV antiarrhythmic heart block, 29-30 CGP 3856011,197-198
agents, 31,32,43-44,167- malignant, 157-164 CGP 71683A
168 and myocardial oxygen de- neuropeptide Y antagonist,
1-4dihydropyridine deriva- mand, 9 212
tives, 20-28 re-entrant, 30-31,161 CGS 4270,131
effect on endothelin, 207 role of extracellular potas- CGS 23425,582-584
glycoprotein IIbIIIIa receptor sium, 163-164 CGS 24128,137-138
antagonists, 29 thyromimetics for, 576 CGS 24592,137-138
termination of ventricular fi- treatment perspective, 33-34 CGS 25462,137-138
brillation, 160 types of, 31 CGS 26214,583-584,587
Calcium channels, 7-8 Cardiac Arrhythmia Suppres- CGS 169494120-121
Calomel, 65 sion Trial, 45 Chemical reactions
Calusterone,698,700 Cardiac contractility, 9 steroids, 604-606
CAMP Cardiac glycoside calcium chan- Chemokines
after myocardial infarction, nel blockers, 28 for atherosclerosis treatment,
158 Cardiac physiology, 2-6 372-374
role in clotting, 306 electrophysiology, 3-4 Chloexolone, 83
TSH effect on, 568,572 excitation and contraction Chlorazanil, 123,124,125
CAMP-dependent protein kinase, coupling, 4-6 Chloride-formate exchanger
158 and ion channels, 6-8 in kidneys, 58-59
Candida albicans Cardiotropin-1,269 6-Chloro-l,3-benzenedisulfon-
iron uptake by, 494-495 Cardiovascular disease, 2 amide, 72
Candoxatril, 136 Cardrase, 70 Chloromerodrin, 66,67
Candoxatrilat, 136 Carinamide, 139 4-Chloro-3-sulfamoyl-N-substi-
Cangrelor, 320,321 Casodex, 717,720 tuted benzenesulfonamides
C-ANP4-,,, 134 Catalase diuretics, 74
Captopril, 198 hydrogen peroxide manage- Chlorotestosterone, 699,700,
Carbamates ment, 502 703,705
synthesis, 525 Catecholamide iron chelators, Chlorothalidone, 81,82
Carbohydrate metabolism 483,485 Chlorothiazide, 70,71-72,73-
androgen effect on, 682 synthesis, 522524 78,81
thyroid hormone effect on, Catecholamines metabolism,80
571 and coronary occlusion, 156 metolazone compared, 86
Carbonate and cytosolic free calcium, 158 Chlorotrianisene, 661
binding to hemoglobin, 386 and malignant arrhythmias, applications, 631,632
Carbon dioxide 157 oxidative metabolism, 636
binding to hemoglobin, 386- TSH modulation, 568,572 Cholestanes, 597,598
387 CD36 Cholesterol, 340,358,594,598
Carbonic acid interaction with sickled cells, in adrenal steroidogenesis,
production in kidneys, 58,68 458 112-113,615
Index

biosynthesis, 606-609 Class I1 antiarrhythmic agents, Collagen, 290


conversion to pregnenolone, 31,32,38-40, 167. See also in clotting, 303
613 p blockers glucocorticoid effects on, 751
conversion to testosterone, Class I11 antiarrhythmic agents, Colloidal plasma volume expand-
604 31,32,40-43,167 ers, 429-433
and coronary artery disease, Class IV antiarrhythmic agents, Colony-forming units, 252, 253
10 31,32,43-44, 167-168. See Colony-stimulating factor-1,
effect of thyroid hormone, 572 also Calcium channel block- 272-273
lowering with thyromimetics, ers Colony-stimulating factors, 252
575 Clazolimine, 124,132-133 Comparative molecular field
reverse transport, 341, 575 Clentiazem, 17
analysis (CoMFA)
role in androgen biosynthesis, bioavailability and half-life, 18
glucocorticoids, 793-796
683-684 cardiovascular effects, 17
Comparative molecular surface
role in atherosclerosis, 342 Climara, 632
Clobetasol propionate, 750, 752, analysis (CoMSA)
role in estrogen biosynthesis, glucocorticoids, 791-792
824,833
642-645 Congestive heart failure, 156
Clocortolone pivalate, 750, 752
role in glucocorticoid biosyn- Clofibrate, 343,346-347 and ventricular remodeling,
thesis, 773-774 allosteric effector of hemoglo- 164
Cholesterol absorption inhibi- bin, 388 Conjugated hemoglobins, 417-
tors, See Bile acid seques- enzyme activity, 365 420
trantslcholesterol absorp- metabolism, 357 Contraceptives, 630,650-651
tion inhibitors sickle hemoglobin modifier, currently marketed, 632
Cholesteryl esters, 340-341 461 ovulation modulation, 654-
Cholesteryl ester transfer pro- side effects, 354 655
tein (CETP) inhibitors, 371 Clofibric acid, 346 Contraction coupling, in heart,
Cholestyramine, 343, 348 Clomid, 632 4-6
effect on hydrothiazide ab- Clomiphene, 661-662,663 CORINA, 792
sorption, 80 applications, 631, 632 Coronary artery disease, 3 , 8
metabolism, 357 in contraceptives, 654-655 causes, 10-11
Chromene, 664 oxidative metabolism, 636 and lipids, 340-342
Chromenoquinoline, 653-654, Clopamide, 81, 82, 84,97, 105 treatment regimens, 298-299
668 Clopidogrel, 29,285, 320 Coronary occlusion, 156-157,
Chylomicrons, 341, 358 ADME, 297 289
CI-1011, 370 clinical trials, 293 Coronary vasodilators, 12
formula weight, mechanism of Corsevin M, 326
CI-1031,324-325
action, and route of admin- Cortexolone, 750
Cigarettes
istration, 287 Cortical nephrons, 57
and coronary artery disease, structure, 289 Corticosteroid-binding a-globu-
10 for thrombolytic therapy, 166 lin, 686, 703, 771, 793-796
Ciliary neutrophic factor, 269 treatment regimen, 298 Corticosteroids, 594, 748. See
Cilnidipine, 21 Clorexolone, 85 also Glucocorticoids
bioavailability and half-life, 23 Clotrimazole metabolism, 619, 620
cardiovascular effects, 24 sickle hemoglobin hydration Corticosterone, 748, 750, 759,
Ciprofibrate, 343,347 agent, 461 783,788
enzyme activity, 365 Coagulation factor inhibitors, binding, 771
metabolism, 357 323-326 synthesis, 612-613,615
Ciprol, 343 Cocaine Corticotropin-releasing factor,
c-Kit factor, See Stem cell factor prevention of ventricular fi- 612-613
Class IA antiarrhythmic agents, brillation caused by, 169, Cortisol, 113, 598, 748, 749, 752,
31,32,34-36,167 171 758-759,772, 782, 793,803
Class I antiarrhythmic agents, Colesevelam, 348 binding, 771
31,32,34-38,167,168 metabolism, 357 cortisol-cortisone conversion,
Class IB antiarrhythmic agents, Colestid 773
31,32,36-37,167 for angina treatment, 11 effect of structural changes on
Class IC antiarrhythmic agents,
- Colestipol, 343, 348 activitv. 796-837
31,32,37-38,167 metabolism, 357 half-life,?76
Cortisol (Continued) Cyproterone acetate, 717, 718, Delayed afterdepolarizations,
Hansch-type analysis, 788- 721 159-160
790 Cytadren, 632 Delestrogen, 632
metabolism, 769 Cytochrome P450 Demulen, 632
synthesis, 612,615 in adrenal steroidogenesis, Deoxycorticosterone, 113,750,
Cortisone, 595, 748, 749, 758 112-113 759
cortisol-cortisone conversion, role in steroid biosynthesis, and chlorazanil, 123
773 610,614,619-620 21-Deoxydexamethasone, 750
effect of structural changes on Cytokines. See also specific cyto- Deoxyhemoglobin, 387
activity, 796-837 kines such as Erythropoie- in sickle-cell anemia, 447-450
metabolism, 769 tin Depolarization, of myocardial
synthesis, 599-602 for atherosclerosis treatment, cells, 3, 5, 159-160
synthesis by microbial trans- 372-374 Depo-Provera Lunelle, 632
formation, 602-604 and glucocorticoids, 779 Depo-Testosterone, 697
Cotransmission, 194 role in blood cell development, Desazadesferrithiocin, 530, 532
Coumadin, 286 252-255 metabolism, 550-551
COX-1 therapeutic implications, 272- Desferal, 507,515
role in clotting, 305 273 Desferri-ferrichrome, 486,487
COX-2 inhibitors and VCAM-1,459 Desferrioxamine B (DFO), 483,
aspirin's effect, 297-298 Cytomel, 576 486,496
CP-96,345 analogs, 530,531
tachykinin antagonist, 224 Daidzein, 652 development of, 514-515
CP-346086,368 Dalteparin and Fenton reaction, 503
CP-529414,371 formula weight, mechanism of formation constants with iron
Crescendo angina, 10 action, and route of admin- and other metals, 506
Crestor, 343, 368 istration, 286 iron chelator, 507-511
Crohn's disease, 780 Danaparoid, 29 for iron overload disorders,
Cromakalim clinical trials, 294 504-505
and extracellular potassium, formula weight, mechanism of synthesis, 524-527
163 action, and route of admin- use with animal models, 534-
Crosslinked hemoglobins, 397, istration, 286 540
405,406-408 mechanism and site of action, Desferrioxamine E, 483,486
Crystalloid plasma volume ex- 309 synthesis, 524-525,528-529
panders, 429-433 treatment regimen, 298 Desferrioxamine G, 483,486,
CS-505,370 Danazol, 697, 713, 715 530
CS-747,320, 321 Danocrine, 697 Desferrithiocin iron chelators,
CT50547,320-321 Daquin, 124 488
C-type natiuretic peptide, 214, Daranide, 70 design, synthesis, and testing,
215 Darusentan 540-545
biological action, 218 endothelin receptor antago- iron-clearance evaluations,
biosynthesis, metabolism, and nist, 206 545-549
structure, 216-217 DBBF-Hb, 397 metabolism of desazadesfeni-
Cushing's syndrome, 748,751 Deep vein thrombosis, 289-290 thiocins, 550-551
CVT-124, 122 Deferiprone, 508,515 synthesis, 530-532
Cyanate formation constants with iron toxicity, 549-550
sickle-cell anemia therapy, and other metals, 506 Desirudin, 29,285,316,317
460-461 iron chelator, 512-514 clinical trials, 294
Cyanomethemoglobin, 401 pM values, 484 structure, 288
Cyclic polynitrogen diuretics, promotion of Fenton reaction, Desmopressin, 200
121-125 503 sickle hemoglobin hydration
Cyclooxygenase 112 inhibitors, Defibrillators, 156 agent, 461
See COX-1 inhibitors; implantable cardioverter defi- Desogen, 632
COX-2 inhibitors brillator, 46 Desogestrel, 632
Cyclopenthiazide, 76,81 Dehydroepiandrosterone biotransformation, 641
8-(Cyclopenty1)-1,3-dipropylxan- (DHEA), 113,616,681,682 in contraceptives, 654
thine, 122 mechanism of action, 693-694 interactions with steroid re-
Cyclothiazide, 77 Delatestryl, 697 ceptors, 633
Index

Desonide, 750, 753,803 Digitalis 1,3-Dipropyl-8-[2-(5,6-ep-


Desoximetasone, 750,752 cardiovascular effects, 28 oxy)norbornyllxanthine,
Dexamethasone, 750,753,758, Digitoxin, 32 122
768,771-772,779,783,786, cardiovascular effects, 28 Dipyridamole, 285
788, 797,802-803,821,824, Diglucomethoxane, 66 clinical trials, 293
826,839 Digoxin, 32 formula weight, mechanism of
half life, 776 cardiovascular effects, 28, action, and route of admin-
side effects, 756 44-45 istration, 287
Dextrans multiple inhibitory mecha- mechanism and sites of action,
for conjugated hemoglobins, nisms, 33 321-322
417 4,5a-Dihydronorethindrone structure, 289
for plasma volume expanders, interactions with steroid re- treatment regimen, 298
429,430,431,432,433 ceptors, 633 Disopyramide, 32,34,167
DFO, See Desferrioxamine B 1,4-Dihydropyridinecalcium cardiovascular effects, 36
DHT, See 5a-Dihydrotestoster- channel blockers, 20-28 multiple inhibitory mecha-
one recent developments, 46-47 nisms, 33
Diabetes Dihydroquinazolinone sulfon- uses and side effects, 35
and coronary artery disease, amides, 85-86 1,3-Disulfonamide,70-71
10 5a-Dihydrotestosterone (DHT), Diurazine, 124
Diacetylformoguanamine, 123 616,618,621 Diuretic agents, 55-61, 142-143.
Diacylglycerol, 158 binding, 686 See also Uricosuric agents
2,4-Diamino-6,7-dimethylpteri- esterification, 714 aldosterone biosynthesis in-
dine, 125,126 most potent endogenous an- hibitors, 120-121
4,7-Diamino-N-(2-mopholin- drogen, 680,681 animal studies, 62
oethy1)-2-phenyl-6-pteridin- relative androgenicity and re- aromatic sulfonamides, 68,
ecarboxamide, 125-126 ceptor binding of analogs, 70-73
4,7-Diamino-2-phenyl-6-pteri- 702 atrial natiuretic peptide, 133-
dinecarboxamide, 125,126 selective retention, 693 138
Diapamide, 83,84 testosterone metabolite, 687, carbonic anhydrase inhibitors,
Diarylethylene estrogen antago- 689 66-70
nists, 660-661 Dihydroxamic acid clinical aspects, 62-63
Diazoxide, 78 synthesis, 524 cyclic polynitrogen com-
DIBRT 2,3-Dihydroxy-N-N1-dimethyl- pounds, 121-125
thyroid antagonist, 585-586 benzamide (DHBA) and edema, 62-63
Dichlorisone, 750 iron-specific ligand, 482-483 high ceiling, 88,89-111
Dichlorophenamide, 69, 70, 71 pM values, 484 hydrothiazides, 78-81
Dicumerol, 311 Diltiazem, 32, 160, 169-170 mercurial, 64-66
8-(Dicyclopropylmethyl-)-1,3- bioavailability and half-life, 18 metabolic and electrolyte dis-
dipropylxanthine, 121-122 cardiovascular effects, 16-17, orders, 63
Didox 44 osmotic, 63-64
for sickle-cell anemia, 464 dihydropyridines contrasted, pharmacological evaluation,
Dienestrol, 632 27-28 61-62
estrogen receptor aflinity, 659 multiple inhibitory mecha- potassium-sparing, 125-133
Diethylenetriamine pentaacetic nisms, 33 steroidal aldosterone antago-
acid (DTPA), 3,507, 508, uses and side effects, 43 nists, 111-120
514 1,2-Dimethyl-3-hydroxypyridin- sulfonamides, aromatic, 68,
formation constants with iron 4-one, See Deferiprone 70-73
and other metals, 506 DIMP, 720-721 sulfonamides other than aro-
iron chelator, 511-512 Dinestrol matic, 68, 81-89
pM values, 484 applications, 630, 632 thiazides, 73-78
Diethylstilbestrol, 656 2,3-Diphosphoglycerate Diuril, 76
biotransformation, 637-638 (2,3-DPG) DMP 504,369-370
estrogen receptor affinity, 659 binding to sickle hemoglobin, DMTl (Divalent Metal Trans-
interaction with estrogen re- 445-446 porter 1; NrampZ), 498
ceptors, 646 hemoglobin allosteric effector, Dofetilide, 32, 41, 167
Diflorasone diacetate, 750, 752 386,387,388-390,395 cardiovascular effects, 42-43
Index

Dofetilide (Continued) Enalaprilat EPO, See Erythropoietin (EPO)


potassium channel subtype covalent joining to thiazide Epoetin alfa, 258
selective, 45 diuretics, 86-87 EPOGEN, 258
uses and side effects, 40 Encainide, 32,34, 167 Epoxyrnexrenone, 117,120
Dolichol, 353 cardiovascular effects, 37 Epristeride, 723-724
Dopamine increased mortality from, 45 Eptifibatide, 29,285, 318
and neurotensin, 230 multiple inhibitory mecha- ADME, 297
TSH modulation, 568 nisms, 33 clinical trials, 293, 294
Dorzolamide, 67 uses and side effects, 35 formula weight, mechanism of
DPC423,324,325 Encapsulated hemoglobins, 398, action, and route of admin-
DRF-2725,370 420-421 istration, 287
DRF-4832,370 Endogenous vasoactive peptides, structure, 289
Dronedarone, 41 193-195 for thrombolytic therapy, 167
potassium channel subtype Endothelin-1,202-208 treatment regimen, 298
selective, 45 substance P opposes, 223 Equilenin, 599, 644
Drostanolone, 709 EndothelinS,202,203 Equilin, 635, 644
anabolic activity, 716 Endothelin-3,202, 203 estrone compared, 658
Drug metabolism, See Metabo- Endothelin antagonists, 206- Equine estrogens, 631, 632
lism 208 fate of conjugated, 635-636
DS 210,128 Endothelin-converting enzyme, genesis of, 644-645
DS 511,128 203,208 Ergosterol, 594
DTPA, See Diethylenetriamine Endothelin-converting enzyme Ersentilide
pentaacetic acid inhibitors, 208 potassium channel subtype
Dyrenium, 124 Endothelin peptide family selective, 45
Dyslipidemia, 340 -341 biological actions, 203-205 Erythrocytes, 252-254,256-257
biosynthesis and metabolism, sickle, 445, 451-452, 453, 454
E. Coli 202-203 Erythroid krupple-like factor
iron uptake by, 494-495 pathological roles, 205-206 OXLF)
Early afterdepolarizations, 159- structure, 201-202 and sickle-cell anemia, 467
160 Endothelin peptide receptors, Erythropoietin (EPO), 255-256
Echogen, 399 203 androgen effects on, 682, 705-
Ecotrin, 287 Endothelium 706
Ectopic beats, 5 and sickle-cell anemia, 458- bioactivity, 256
Edecrin, 88 459 and decline in need for trans-
Edema, 9 Endothelium-derived relaxation fusions, 272
and renal excretion, 62-63 factor, 12,205. See also Ni- with hydroxyurea for sickle-
EDTA, See Ethylenediaminetet- tric oxide cell anemia, 464
raacetic acid (EDTA) Enduron, 77 preparations, 258
Efaproxiral, 391-394 Enovid, 632 role in blood cell development,
Efonidipine, 21 Enoxaparin, 309 252-254,445,447
bioavailability and half-life, 23 clinical trials, 292 secretion in kidneys, 56
cardiovascular effects, 24 formula weight, mechanism of with stem cell factor, 268
Eglodipine, 21 action, and route of admin- therapeutic implications, 272,
bioavailability and half-life, 23 istration, 286 273
Elecon, 767 Enrasentan therapeutic indications, side
Electrocardiogram (ECG), 4 , 5 endothelin receptor antago- effects, and pharmacokinet-
Electrophysiology, 3-4 nist, 206 ics, 256-258
Elgodipine Enterobactin, 482-483,490 ES 37,135-136
cardiovascular effects, 24-25 pM values, 484 Esidrix, 76
Elkpain, 110 Eosinophilic granulocytes, 252, Esmolol, 32
EM-652,664 253 cardiovascular effects, 39,40
EM-800,664 and granulocyte-colony stimu- uses and side effects, 38
EMD 122946 lating factor, 263 Estinyl, 632
endothelin receptor antago- and granulocyte-macrophage l7P-Estradiol, 599
nist, 206 colony stimulating factor, biosynthesis, 617, 618-620,
Emulsion No. 11,422 259 633-634,642-645
Enalapril, 198 Eplerenone, 112,117, 120 in contraceptives, 654
Index

enterohepatic recycling, 640 oxidation and reduction, 633- F12511,370


estrogen receptor affinity, 635 634 Factor I1
fate of l7a-ethynyl analogs, side effects, 631-633 hemostatic preparations, 292
634-635 temperature-dependent up- plasma concentrations and
metabolism, 621, 622 take, 686 half-life, 296
nonsteroidal analogs of, 659- tissue-selective, 652-654 Factor V
666 Estrone, 594 in clotting, 301-308
steroidal analogs, 656-659 biosynthesis, 633-634 plasma concentrations and
testosterone metabolite, 687 enterohepatic recycling, 640 half-life, 296
Estradiol valerate equilin compared, 658 Factor Va
applications, 630, 632 estrogen receptor affinity, 635 in clotting, 300-308
Estranes, 597, 598 with medroxyprogesterone, Factor VII
Estrenol, 712 650 in clotting, 300-308
anabolic activity, 716 metabolism, 621, 622 hemostatic preparations, 292
Estriol Estrone sulfate, 621 plasma concentrations and
estrogen receptor affmity, 635 Estropipate half-life, 296
Estrogen modulator antihyper- applications, 630, 632 Factor VIIa
lipidemics ETC-216,374 in clotting, 299-308
clinical applications, 351 ETC-588,374 hemostatic preparations, 292
side effects, adverse effects, Ethacrynic acid, 142 Factor VIIa inhibitors, 325
drug interactions, and con- animal studies, 62 Factor VIII
traindications, 355 diuretic, 88,89-92 in clotting, 301-308
Estrogen-progestin combina- sickle hemoglobin modifier, and hemophilia A, 291
tions, 630,631,654-655 461 hemostatic preparations, 292
currently marketed, 632 Ethamide, 70 plasma concentrations and
Estrogen receptor a, 645-647, Ethoxzolamide, 69, 70 half-life, 296
669 Ethyldienolone, 708 Factor VIIIa
Estrogen receptor P, 351, 645- Ethylenediaminetetraacetic acid in clotting, 299308
647,669 (EDTA) Factor IX
Estrogen receptors formation constants with iron in clotting, 299-308
activity assessment, 648-649 and other metals, 506 and hemophilia B, 291
affinity assessment, 648-649 history of use for iron chela- hemostatic preparations, 292
distribution of isoforms, 648 tion, 514 plasma concentrations and
electrophilic metabolites, 638- pM values, 484 half-life, 296
639 promotion of Fenton reaction, Factor IXa
molecular endocrinology, 648 503 in clotting, 299-308
topography, 645-648 Ethyndiol diacetate, 632, 666 Factor Ma inhibitors, 325
Estrogen-replacement therapy, Ethynyl estradiol, 634 Factor X
350-351,630 applications, 631, 632 in clotting, 299-308
and SERMs, 655 in contraceptives, 654 hemostatic preparations, 292
side effects, adverse effects, enterohepatic recycling, 640 plasma concentrations and
drug interactions, and con- steroidal analogs, 656, 657 half-life, 296
traindications, 355, 631-633 Etiocholane Factor Xa, 285
Estrogen response elements, testosterone metabolite, 687 in clotting, 299-308
648,653 Etozolin, 109-110 hemostatic preparations, 292
Estrogens, 595,598-599 Eulexin, 717, 719-720 Factor Xa inhibitors, 324-325
androgen conversion to, 690- Eunephran, 76 Factor XI
692 Evista, 343, 632 in clotting, 301-308
biosynthesis, 617,618-620 Excitation, in heart, 4-6 plasma concentrations and
currently marketed, 632 Exemestane, 632,643, 728 half-life, 296
environmental and dietary, Exna, 76 Factor XIa inhibitors, 308
651-652 Extertional angina, 9 Factor XIIa
fate of conjugated equine, Extrinsic tenase complex, 299- and bradykinin production,
635-636 300 225
first antiandrogens, 718 Ezetimibe, 343,348-349,366 in clotting, 299-308
genesis of equine, 644-645 metabolism, 357 Factor XIII, 285
metabolism, 620-622 side effects, 355 in clotting, 303
Index

Factor XIIIa, 284,285 Ferrioxamines, 514 6a-Fluoro-16a-hydroxycortisol,


in clotting, 303 Ferriprox, 507 750
Factor XIIIa inhibitors, 329-331 Ferritin, 495-496,498-499 Fluoromethalone, 750, 753,803,
Fadrozole, 643,668 regulation, 499-501 828
Familial hypercholesterolemia, Ferroportin 1,501 9a-Fluoroprednisolone, 751,797,
11 Fetal hemoglobin, 452-453 800,802
Fareston, 343, 632 hereditary persistence of, 457 Fluosol, 398,421-422
Faslodex, 605-606 "sparing" effect in sickle-cell emulsion stability, 428
Feiba anemia, 456-457 pharmacokinetics, 423
hemostatic preparation, 292 Fibric acid antihyperlipidemic pharmacology, 425-426
Felodipine, 21 agents side effects, 423
bioavailability and half-life, 23 ADME, 357 Fluoxymesterone, 697,699-700
cardiovascular effects, 25
for angina treatment, 11 Flurandrenolide, 750, 752,803
vascular-to-cardiac selectivity
clinical applications, 346-347 Flutamide, 717, 719-720,721,
ratio, 172
Female sex hormones, 598,629- marketed agents, 343 722
630. See also Contracep- right shifters for hemoglobin, Fluticasone propionate, 750,
tives; 17p-Estradiol;Estro- 389-394 753,827,832-833
gens; Progesterone side effects, adverse effects, Fluvastatin, 343-344,345
aromatase inhibitors, 642-645 drug interactions, and con- ADME, 356
biosynthesis, 617, 642-645 traindications, 354-355 for angina treatment, 11
clinical applications, 630-633 structure-activity relation- enzyme activity, 364
discovery of SERMS, 655-656 ships, 364365 D-Fluviabactin, 489,492
drug metabolism, 633-642 Fibrillation, 4, 31 L-Fluviabactin, 483,485,490,
nonsteroidal analogs of estra- and reentry, 30 495
diol, 659-666 Fibrin, 165-166,284-285 synthesis, 522
nonsteroidal analogs of pro- in clotting, 299-308 Fluxon, 423
gesterone, 667-668 Fibrinogen, 285,303,318 Foldsome, 778
physiology and pharmacology, Fibrinolysis, 299308 Follicle-stimulating hormone,
645-655 Fibrinolytic agents 616
recent and future develop- bleeding and other adverse and androgen synthesis, 682-
ments, 668-669 effects, 291-294 683
steroidal analogs of estradiol, clinical uses, 284-299 and estrogen synthesis, 642,
656-659 marketed, 290 654-655
steroidal analogs of progester- treatment regimens, 298-299 Fondaparinux, 285
one, 666-667 Filgrastim, 263, 264 clinical trials, 292, 294, 310
structure-activity relation- Finasteride, 717, 722 formula weight, mechanism of
ships, 656-668 reactions of, 604,605 action, and route of admin-
Femara, 632 FK143, 724 istration, 286
Fendiline, 17 Flecainide, 32, 167 structure, 288
bioavailability and half-life, 18 cardiovascular effects, 37-38 Formestane, 727
cardiovascular effects, 19 increased mortality from, 45 Formoguanamine, 123
Fenofibrate, 343,347 multiple inhibitory mecha- FR 139317
for angina treatment, 11 nisms, 33 endothelin receptor antago-
enzyme activity, 365 uses and side effects, 35 nist, 206
metabolism, 357 Flonatril, 83 FR 171113,327,328
side effects, 354 Flumethasone, 751,796,802- FR 173,657
Fenquizone, 86 803 bradykinin antagonist, 227
Fenton reaction, 499,502-503 Flunarizine, 160, 170 FR 181877,328,329
Ferrichrome, 490 Flunisolide, 750, 753, 821 FR 901533
pM values, 484 Fluocinolone, 750, 752, 761, 784, endothelin-converting enzyme
Ferric hydroxide, 481 797,803 inhibitor, 208
Ferrimycin, 514 Fluocinolone acetonide, 750, Fragmin, 286
Ferrioxamine B, 514 752, 761,797,803,824,834 Friedreich's ataxia
pM values, 484 Fluocinonide, 750, 752,803,824 role of iron, 504,505
Ferrioxamine E 6a-Fluorocortisol, 750,800,802 Furosemide, 88,94-95,142
pM values, 484 9a-Fluorocortisol, 749, 776,802 animal studies, 62
Index

Gallopamil, 17 Glucose 6-phosphate dehydroge- therapeutic indications, side


bioavailability and half-life, 18 nase effects, and pharmacokinet-
cardiovascular effects, 19 inhibition by carbonic anhy- ics, 259-261
Gap junctions, 3 drase, 68 Granulocytes, 252-255
Gastrin-releasing peptide, 229- Glucuronidase, 640 Granulokine, 264
230 Glucuronidation, 640 Grave's disease, 574
Gelatin Glucuronide conjugation Grimatin, 264
for plasma volume expanders, androgens, 687-688 GT-102279,370
429-430,431 estrogens, 638,639-640 Guy's Hospital pill, 65
Gemfibrozil, 343,346,347 Glutathione peroxidase GW 532,369
for angina treatment, 11 hydrogen peroxide manage- GW 707,369
enzyme activity, 365 ment, 502 GW 5638,663
metabolism, 357 Glycerine GW 7604,663
osmotic diuretic, 64 GW 501516,370,372
side effects, 353, 354
Glycerol trinitrate, 13-14 GW 590735,370
Gene transfer
pharmacokinetics, 15
recombinant globin gene vec-
Glycoprotein IB Halbetasol propionate, 750, 752
tors, 468-469 interaction with sickled cells, Halcinonide, 750, 752, 803, 823-
technology improvements, 470 458458 824,831
Genistein, 647, 652 Glycoprotein IIb/IIIa (GpIIb/ Halotestin, 697, 699-700
Gestodene, 641,658 IIIa) inhibitors Hansch approach, to QSAR
Glibenclamide, 176 -178 calcium channel blockers, 29 for anti-inflammatory ste-
and extracellular potassium, for thrombolytic therapy, roids, 788-791
162-163,164 166-167 Haptoglobin, 403
a-Globin chain, 445 Glypressin, 200 Hashimoto's thyroiditis, 574
P-Globin chain, 445 Goiters, 565 HBED, See N-,N'-Bis(2-hydroxy-
y-Globin chain, 456,457 Gonadotropin releasing hor- benzy1)enylenediamine-N-,
P-Globin gene mone, 654,683,698 N'-diacetic acid (HBED)
abnormal, 445-446 Gonanes, 597,598 HBOC-201,398,400,413-414
geographic distribution in Af- Gossypol, 728-729 HDL, See High density lipopro-
rica, 452,455 Gossypolone, 729 teins
recombinant vectors, 468-469 gp130 cytokines Heart
regulation, 466-469 and interleukin-6,269 anatomy, 2-6
Glomerular filtrate, 57 and interleukin-11,265 thyroid hormones effect on,
Glomerulus, 57-58 GPR14 receptor 571-572
urotensin-I1 as ligand for, 213 Heartbeat, 3-4
Glucocorticoid receptors, 749
Granocyte, 263,264 Heart block, 9,29-30
structure, 776
Granulocyte-colony stimulating HemAssist, 397,400,406-408
Glucocorticoid response ele- factor, 261-262 clinical trials, 399
ments, 749,778-779 bioactivity, 262 Hematopoiesis, 252-255
Glucocorticoids, 612. See also preparations, 264 Hematopoietic agents, 251-255
Anti-inflammatory steroids role in blood cell development, investigational agents, 268-272
antiglucocorticoids, 837-845 252-255 Hematopoietic stem cells, 252,
biosynthesis and metabolism, with stem cell factor, 268 253
773-776 therapeutic implications, 272, response modification as sick-
functions of, 748 273 le-cell therapy, 469-470
non-steroidal, 845-846 therapeutic indications, side Heme iron, 501
temperature-dependent up- effects, and pharmacokinet- Hemochromatosis, 504,514
take, 686 ics, 262-264 Hemofil
Glucose Granulocyte-macrophage colony hemostatic preparation, 292
osmotic diuretic, 64 stimulating factor, 258-259 HemogFetal hemoglobin
for prevention of ventricular bioactivity, 259 therapeutic induction, 463-
remodeling, 181 preparations, 261 465
thyroid's effect on metabo- role in blood cell development, Hemoglobin. See also Blood sub-
lism, 571 252-255 stitutes; Fetal hemoglobin
transport mechanism in renal therapeutic implications, 272, abnormal, in sickle-cell ane-
tubules, 57-58 273 mia, See Sickle hemoglobin
Index

Hemoglobin (Continued) Heparin, 285 3p-HSD, 614-615


allosteric effectors, 386,388- activated partial thromboplas- 5-HT, See Serotonin
394 tin time, 291 Human Genome Project
autooxidation, 404 ADME, 295 draft map and understanding
digestion, 501 clinical trials, 291-292,294 of diseases, 2
left shifters, 386, 388 formula weight, mechanism of Human serum albumin, See Al-
molecular size and nonideal- action, and route of admin- bumin
ity, 446-447 istration, 286 Humate
oxygen binding curve, 387 limitations of use, 323 hemostatic preparation, 292
~50,386 mechanism and sites of action, H029953XX, 330,331
right shifters, 386,388-394 308-310 Hydrochlorothiazide, 76, 78-81,
structure, 387-388 structure, 288 84,142
Hemoglobin A (HbA; normal for thrombolytic therapy, 166 metabolism, 80
hemoglobin), 444-445 Heparin-induced thrombocyto- MK-196 compared, 92
penia, 294 Hydrochlorothiazide-amiloride,
oxygen binding to, 386,446
Heparin-induced thrombocyto- 78
Hemoglobin C, 457
penia with thrombosis, 294 Hydrochlorothiazide-triam-
Hemoglobin F, See Fetal hemo- Heparin rebound, 308 terene, 78
globin Hephaestin, 501 Hydrocortisone, See Cortisol
Hemoglobinopathy database, Hereditary persistence of fetal Hydrocortisone esters, 749, 752,
453 hemoglobin, 457 791,803,808
Hemoglobins, conjugated, 417- Hexestrol effect of structural changes on
420 eledrophilic metabolites, 638 activity, 796-837
Hemoglobins, crosslinked, 397, estrogen receptor affinity, 659 HydroDiurit, 76
406-408 High ceiling diuretics, 88, 89- Hydroflumethiazide, 77
autooxidation, 405 111,142 Hydrogen peroxide
Hemoglobins, encapsulated, 398, High density lipoproteins reaction with iron(II), 502
420-421 (HDL), 340-341 Hydromox, 83
Hemoglobins, modified and coronary artery disease, Hydrothiazide diuretics, 78-81
clinical use, 398-420 10 Hydroxamate iron chelators,
development, 396 current drugs to raise, 342- 483,486
recent developments, 420-421 352 synthesis, 524-530
Hemoglobins, polymerized, 397, new treatments for raising 19-Hydroxyandrostenedione,
412-417 while lowering TG, 370-372 690
Hemoglobins, recombinant, 396, transport, 359-361 1,5-Hydroxycholecalciferol
Hirudin and analogs, 29,285, secretion in kidneys, 56
397-398,408-412
316318 Hydroxyethyl starch
Hemoglobin S (HbS; sickle he-
marketed antithrombotics, for plasma volume expanders,
moglobin), See Sickle hemo- 286 429,430,431,432,433
globin HMG-CoA reductase, 667 Hydroxymethylglutaryl-CoA
Hemolink, 400,412,415-417 HMG-CoA reductase inhibitors, (HMG-CoA) reductase in-
Hemophilia, 291 See Statins hibitors, See Statins
hepatitis vaccine recom- HMR 1098,163,177,178 17a-Hydroxyprogesteronecap-
mended, 295 HMR 1453,369 roate, 641
Hemopure, 397,400,412,413- HMR 1883,177,178-179 4-Hydroxytamoxifen, 647,662
414 HOE-140 Hydroxyurea
clinical trials, 399 bradykinin antagonist, 226 for sickle-cell anemia, 464
Hemorrhage, 284 L-Homofluviabadin, 489,490 Hygroton, 82
Hemospan, 419 L-Homoparabactin, 489,490 Hypercholesterolemia,11
Hemostatic plugs, 284 Homopoietin, 255 Hypertension
Hemostatic preparations Hormone-responsive elements, and coronary artery disease,
ADME, 295-298 623 10
adverse effects, 294-295 Hot flashes, 631 Hyperthyroidism, 565,574,575
challenges, 284 HP 522,93 Hyperuricemia, 56
clinical use, 284-299 HS-142-1 Hypogonadism, 698
marketed, 292 binding to atrial natiuretic Hypothyroidism, 565,574, 576
HemoZyme, 405 peptide receptor, 217 Hypoxanthine, 141
Index

Hypoxia Interleukin-2 IRL 3630


and multistep carcinogenesis, role in blood cell development, endothelin receptor antago-
392 253 nist, 206
and VCAM-1,459 Interleukin-3,268-270,271-272 Iron
Hypoxia inducible factor-la, 256 role in blood cell development, absorption, 501
253,254-255 in biosphere, 480-481
Ibutilide, 32, 41, 167 with stem cell factor, 268 chelate effect, 481-483
cardiovascular effects, 42 Interleukin-4 dynamics in humans, 495403
potassium channel subtype role in blood cell development, dynamics in microorganisms,
selective, 45 253 481-495
uses and side effects, 40 Interleukin-5 molecular control of uptake,
ICI 118,552,180 role in blood cell development, processing, and storage,
ICI 147798,131 253 499 -501
Interleukin-6,268-270 non-transferrin bound, 501
ICI 164,384,585,635,657
with IL-11,265 oxidation states, 480-481
ICI 182,780,604
role in blood cell development, siderophores, 483-488
reactions of, 604,605-606
253,254-255 storage and transport, 495-499
ICI 206970,131 with stem cell factor, 268 Iron chelators. See also Desferri-
ICI (ZN) 182,780,635,657,668 Interleukin-7 thiocin iron chelators
molecular endocrinology, 648 role in blood cell development, animal models, 534-540
Idraparinux, 310 253 clinical use, 505-514
Ifetroban, 326,327 Interleukin-11,264-265,269, future developments, 522551
Iloprost, 328 272 HBED, 515-522
Imavist (Imagent),399 bioactivity, 265 history of chelation therapy,
Implantable cardioverter defi- preparations, 266 514-515
brillator, 46 role in blood cell development, pM values for selected ligands,
Increased pulmonary relative 253 484
volume, 424 therapeutic indications, side recent developments, 515-522
Indacrinone, 92-93 effects, and pharmacokinet- synthetic approaches, 522-534
Indapamide, 83,84 ics, 265-266 Iron(II), 480-481
metabolism, 80 Interleukin-12 absorption, 501
Inderal, See Propranolol role in blood cell development, reaction with hydrogen perox-
Inhibin, 683 253 ide, 502
Innohep, 286 Interleukin-17 Iron(III), 480-481
Inositol hexaphosphate role in blood cell development, absorption, 501
allosteric effector of hemoglo- 253 Iron-mediated damage, 501403
bin, 388 Interleukins Iron-mediated diseases, 503-505
Inositol hexasulfate role in blood cell development, Iron overload disorders, 503-505
allosteric effector of hemoglo- 252-255 animal models for, 534-540
bin, 388 Intermediate density lipopro- Iron-processing proteins, 495
Inositol triphosphate, 158 teins (IDL),340-341 Iron regulatory proteins, 500-
Insulin Intrinsic tenase complex, 299- 501
for prevention of ventricular 300 role in Alzheimer's disease,
remodeling, 181 In vitro studies 504-505
Integrilin, 29,287 shortcomings of, 62 Iron-responsive elements, 499-
Integrin a4pl Inward-rectifying K+ current, 7 501
interaction with sickled cells, Iodine Ismotic
458 incorporation in thyroid hor- osmotic diuretic, 64
Integrin aVp3 mone biosynthesis, 568-569 Isosorbide
interaction with sickles cells, and thyroid gland, 565 osmotic diuretic, 64
458 Ion channel gates, 6-7 Isosorbide dinitrate, 13,14
Intercalated disk, 3 Ion channels, 6-8 Isosorbide mononitrate, 13, 14
Interleukin-1 Ionotropic state, 9 Isoxsuprine, 13, 14
enhances IL-11,264 Ipamix, 83 Isradipine, 21
role in blood cell development, IRL 1038 bioavailability and half-life, 23
253,254-255 endothelin receptor antago- cardiovascular effects, 25-26
and stem cell factor, 267 nist, 206 Itavastatin, 343
Index

JTT-705,371,372 formula weight, mechanism of Liposomes


Junctional rhythm, 9 action, and route of admin- hemoglobin encapsulation,
Juxtamedullary nephrons, 57 istration, 286 420,421
structure, 288 Liquivent, 399
Kallikrein, 225 treatment regimen, 298 Liver
KB-R7943,173-175 Lercanidipine, 22 thyroid hormones effect on,
Ketanserin, 328,329 bioavailability and half-life, 23 572
Ketoconazole, 724 cardiovascular effects, 26 Liver x-receptor agonists, 371-
Kidneys fastest growing class of cal- 372
and edema, 62-63 cium channel blocker, 23 Long QT syndrome
functions, 56-57 Lescol, 343 anti-arrhythmic drugs, 168
operation, 57-61 Letrozole, 632, 643 Loop diuretics, See High ceiling
Kininase 11, See Angiotensin- Leukemia inhibitory factor,
diuretics
converting enzyme 268-269
Loop of Henle, 57-58,60
Kininogen, 225 Leukine, 260
Kit ligand, See Stem cell factor Levonorgestrel Lopid, 343
Klinefelter's syndrome, 698 applications, 631, 632 Lorcainide, 32, 34
Koate Levormeloxifene, 663 cardiovascular effects, 38
hemostatic preparation, 292 Levothroid, 576 uses and side effects, 35
Kogenatemelixate Levothyroxine, 580-581 Losartan, 198,199
hemostatic preparation, 292 LF16-0687 Lotrafiban, 319
Konyne bradykinin antagonist, 227 Lovastatin, 343,345,609
hemostatic preparation, 292 Liarazole, 725 ADME, 356
Koshland-Nemethy-Filmer/Dalz- Lidocaine, 32,34, 167 for angina treatment, 11
iel-Engek model, of allostery ATP-sensitive potassium with niacin, 350
hemoglobin, 386 channel inhibition, 178 with Niaspan, 368
KFP-297,370,371 cardiovascular effects, 36 Lovenox, 286
KW-3902,122 multiple inhibitory mecha- Low density lipoproteins (LDL),
Kyorin, 370,371 nisms, 33 340341
uses and side effects, 35 and coronary artery disease,
L-35, 390 Ligands 10-11
L-345,390 siderophores (iron-specific), current drugs to lower, 342-
L-375,378, 314, 316 483-488 352
L-733,060 Lineweaver-Burk plot
new treatments for lowering,
tachykinin antagonist, 224 iron transport in bacteria, 491
L-749,329 368-370
Liothyronine, 580-581
endothelin receptor antago- Lipantil, 343 reduced by thyroid hormones,
nist, 206 Lipid-membrane artificial red 571
Lacidipine, 22 blood cells, 420-421 transport, 358-359
bioavailability and half-life, 23 Lipid metabolism, 358-362 Low molecular weight heparin,
cardiovascular effects, 26 androgen effect on, 682 285
Larnifiban, 29,319 thyroid hormone effect on, ADME, 295
Laminin 571 adverse effects, 294
interaction with sickled cells, Lipids. See also Antihyperlipi- clinical trials, 292
459 demic agents mechanism and sites of action,
Lasix, 88 lowering with thyromimetics, 309-310
Lasofoxifene, 662, 664-665, 668 575 structure, 288
LDL, See Low density lipopro- role in artherogenesis, 342 treatment regimen, 298
teins Lipid transport, 358-362 Lown-Ganonge-Levine syn-
LDL-receptor upregulators, 369 Lipitor, 343,609 drome, 19
LDR receptor, 11 discovery, 362-363 L-type calcium channels, 7-8,
Left shifters, for hemoglobin, Lipoprotein-associated phospho- 168
386 lipase 4 inhibitors, 374 LU135252
Lenograstim, 263-264 Lipoproteins endothelin receptor antago-
Lepirudin, 29,285,316 and coronary artery disease, nist, 206
ADME, 296 10 Lunetron, 88
clinical trials, 293 metabolism, 358-362 Luret, 88
Index

Luteinizing hormone, 616 structure-activity relation- Metabolic rate


and androgen synthesis, 682- ships for androgen antago- thyroid hormone effect, 570-
683 nists, 718-721 571
and estrogen synthesis, 642, structure-activity relation- Metabolism. See also Absorp-
654-655 ships for synthetic, 699-703 tion, distribution, metabo-
Luteinizing hormones releasing synthetic androgens, 697-706 lism, and excretion (ADME)
factor, 616, 683 toxicities of anabolic steroids, thyroid hormones, 569-570
LY117018 715-716 Methadriol, 699
estrogen receptor afhity, 659 toxicities of androgen antago- Methahydrin, 77
LY519818,370 nists, 721-722 Methandrostenolone, 708
17,20-Lyaseinhibitors, 724-726 toxicities of synthetic andro- anabolic activity, 716
Lymphocytes, 252-254 gens, 705 Methazolarnide, 69, 70
Lymphoid progenitors, 253 Malignant arrhythmias, 157-164 Methemoglobin, 404
Methenolone, 709, 714, 715
Manidipine, 22
Macrophage colony-stimulating Methoxychlor, 651-652
bioavailability and half-life, 23
Methyclothiazide, 77
factor, 252 cardiovascular effects, 26 6a-Methylprednisolone,750,753,
Macrophages, 252-254,259 Mannitol 758,768,770,772,797,800
role in atherosclerosis, 342 osmotic diuretic, 63-64 half life, 776
Magace, 632 with xipamide, 105-106 Methyltestosterone, 681, 697
Magnesium, 160 Mast cell growth factor, See Methyltrienolone, 708
as antiarrhythmic agent, 171 Stem cell factor Metolazone, 83,86
Magnesium supplements Mast cell mediators, 267 Metoprolol, 28, 179
sickle hemoglobin hydration MDL 14962,728 cardiovascular effects, 39,40
agent, 461 Medrogesterone, 721-722 uses and side effects, 38
Malaria Medroxyprogesterone, 641,654 Metyrapone, 120
and sickle-cell anemia, 452 applications, 631, 632 Mevacor, 343,609
Male pseudohermaphroditism, with estrone, 650 Mevastatin, 342-343
698 interactions with steroid re- discovery, 362
Male sex hormones, 598,679- ceptors, 633 Mexiletine, 32, 34
681. See also Anabolic ste- Medrysone, 750,753,827-828 cardiovascular effects, 36
roids; Androgens; 5a-Dihy- Mefruside, 72-73,82 increased mortality from, 45
drotestosterone (DHT); Megakarocyte growth and devel- multiple inhibitory mecha-
Testosterone opment factor, 270 nisms, 33
absorption, distribution, and Megakarocytes, 253,254,256 uses and side effects, 35
metabolism of anabolic ste- Megestrol, 632 Mexrenone, 120
interactions with steroid re- Mibefradil, 17, 160,171-173
roids, 707-715
ceptors, 633 ATP-sensitive potassium
absorption, distribution, and
Melagatran, 314,315 channel inhibition, 178
metabolism of androgen an- Melamine, 123 bioavailability and half-life, 18
tagonists, 721 Membrane-depressant agents cardiovascular effects, 19
absorption, distribution, and (Class I antiarrhythmics), Microemulsified perfluorochemi-
metabolism of endogenous, 31,32,34-38,167,168 cal blood substitutes, 428-
685-692 Meralluride, 67 429
absorption, distribution, and Merbaphen, 65 Microsomal triglyceride transfer
metabolism of synthetic, Mercuhydrin, 67 protein (MTP) inhibitors,
703-705 Mercurial diuretics, 64 - 66 368-369
biosynthesis, 616-618,682- animal studies, 62 Mictine, 124
685 Mercurophylline NF XII,67 Mifepristone, 651
currently marketed anabolic Mercuropurin, 67 interactions with steroid re-
steroids, 705 Mersalyl, 65 ceptors, 633
currently marketed synthetic Mersoben, 66 Mineralcorticoid receptor, 120
androgens, 697 Mespirenone, 118, 119 Mineralcorticoids, 612
endogenous, 681-697 Mesterolone, 709 Mircette, 632
structure-activity relation- Mestranol, 634, 635 Mined dyslipidemia, 342,344
ships for anabolic steroids, applications, 631, 632 MK-196,92-93
707-715 oxidative metabolism, 636 MK-447,108-109
Index

MK-473,93 treatment of arrhythmias in- Neutral endopeptidase inhibi-


MM-25,390 duced by, 167-180 tors, 134-138
MoAb 7E3,462 Myocardial oxygen demand, 9 natriuretic peptides, 218
MoAb LM609,462 Myocardial oxygen supply, 8-9 Neutrophilic granulocytes, 252,
Molgramostim, 260,261 Myocardial pacemaker cells, 4, 253
Mometasone furoate, 750, 753, 29 and granulocyte-colony stimu-
767 Myocardium, 3 , 4 lating factor, 262-264
Monoclate and granulocyte-macrophage
hemostatic preparation, 292 Nadolol colony stimulating factor,
Monocytes, 252-254 cardiovascular effects, 39,40 259
Monod-Wyman-Changeaux uses and side effects, 38 Niacin, 343,349-350,366
model Nafoxidine, 662,664 metabolism, 357-358
and hemoglobin allostery, 386 Nandrolone, 707,714 side effects, 355
Nannochelin, 486,487 Niaspan, 343,350
Mononine
synthesis, 524 Nicardipine, 22
hemostatic preparation, 292
NAPAP, 314 bioavailability and half-life, 23
Moricizine, 32 Napsagatran, 314,315 cardiovascular effects, 26
cardiovascular effects, 38 Naqua, 77 Nicorandil, 13, 15, 178
increased mortality from, 45 Natrilix, 83 Nicospan, 368
multiple inhibitory mecha- Natriuretic peptide family Nicostatin, 350,368
nisms, 33 biological actions, 217-218 Nicotinic acid antihyperlipi-
uses and side effects, 35 biosynthesis, metabolism, and demic agents
Morphine structure, 214-217 ADME, 357-358
for pain relief after myocardial therapeutic potential, 218 clinical applications, 349-350
infarction, 165 Natriuretic peptide receptors, marketed agents, 343
Moxestrol, 634, 635, 658 217 side effects, adverse effects,
MRS2279,321 Naturetin, 76 drug interactions, and con-
Muzolimine, 108 Navidrix, 76 traindications, 355
Mycobactin S, 486,487,493 Necrosis structure-activity relation-
synthesis, 524 apoptosis contrasted, after ships, 366-367
Myeloid progenitors, 253,254 myocardial infarction, 157 Nifedipine, 22, 160, 170
Myocardial cells, 3-4 Nefrolan, 83 bioavailability and half-life, 23
Myocardial infarction, 3, 289 Neohydrin, 67, 70 cardiovascular effects, 26
coronary occlusion, 156-157 Nephrons, 57-58 vascular-to-cardiac selectivity
and lipids, 340 Neptazane, 70 ratio, 172
malignant arrhythmias, 157- Neumega, 266 Nilandron, 720
164 Neupogen, 263 Nilutamide, 717, 720
pathophysiology, 156-164 Neural networks, See Artificial Nilvadipine, 22
preconditioning, 157 neural networks bioavailability and half-life, 23
AT, receptor expression in, Neurogenic inflammation, 223 cardiovascular effects, 26-27
197 Neurokinin A, 221 Nimodipine, 22
treatment regimens, 298 Neurokinin A receptor, 222 bioavailability and half-life, 23
ventricular remodeling, 164 Neurokinin B, 221 cardiovascular effects, 27
Myocardial infarction agents, Neurokinin B receptor, 222 Nisoldipine, 22
155-156. See also Antiar- Neuromedin B, 229 bioavailability and half-life, 23
rhythmic agents Neuromedin L, 221 cardiovascular effects, 27
for pain relief, 164-165 Neuropeptide Y Nitrate reductase, 15
for remodeling prevention, biological actions, 210-212 Nitrates
180-181 biosynthesis and metabolism, pharmacokinetics and toler-
for treatment of arrhythmias, 208-209 ance of organic, 15
167-180 structure, 208 as vasodilators, 11-15
for treatment of thrombolysis, Neuropeptide Y antagonists, 212 Nitrendipine, 22
165-167 Neuropeptide Y receptors, 209- bioavailability and half-life, 23
Myocardial ischemia, 3, 157,289 210 cardiovascular effects, 27
extracellular potassium accu- Neurophysin, 199,200 Nitric oxide
mulation during, 162-163 Neurotensin, 229,230 binding to hemoglobin, 386,
and oxygen supply, 8 , 9 Neutral endopeptidase, 134 387
Index

cardioprotection role, 46 NovoSeven 3-(3-Oxo-17P-hydroxy-4-andro-


from nitratelnitrite vasodila- hemostatic preparation, 292 sten-17-a-y1)-propanoicacid
tors, 11-12 Novurit, 67 lactone, 114
scavenging by hemoglobin, NS3623 1-Oxoisoindolines,84-85
401-402 sickle hemoglobin hydration Oxyfluor, 422,423
and sickle-cell anemia, 462- agent, 461 Oxygen
463 N-type calcium channels, 7 binding to hemoglobin A, 386,
Nitric oxide synthase, 779 Nuclear factor-erythroid 2 (NF- 446
Nitric oxide synthase inhibitors, E2) binding to sickle hemoglobin,
420 and sickle-cell anemia, 467 445-446
Nitrites Nuclear hormone receptors Oxygen extraction, 8-9
as vasodilators, 11-15 transcription regulation by Oxygen supply
and hemoglobin, 386
Nitrogen mustards thyroid hormones, 567
myocardial, 8-9
sickle hemoglobin modifiers, Nuclear receptor corepressor,
Oxygent, 422-423
461 567 clinical trials, 427
Nitroglycerine pharmacokinetics, 423
for pain relief after myocardial Ogen, 632 pharmacology, 425-426
infarction, 165 4-OHA, 727 Oxyglobin
pharmacokinetics, 15 Onapristone, 651 clinical trials, 399
S-Nitrosylated PEG-hemoglobin Oncostatin M, 269 Oxyhemoglobin, 387
(SNO-PEG-Hb), 419 OPC-21268,201 in sickle-cell anemia, 447-448
Nocardamine, 483,486,524 Optro, 398,400,408-412 Oxymesterone, 710
synthesis, 527-528 clinical trials, 399 Oxymetholone, 710
Nolvadex, 343,632 Oratrol, 70 anabolic activity, 716
Nomegestrol, 654 ORG 2058,666 Ozolinone, 110
in contraceptives, 654 interactions with steroid re-
8-(Noradamantan-3-y1)-1,3- ceptors, 633 Pacemaker cells, 4,29
dipropylxanthine, 122 ORG 5020 Pancreatic polypeptide family,
Norbolethone, 710 interactions with steroid re- 208-209
Norethandrolone, 707 ceptors, 633 D-Parabactin, 488,489
anabolic activity, 716 Organomercurial diuretics, L-Parabactin, 483,485,488-495
Norethindrone, 632, 650,666 64- 66 pM values, 484
biotransformation, 641 Orgaran, 29,286 synthesis, 522
in contraceptives, 654 Ornipressin, 200 D-Parabactin A, 489,492
interactions with steroid re- Orpisin, 124 L-Parabactin A, 489,492
ceptors, 633 Ortho-Cept, 632 Paracoccus denitrificans
reactions of, 604-605 Ortho Cyclen-21,632 iron uptake by, 488-495
Norethynodrel, 632,666 Ortho-Novum, 632 Paramethasone, 750,797,802
interactions with steroid re- Osmitrol Parathyroid hormone
ceptors, 633 osmotic diuretic, 64 and calcium reabsorption in
Norgestimate, 632,667 Osmoglyn kidneys, 59
interactions with steroid re- osmotic diuretic, 64 Parkinson's disease
ceptors, 633 Osmotic diuretics, 63-64 role of iron, 504,505
Norgestrel, 632,666-667 Ouabain Parovirus B19,295
biotransformation,641 suppression of arrhythmias Passive diffusion
interactions with steroid re- caused by, 174 in kidneys, 57-60
ceptors, 633 Outward-rectifying K t current, PD 142893
Norinyl, 632 7 endothelin receptor antago-
Normethandrone, 707 Ovral, 632 nist, 206
anabolic activity, 716 Ovulation PD 145065
Normodyne, 28 modulation of, 654-655 endothelin receptor antago-
Norplant, 632 Oxamic acid thyromimetics, nist, 206
18-Nortestosterone, 707 582-583 Pedi-pred, 770
19-Nortestosterone, 702-703 Oxandrolone, 706, 711,714 PEG-hemoglobin, 400,417-419
anabolic activity, 716 anabolic activity, 716 clinical trials, 399
Novastan, 29,286 Oxendolone, 719 immunogenicity, 406
Pentaerythritol tetranitrate, 13, Pitavastatin, 343, 345, 353-354, extracellular-intracellularpro-
14 368 portion, 125
Peptides ADME, 356 for prevention of ventricular
endogenous vasoactive, See enzyme activity, 364 remodeling, 181
Vasoactive peptides Pituitary adenylate cyclase-acti- role in heartbeat, 4-5
Peptide YY, 208-210 vating peptide, 226-228 role in kidneys, 59
Percutaneous intervention, 156 PIXY321,271-272 role of extracellular in ar-
Perflubron-based fluorocarbon Plant sterols rhythmias, 163-164
emulsion ADME, 358 Potassium canrenoate, 117-118
for sickle-cell therapy, 462 antihyperlipidemic agents, Potassium channel, ATP-sensi-
Perfluorochemicals, 396,421- 351-352 tive, See ATP-sensitive po-
side effects, adverse effects, tassium channel
429
drug interactions, and con- Potassium channel antagonists,
clinical use, 398-399
traindications, 355 ATP-sensitive, 175-176
currently used, 421-424 Potassium channel blockers, 6
structure-activity relation-
pharmacokinetics, 424-425 ships, 367 Potassium channels, 7
Perftoran, 422 Plasma cells, 253 Potassium mexrenoate, 118
emulsion stability, 428 Plasma volume expanders, 429- Potassium prorenoate, 117,118
pharmacology, 425 432 Potassium-sparing diuretics,
Peripheral vascular disease, 340 Plasmin, 166,284,285 125-133,142
Peripheral vasodilators, 12 in clotting, 304, 307 Pravachol, 343,609
Peroxisome proliferator-acti- Plasminogen, 166,307 Pravastatin, 343,345,609
vated receptor (PPAR) ago- Plasminogen activator inhibi- ADME, 356
nists, 370-371 tor-1, 166 for angina treatment, 11
Persantine, 287 role in clotting, 307-308 enzyme activity, 364
Perutz stereochemical mecha- Plasminogen activators, 165-166 Prednisolone, 750, 753, 756, 758,
nism Plasmodium falciparum, 452 770, 772, 797,800,802,803,
hemoglobin allostery, 386 Platelet-activating factor, 304 820,821,835-836,845
Pharmacophores Platelet ADP receptor antagonists half-life, 776
thyroid hormone, 578 mechanism and sites of action, Prednisone, 750,753, 758, 797
Phenprocoumon, 311 320-321 half-life, 776
Phenylbutazone, 140 Platelet glycoprotein IIblIIIa side effects, 756
receptor, 285 Pregnanes, 597,598,749
Phenytoin, 32
in clotting, 303-304 Pregnenolone
cardiovascular effects, 36-37
Platelet glycoprotein IIblIIIa formation, 609-612
uses and side effects, 35 precursor for adrenal steroids,
receptor antagonists, 29,
Phosphate right shifters, for he- 318-320 612,615
moglobin, 389 adverse effects, 294 precursor for androgens, 616
Phosphatidylserine Platelet PAR-1 receptor, 304- Preinfarction angina, 10
interaction with sickled cells, 305 Preload, 9
458 Platelet PAR-4 receptor, 304-305 Premarin, 632
Phospholipase C Platelet PAR-1 receptor antago- Premature atrial contractions, 9
role in clotting, 305-306 nists, 326 Premature ventricular contrac-
Phospholipids, 340-341 Platelet PAR-4 receptor antago- tions, 9, 31
Phosphoroamidon nists, 326-327 Prenylamine, 17
diuretic effects, 134-135, 137 Platelets, 284-285 bioavailability and half-life, 18
Phosphoryl-Leu-Phe, 137 in clotting, 299-308 cardiovascular effects, 19
PHP, See Pyridoxalated hemo- Plavix, 29,287 Primary hypothyroidism, 574
globin-polyoxyethylene con- PolyHeme, 397,400,412,414- Prinzmetal's angina, 9
jugate 415 Probasin, 696
Phytoestrogens, 652 Polymerized hemoglobins, 397, Probenecid, 139-140
Phytoprogestins, 652 412-417 with bumetanide, 103
Pinacidil, 177-178 Polythiazide, 77 with hydrochlorothiazide, 78
and extracellular potassium, Potassium with metolazone, 86
163 accumulation of extracellular Probucol, 343
Piprozoline, 109 during myocardial ischemia, ADME, 358
Piretanide, 88, 103-104 162-163 clinical applications, 350
Index

side effects, adverse effects, Proscar, 717,722 Raloxifene, 343,351


drug interactions, and con- Prostacyclin, 205,328 applications, 631,632
traindications, 355 Prostaglandins discovery, 655-656
structure-activity relation- role in clotting, 306 estrogen receptor affinity, 659
ships, 367 Prostate-specific antigen (PSA), glucuronidation, 639-640
Procainamide, 32,34,167 696 interaction with estrogen re-
cardiovascular effects, 35-36 Protein C ceptors, 646
multiple inhibitory mecha- plasma concentrations and side effects, 355,631
nisms, 33 half-life, 296 tissue-selectivity, 652-653
uses and side effects, 35 Protein kinase C Recombinant globin gene vec-
PROCRIT, 258 after myocardial infarction, tors, 468-469
Profibrinolytics, 3 157,158 Recombinant hemoglobins, 396,
Progesterone, 594,598,750, Protein S 397398,408-412
756,759,842-844 plasma concentrations and Recombinate
binding, 771 half-life, 296 hemostatic preparation, 292
biosynthesis, 615,616,642- Proteins 5a-Reductase, 616,618,689
645 thyroid's effect on metabo- 5a-Reductaseinhibitors, 605,
biotransformation, 640-642 lism, 571 722-724
functions of, 645 Prothrombinase complex, 299- Reentry excitation, 3031,160-
interactions with steroid re- 300 161
ceptors, 633 Prothrombin time, 291 ReFacto
metabolism, 620 Pulmonary embolism, 289-290 hemostatic preparation, 292
nonsteroidal analogs of, 667- Purkinje fibers, 3-4,5 Refludan, 286,316
668 Pyochelin, 488 Refractory period, of myocardial
receptor affinity, 780-786 F'yrido-1,2,4-thiadiazines,75 cells, 4
steroidal analogs of, 666-667 Pyridoxalated hemoglobin- and reentry, 30
Progesterone antagonists, 651 polyoxyethylene conjugate Regimen Bayer, 287
Progesterone receptors, 649- (PHP), 397,400,419-420 Regramostim, 260
650 clinical trials, 399 Reifenstein syndrome, 698
activity assessment, 650 Relaxin, 229,230
Progestins, 595,650-651.See Quantitative structure-activity Renal tubules, 57-60
also Estrogen-progestin relationships Renese, 77
combinations anti-inflammatory steroids, Renin
binding to glucocorticoid re- 786-792 mediation of release, 195
ceptors, 841 Questran, 343 secretion in kidneys, 56
currently marketed, 632 for angina treatment, 11 Renin-angiotensin-aldosterone
environmental and dietary, Quinazolinone sulfonamides, system, 114,195
651-652 85-86 and endothelin, 203
first antiandrogens, 718 Quinestrol, 634 inhibitors and antagonists,
side effects, 631-633 applications, 631,632 197-199
tissue-selective, 652-654 Quinethazone, 83,86,97 Renin inhibitors, 197-198
Promegestrone, 666,725 Quinidine, 32,167 ReoPro, 29,287,318
Propafenone, 32 ATP-sensitive potassium Reperfusion injury, 405
cardiovascular effects, 38 channel inhibition, 178 Reperfusion therapy, 156
increased mortality from, 45 cardiovascular effects, 3435 Repolarization, of myocardial
multiple inhibitory mecha- multiple inhibitory mecha- cells, 5
nisms, 33 nisms, 33 Repolarization prolongators
uses and side effects, 35 uses and side effects, 35 (Class 111 antiarrhythmics),
Propecia, 717 31,32,40-43,167
Proplex R-2956,719 RES 701-1
hemostatic preparation, 292 R-5020,666 endothelin receptor antago-
Propranolol, 28,32,179 R-102444,328,329 nist, 206
cardiovascular effects, 39-40 R-103757,368,369 Resveratrol
multiple inhibitory mecha- Radiation therapy for sickle-cell anemia, 464
nisms, 33 hypoxia improves efficacy, 392 Reteplase, 166,167
uses and side effects, 38 0-Raff&ose-crosslinkedhemo- clot selectivity, 290
Prorenone, 120 globin, 415-417,418 Reticulocytes, 253
Reticuloendothelial macro- SA node, See Sinoatrial (SA) Flucor for, 462
phages, 496 node globin gene regulation, 466-
Revasc, 285 Sarafotoxin-6c, 202 469
rHbl.l,400,408-412 Saralasin, 198-199 hematopoietic stem cell re-
rHb3011,404 Sargramostim, 260,261 sponse modification, 469-
Rheumatoid arthritis, 780 Sarpogrelate, 328, 329 470
Rhizobactin, 488 SB-209670 and hereditary persistence of
Rhizoferrin, 488 endothelin receptor antago- fetal hemoglobin, 457
synthesis, 530432 nist, 206 hydroxyurea therapy, 464
Rhodotorulic acid, 486,487 SB-217242 modifiers of, 452-454
Right shifters, for hemoglobin, endothelin receptor antago- and nitric oxide, 462-463
386,388394 nist, 206 pathophysiology, 443-452
SB-273779 sickle hemoglobin polymeriza-
Rimexolone, 750, 753,768,821
calcitonin gene-related peptide tion and disease manifesta-
RNA
antagonist, 222 tion, 448-449
T, effect on synthesis, 565
SB-480848,374 and a-Thalassemia, 454-456
Ro-42-5892,197-198 SC-46542,134 and P-Thalassemia, 456
Ro-46-2005 SCH 34826,136-137 therapeutic decrease of micro-
endothelin receptor antago- SCH 39370,136-137 vascular entrapment, 462-
nist, 206 SCH 42495,136 463
Ro-47- 0203 SCH 48461,366 therapeutic induction of fetal
endothelin receptor antago- SCH 57050,664,668 hemoglobin, 463-465
nist, 206 SCH 57068,664 therapy approaches, 459-461
Ro-61-0612 Schizokinen, 486,487 Sickle hemoglobin. See also Fe-
endothelin receptor antago- synthesis, 524 tal hemoglobin
nist, 206 SD/Ol (Filgrastim),264 chemical modifiers, 461
Rolicton, 124 Sectral, 28 low deoxygenated solubility
Rosuvastatin, 343,345,368 Selective estrogen receptor mod- and polymerization, 450-
ADME, 356 ulators (SERMs), 631,652- 451
enzyme activity, 364 654 polymerization, 447-450
side effects, 354 discovery of, 655-656 polymerization inhibition,
Roxfiban, 319,320 glucuronidation, 640 459-460
RPR 111844 Selestoject, 770 polymerization kinetics, 450,
endothelin receptor antago- SERCA, 572,575 451
nist, 206 Serotonin "sparing" effect of fetal, 456-
RPR 209685,324,325 role in clotting, 304, 306 457
RSR4,388,390 Serotonin receptors structure, 444-445
RSR-13,388,390 antiplatelet agent target, 327- Sickle syndromes, 453,456
RTI 012,842,843,844 329 Sickle trait, 457-458
RTI 6413-003,843 Serpins, 307-308 Sick sinus syndrome, 31
RTI 6413-018,843 Serum albumin, See Albumin Siderophores, 483-488
RTI 6413-033,843 Sex hormones. See also Female Silandrone, 714
RU-486,651 sex hormones; Male sex hor- Silencing mediator of retinoic
anti-inflammatory properties, mones and thyroid receptor
839,841-845 biosynthesis, 616-618 (SMRT),567
RU-39305,839-840 Sex steroid binding P-globulin, Simvastatin, 343,345, 609
RU-43044,839 686,703 ADME, 356
RWJ-58259,327,328 Sibrafiban, 319 for angina treatment, 11
RXR (retinoid X receptor), 566- Sickle-cell anemia enzyme activity, 364
567 animal models, 459 with ezetimibe, 349
Ryanodine, 160,171 5-azacytidine therapy, 464 Sinlestal, 343
bone marrow transplantation, Sinoatrial (SA) node, 3,4, 5
S-18886,326,327 465-466 and disorders of signal genera-
Salicylic acid butyrate therapy, 465 tion, 29
uricosuric agent, 138-139 and cyanate, 460-461 and reentry excitation, 30-31
Saltucin, 76 cyanate for, 460-461 Sinus arrhythmia, 31
Saluron, 77 and endothelium, 458-459 Sinus bradycardia, 9
Index

Sinus tachycardia, 9,31 multiple inhibitory mecha- 5a-Steroid, 596


Sitaxsentan nisms, 33 5p-Steroid, 596
endothelin receptor antago- uses and side effects, 40 Steroidal aldosterone antago-
nist, 206 L-Sotalol, 32 nists
Sitostanol, 352,367 cardiovascular effects, 39,40 diuretic agents, 111-120
p-Sitosterol, 351-352,367 uses and side effects, 38 Steroid aromatization, 619-620,
SKF 94901,581482,587 Spantide, 224 642-643
tissue-selective thyromimetic, Spirolactams, 115-116 androgens, 690
570 Spironolactone, 111-120, 142 estrogens, 642-643
SKF 105,657,723-724 SQ 28603,136 Steroid biochemistry, 606
SL 65.0472,328,329 SQ 29072,136 hormone action, 622-624
SM-19712 SQ 32547,46 Steroid hormones, 593-595. See
endothelin-converting enzyme SQ 32926,46 also Anti-inflammatory ste-
inhibitor, 208 Squalene roids; Female sex hormones;
endothelin receptor antago- conversion to cholesterol, Male sex hormones
nist, 207 608-609,612 chemical reactions, 604-606
Small dense LDL (sdLDL), 341 formation, 611 chemical synthesis, 599-602
Sodium SR-140333,223 microbial transformation,
and coronary artery disease, SR-142801 602-604
11 tachykinin antagonist, 224 nomenclature, 597-599
reabsorption in kidneys, 58- SR-121463A, 201 structure and physical proper-
59,62-63 Stable angina, 9,290 ties, 595-597
role in heartbeat, 4-5 Stanazolol, 712,715 Steroid metabolism, 620-622
Sodium+/Calcium2+exchanger anabolic activity, 716 Steroidogenesis, 606-620
after myocardial infarction, Stanolone, See 5a-Dihydrotes- and trophic hormones, 614
156,158 tosterone Steroidogenic acute regulatory
in kidneys, 59 Staphlokinase, 323 protein, 610
Sodium+/Calcium2+exchanger Staphylococcus aureus Steroid receptors, 622-624
antagonists, 173-175 iron uptake by, 494-495 Streptokinase, 165,322
Sodium channel blockers, 6 Statins, 609 clinical trials, 294
increased mortality from, 45 ADME, 355357 clot selectivity, 290
Sodium channels, 7 for angina treatment, 11 treatment regimens, 298
Sodium+/HydrogenCexchanger and cholesterol synthesis, 608 Stroma-free hemoglobin, 396-
in kidneys, 58 clinical applications, 342346 397,403
Sodium mercaptomerin, 67 history of development of, Substance K, 221
Sodium /Potassium -ATPase
f f

362-363 Substance P
P-P new treatments for lowering biological action, 222-224
after myocardial infarction, LDL and TG, 368-370 biosynthesis, 221-222
159,162 with niacin, 350 discovery, 194
in kidneys, 58 side effects, adverse effects, Substance P receptors, 222
up-regulation by thyroid hor- drug interactions, and con- Sucrose
mones, 570 traindications, 352-354 osmotic diuretic, 64
Sodium salicylate, 138-139 structure-activity relation- m-Sulfamoylbenzoic acid hydra-
Soldactone, 117-118 ships, 363364 zides, 81-84
Solu-Cortef, 770 Steel factor, See Stem cell factor Sulfanilamide, 56, 69
Solu-Medrol, 771 Stem cell factor, 266-267 makes urine alkaline, 68
Somastatin-14 bioactivity, 267-268 Sulfated glycolipids
similarity to urotensin-11, preparations, 268 interaction with sickled cells,
212-213 role in blood cell development, 458
Somatostatin, 228-229 252,253,255 Sulfinpyrazone, 139,140-141
TSH modulation, 568 therapeutic implications, 272 Sulfonamide diurectic-antihy-
Sorbitol therapeutic indications, side pertensive agents, 86-87
osmotic diuretic, 64 effects, and pharmacokinet- Sulfonamides
D-Sotalol, 167 ics, 268 aromatic diuretic agents, 68,
D,L-Sotalol, 32,41 Stem cell leukemia (SCLITal-1) 70-73
cardiovascular effects, 42 transcription factors nonaromatic diuretic agents,
increased use of, 45 and sickle-cell anemia, 467 68,81-89
Index

Sulfonylurea receptors, 177 Testopel, 697 Thrombin inhibitors


Superoxide, 502503 Testosterone, 598-599,681 from concept to drug, 310-318
Superoxide dismutase, 502 absorption, distribution, and hirudin and hirudin-like, 316-
Supraventricular arrhythmias, metabolism, 620, 621, 685- 318
31 692 small-moleculedirect, 311-
Supraventricular tachycardia, 9, biosynthesis, 616- 618,682- 316
31,161 685 for thrombolytic therapy, 166
Synthokine, 271 and estrogen biosynthesis, 642 Thrombolysis, 156
Synthroid, 576 mechanism of action, 692-697 Thrombolytic agents (clot-dis-
occurrence and roles, 681-682 solving), 165-167
T,, See Thyroid hormone T, relative androgenicity and re- calcium channel blockers, 29
T,, See Thyroid hormone T, challenges, 284
ceptor binding of analogs,
Tace, 632 mechanisms, 322-323
702
Tachycardia, 9,31,161 Thrombomodulin, 301
from hyperthyroidism, 574 synthesis from cholesterol,
Thrombopoietin, 252
and reentry, 30 604
therapeutic uses, 270-271
Tachykinin antagonists, 224 Testosterone butyrate, 714
Thrombosis, 156,284,342
Tachykinin receptors, 222 Testosterone cypionate, 697, 703 antithrombotic treatment reg-
Tachykinins Testosterone enathate, 697, 703 imen, 298-299
biological actions, 222-224 Testosterone-estradiol binding molecular mechanisms, 299-
biosynthesis, metabolism, and globulin, 686 308
structure,. 221-222 Testosterone gel, 697 Thrombospondin
TAK-044 Testosterone pellets, 697 interaction with sickled cells,
endothelin receptor antago- Testosterone propionate, 703 458,459,462
nist, 206 Testosterone valerate, 714 Thrombotic thrombocytopenic
Tamoxifen, 343,351,630, 661- Testred, 697 purpura, 294
665 D,L-Tetramethylparabactin, 494 Thromboxane, 303
applications, 631, 632 Tezosentan Thromboxane A,, 326
discovery, 655-656 endothelin receptor antago- Thromboxane receptor inhibi-
electrophilic metabolites, 638- nist, 206 tors, 326
639 a-Thalassemia, 456 Thromboxane synthase inhibi-
interaction with estrogen re- animal models, 534 tors, 326
ceptors, 646 and sickle-cell anemia, 454- Thyroglobulin, 568
oxidative metabolism, 636, 456 Thyroidal hypothyroidism, 574
637 and sickle trait, 458 Thyroid diseases, 574
side effects, 355, 631 P-Thalassemia, 456 Thyroid gland, 565
tissue-selectivity, 652-653 animal models, 534-535 Thyroid hormone antagonists,
TAP (tick anticoagulant pep- bone marrow transplantation, 585-586
tide), 324, 325 465 Thyroid hormone receptors, 564,
TBC-11251 and sickle-cell anemia, 456 565-566
endothelin receptor antago- Theophylline, 124 isoform knockouts, 573-574
nist, 206 diuretic action, 121 Thyroid hormone response ele-
Tedisamil Thiazide diuretics, 73-78, 142 ments, 566
potassium channel subtype with triamterene, 128 Thyroid hormones, 563-564
selective, 45 Thiomerin, 67 biological actions, 570-571
Tenecteplase, 166,323 Thiomesterone, 711 biosynthesis, 568-569
clot selectivity, 290 Thiorphan coactivators and corepressors,
Tenormin, 28 diuretic effects, 134-135 567
Terbogrel, 326, 327 3D-MORSE(Molecular Repre- effect on bone and skeletal
Terlipressin, 200 sentation of Structures muscle, 572-573
Terodiline based on Electron Diffrac- effect on carbohydrate metab-
bioavailability and half-life, 18 tion), 792 olism, 571
cardiovascular effects, 19-20 3D Quantitative structure-activ- effect on heart and cardiovas-
Testes, 680 ity relationships (3D-QSAR) cular system, 571-572
Testlac, 697, 700 anti-inflammatory steroids, effect on lipid metabolism, 571
Testoderm, 697 793-796 effect on liver, 572
Testolactone, 697, 698 Thrombin, 284,285 effect on metabolic rate, 570471
Index

effect on protein metabolism, Tifocogin, 325 Transferrin, 496-498


571 Tinzaparin pit4 values, 484
molecular mechanism of hor- formula weight, mechanism of regulation, 499-501
mone action, 565-567 action, and route of admin- Transferrin receptor, 496-498
physiology of hormone action, istration, 286 Transhsional iron overload, 504
568-570 Tirofiban, 29,285, 318 Transthyretin, 569
recent developments, 585-587 ADME, 297 Trecetilide, 41
regulation, 568 clinical trials, 293, 294 cardiovascular effects, 42
structure-activity relation- formula weight, mechanism of potassium channel subtype
ships, 576-585 action, and route of admin- selective, 45
therapeutic potential of ana- istration, 287 Triac, 579, 581
logs, 575-576 structure, 289 Triamcinolone, 750, 752, 758-
transcriptional regulation, 759,768,775-776,789,797,
for thrombolytic therapy, 167
566-567 800,803
treatment regimen, 298
transport, 569 half life, 776
uptake and metabolism, 569- Tissue factor, 290
Triamcinolone acetonide, 750,
570 in clotting, 299-308 752, 759, 772, 786, 788, 797,
Thyroid hormone T,, 564,565 Tissue factor pathway inhibitor, 800,803,808-809,821,824
analogs, 578-585 300,301 2,4,7-Triamino-6-phenylpteri-
biosynthesis, 568-569 Tissue plasminogein activator dine, 126
regulation, 568 (t-PA) 2,4,7-Triamino-5-phe-
synthetic, 576 clinical trials, 293, 294 nylpyrimido[4,5d]pyrimi-
transport, uptake, and metab- in clotting, 307 dine, 127
olism, 569570 for thrombolytic therapy, 165- Triamterene, 124,125-128,132,
Thyroid hormone T,, 564,565 166 142
analogs, 578-585 treatment regimen, 298 hydrochlorothiazide-triam-
biosynthesis, 568569 Tizolemide, 87, 89 terene, 78
regulation, 568 T-lymphoblasts, 253 with xipamide, 107
synthetic, 576 T-lymphocytes, 253,254 Triarylethylenes, 655, 661-665
transport, uptake, and metab- TMC-66 oxidative metabolism, 636-
olism, 569-570 endothelin-converting enzyme 637
uptake and metabolism, 569- inhibitor, 208 Triazines, 123-125
570 Tocainide, 32,34, 167 Trichlormethiazide, 77, 81
Thyroid releasing hormone cardiovascular effects, 36 Tricor, 343
(TRH),568 multiple inhibitory mecha- Tridegin, 330
Thyroid stimulating hormone nisms, 33 Triflocin, 107
(TSH),567-569 uses and side effects, 35 Triggered activity, 159
Thyromimetics, 564, 575-576 a-Tocopherol, 372 Triglycerides, 340-342
design, 586-587 Toprol, 28 androgen effects on, 706
structure-activity relation- Toradiur, 89 and coronary artery disease,
ships, 576585 Torasemide, 89,107-108 10
Thyroperoxidase, 568,569 Toremifene, 343,351,662 current drugs to lower, 342-
Thyrotoxicosis, 565, 574 applications, 631, 632 352
Thyrotropin, 568 oxidative metabolism, 636 new treatments for lowering
Thyroxine, 565. See also Thyroid side effects, 355 while lowering LDL, 368-
hormone T, Torsade de pointes, 168, 170- 370
Thyroxine-bindingglobulin, 569 171 new treatments for lowering
Tibolone, 650, 668 Toxicity while raising HDL, 370372
Ticlid, 287 and physiological changes in- reduced by thyroid hormones,
Ticlopidine, 285, 320 duced by compound, 62 571
ADME, 297 Trandate, 28 Trioxifene, 655
clinical trials, 293 Trandolapril Troxolon
formula weight, mechanism of with verapamil, 169 for crosslinked hemoglobin,
action, and route of admin- Transcortin, 686, 703 417
istration, 287 Transcription Trusopt, 67
structure, 289 control by thyroid hormones, TSAA21,719
for thrombolytic therapy, 166 565567 TSE-424,660
Index

TTA-386 Vasoactive peptides, 193-195. von Willebrand disease, 291


endothelin receptor antago- See also specific peptides von Willebrand factor, 290
nist, 206 Vasodilators, 11-15. See also in clotting, 303
T-type calcium channels, 7,8, Antianginal agents in hemostatic preparations, 292
168 future directions, 46-47 interaction with sickled cells,
Tumor necrosis factor-a pharmacokinetics and toler- 459,462
enhances IL-11,264 ance of organic nitrates, 15 Vorozole, 643,668
and stem cell factor, 267 Vasopressin, See Arginine vaso- L-Vulnibactin, 483,485
pressin
U466619,326 synthesis, 522-524
Vasospastic angina, 9-10
Ubiquinone, 353-354 Vectren, 83
Unat, 89 Warfarin, 285
Venous thrombosis, 289-290
Uncoupling proteins antithrombotic treatment reg- ADME, 295-296
effect of thyroid hormones, 570 imen, 298 clinical trials, 292-293
Unfractionated heparin, 308- Ventricles, 3, 4 formula weight, mechanism of
309 Ventricular arrhythmias, 31 action, and route of admin-
Unidirectional conduction block, Ventricular fibrillation, 9, 31, istration, 286
29,30-31 157,159-164 limitations of use, 323
Unstable angina, 10,290 Ventricular remodeling, 164 mechanism and sites of action,
Upstroke, of heartbeat, 5 prevention, 180-181 310
Urea Ventricular tachycardia, 9,31 prothrombin time, 291
osmotic diuretic, 64 Verapamil, 15, 18,32,160, 161, structure, 288
Urea cycle, 138 168-169 treatment regimen, 298
Ureaphil ATP-sensitive potassium and variant CYP2C9 alleles,
osmotic diuretic, 64 channel inhibition, 178 297
Uric acid, 56, 138-142 bioavailability and half-life, 18 Welchol, 343
effect of diuretic therapy on, 63 cardiovascular effects, 17, 19, for angina treatment, 11
Uricase, 62 43-44 Wolff-Parkinson-Whitesyn-
Uricosuric agents, 56,138-142. dihydropyridines contrasted, drome, 19,31,38
See also Diuretic agents 27-28
animal studies, 62 multiple inhibitory mecha- Xanthine oxidase, 138, 141
Uridine diphosphate (UDP) gluc- nisms, 33 Xanthines, 121-122
uronyl transferases, 640 uses and side effects, 43 Xanthopterin, 125
Urokinase, 308 vascular-to-cardiac selectivity Xemilofiban, 319
Urotensin-I1 ratio, 172 Ximelagatran, 314, 315
biological actions, 213-214 Very late antigen activator 4 Xipamide, 88,104-107
biosynthesis, metabolism, and w - 4 ) XR5118,330,331
structure, 212-213 interaction with sickled ells,458
Urotensin-I1 receptor, 213 Very low density lipoproteins
(VLDL),340-341
Valsartan, 198,199 and coronary artery disease, 10 Zanoterone, 719
Vanillin transport, 358-359 Zaroxolyn, 83
allosteric effector of hemoglo- Vesicles ZD 1611
bin, 388,394-395 hemoglobin encapsulation, 420, endothelin receptor antago-
sickle hemoglobin modifier, 461 421 nist, 206
Variant angina, 9 L-Vibriobactin, 483, 485,490 Zebeta, 28
Vascular adhesion molecule 1 synthesis, 522-524 Zetia, 343
(VCAM-1) Virilon, 697 ZK 91587,118
interaction with sickled cells, Vitamin E ZK 98299,651
458-459,464 for lipid lowering, 372-373 ZK 119,010
Vascular receptor, 200 Vitamin K estrogen receptor affinity, 659
Vascular tone, 194 and clotting, 300 ZK 169,978
Vasoactive intestinal constrictor, Vitamin-K dependent inhibitors, estrogen receptor affinity, 659
202 285,310 ZK 807834,324
Vasoactive intestinal peptide, VLDL, See Very low density li- ZM 189,154
226-228 poproteins estrogen receptor affinity, 659
discovery, 194 Voltage gating, 6 Zocor, 343,609
ition to the .
..
ng work . highly praised as a fountain of
-

;studies and research."

This new edition of Dr. Alfred Burger's internationally celebrated


classic helps researchers acquaint themselves with both traditional
and state-of-the-art principles and practices governing new
medicinal drug research and development. Completely updated
and revised to reflect the many monumental changes that
have occurred in the field, this latest edition brings together
contributions by experts in a wide range of related fields to explore
recent advances in the understanding of the structural biology
of drug action, as well as cutting-edge technologies for drug
discovery now in use around the world.
This Sixth Edition of Buver's Medicinal Chemistry and D r u ~
Discovery has been expanded to six volumes:
Volume 1: Drug Discovery
Volume 2: Drug Discovery and Drug Development
Volume 3: Cardiovascular Agents and Endocrines
Volume 4: Autocoids, Diagnostics, and Drugs from New Biology
Volume 5: Chemotherapeutic Agents
Volume 6 : Nervous System Agents

"ONALD A. ABRAHAM, PHD, is Professor and Chairman


"1 the Department of Medicinal Chemistry at the Virginia
Commonwealth University School of Pharmacy. A world-renowned
leader in medicinal chemistry and biotechnology, he is the author
of more than 140 journal citations and twenty-five patents. H e
was selected by the AACP Board of Directors as the recipient of the
2002 AACP Paul Dawson Biotechnology Award.

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