Professional Documents
Culture Documents
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
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
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
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
Sinoatrial node
lnternodal pathways
Atrioventricular node
+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
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
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.
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)
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
"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)
OMe
Gallopamil (12)
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)
..
Me Me Me
Me
\
MeOOCg O C H 2 M e MeOOC
F
CI
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
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
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
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
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
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
0 A.CH3
Acetobutolol (51)
CH3
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
Diltiazem (9)
Bepridil (8)
Figure 1.16. Chemical structures of class IV antiarrhythmic agents: calcium channel blockers.
44 Cardiac Drugs: Antianginal, Vasodilators, and Antiarrhythmics
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
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
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
w
Basolateral
membrane
Na+ +
ATP
K+
* I
CI-
---------- +
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
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
(3) Glucose
(6) Sorbitol
(7) Sucrose
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
0
I
R = theophylline
(16) Sodium mercaptomerin Thiomerin
HO N/Y\H
H 0
0 I
R = theophylline
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-
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
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
~ ~ ~ 0 , s '
Increase in Na+
Excretion at 80
Threshold Dose, p.0. &kg P.0.
No. R (mg/kg) [peq/kg/6 h (Rat)]
(39) -3 6 3910
(40) 18 2900
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
(29) Hydrochlorothiazide
V
Q,
(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
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
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
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)
(119) Piretanide
I 2 Clinical Applications 89
0 0 HN
N
H
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
"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-
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
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
OH
NHBu
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
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
54- H
-Q
(179)
H3C
:>
H3C
H3C
"From Ref. 242.
bDose(in m&g) that increases 5-h urine volume by 50%.
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
yNH2 (187)
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
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
Glucocorticoids
Mineralocorticoids
C19
Cholesterol
C27
Cholesterol
Aldosterone Esterone
Figure 2.9. Pathway of adrenal steroidogenesis. 11 = llp-hydroxylase;17 = l7a-hydroxylase;18 =
18-hydroxylase; 21 = 2la-hydroxylase.
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
0
I
0
(194) Epoxymexrenone Eplerenone
0
(209) Potassium Soldactone 336
canrenoate
0
(210) Potassium 0 337
prorenoate
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)
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
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)
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
I
(229) Aminometradine Mictine 376
0
(230) Amisometradine Rolicton
P 376
0
(235) Chlorazanil Daquin, 382
Diurazine,
Orpisin
HN
C1
(240) Triameterene Dyrenium 396
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.
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
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
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
1I (457).
(264) R = H, candoxatrilat
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
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
/
R-N
I
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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CHAPTER THREE
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
(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
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
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
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
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
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
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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
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.
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.
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-
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- ~ ~
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)
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
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)
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.
30
1Carboxypeptidase 272).
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
21 114
I Signal peptidase
124
U-ll
1 Pro-hormone convertases
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
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).
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!
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
Alternative splicing
3 distinct mRNAs
Neurokinin B
Substance P
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
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.
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
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
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
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
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(1991). MacNeil, A. W . Derrick, K. A. Schneck, R. W.
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chem. Biophys., 160, 185 (1974). Siegel, G. Keller, H. D. Liggitt, E. A. Bauer,
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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
'
I SCF
IL-1
I" IL-11
\
Lymphoid
progenitor progenitor
IL-1 @-
CFU-GEMM
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
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
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
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
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
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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promotes endothelial cell angiogenic activity type-specific effects grows, the identification
(279). As our understanding of the biological of more effective therapeutic agents will fol-
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References
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
Thrombin
FXlll - FXllla
Stabilized clot
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
(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
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
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
-
Factor ZX Preparations
Mononine Aventis Behring FIX, purified from human plasma
Beneh Genetics Institute FIX, recombinant
Other
NovoNordisk FVIIa, recombinant
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
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
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
DS ADP (P2Yj2)
platelet receptor
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
(b)
Prothrombin
FXa
or FlXa n '1 \ 1 Thrombin
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
,- - - - - - -
\
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)
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.
- Covalent bond
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
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-
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-
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
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
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~-
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
-Gly-Pro-Arg-Val-
I
Cleavage site
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
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
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
(21) roxifiban
Figure 6.26. Several platelet GPIIbflIIa inhibitors investigated in clinical trials by IV (17)and by
oral administration(18-21).
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
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
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)
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
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)
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
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-
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%)
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
"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
(4)
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
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
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).
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
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 ?
Fibrates
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
-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)
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.
4.1 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
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.
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
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.
H p c l
H G N 0
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
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
tively remove lipid from atherosclerotic le- library online services, especially Current
sions leading to plaque regression in a manner Contents, Medline, and/or SciFinder. Key ref-
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CHAPTER EIGHT
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
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).
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
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
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 [
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
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
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
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
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
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~
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
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
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
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
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
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restoring (251). Solutions isooncotic with 0 Northfield Laboratories Inc. (Evanston, IL,
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References 441
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
- +
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
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
- -
-02
+02
7
lntracellular HbS
polymerization
Bone marrow
(b) Neurological
complications Ophthalmological
complications
= Cardiovascular
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
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
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
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
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
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.,
~
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
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
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.
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
Normal gene
w\
Red
blood
cells
Bone marrow
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
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Through use of a drug-binding extracellular (1910).
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thrombopoietin or erythropoietin, the hybrid 335344(1923).
receptors could be activated by drug binding to 3. E. V. Hahn and E. B. Gillespie, Arch. Intern.
mimic cytokine activation (268). In another Med., 39,233-254(1927).
strategy, cells expressing another hybrid re- 4. T. H. Ham and W. B. Castle, Trans. Assoc. Am.
ceptor created by fusing the G-CSF cytoplas- Physicians, 55, 127-132(1940).
mic domain with the estrogen receptor extra- 5. J. V. Neel, Science, 110,64-66 (1949).
cellular domain became hormone responsive 6. J. F. Bertles and J. Dobler, Blood, 33,884-898
(273). These strategies provide a means of se- (1969).
lective stimulation of the transduced hemato- 7. W. N. Jensen and L. S. Lessin, Semin. Hema-
poietic stem cells or progenitor cell popula- tol., 7,409-426(1970).
tion. However, host immune response to the 8. L. Pauling, H. A. Itano, S. J. Singer, and I. C.
expression of new or foreign genes may limit Wells, Science, 110,543-548(1949).
the application of these strategies dependent 9. T.H. Huisman, Am. J. Hematol., 6, 173-184
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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
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.
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
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
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
(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)
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
Transferrin receptor
-5' AUG
High iron
Aconitase
Ferritin UH
3'
I Efficient translation
Transferrin receptor
5'AUG
I Decreased stability of mRNA
~ndonuclease\L)
(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
CH3
(33)
HOOC-, n ~ C O O H
N N N
H O O C ~
I COOH
-COOH
(34) (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
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
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
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
4 RECENT DEVELOPMENTS
-
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
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.]
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.]
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.)
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
..
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%).
..
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)
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.
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
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
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: 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
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
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
(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
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]
(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
(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)
_+
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.
(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
(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.
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
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
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Iron Chelators and Therapeutic Uses
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
(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
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
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
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
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.
*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
(10) Triac
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
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).
References
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15. C. Weinberger, C. C. Thompson, E. S. Ong, R.
Lebo, D. J. Gruol, and R. M. Evans, Nature,
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'i
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drugs from a class of agents with the diversity J. Biol. Chem., 267,13014(1992).
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23. W. Bourguet, M. Ruff, P. Chambon, H. Grone-
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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
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
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
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
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
-.
XVll XVlll XIX XX
0 CH20Ac
&
,,\OH
CH~COCOZH,HOAc
CHCI,, HBr (GO2)
*
O H (NOZ)~C~H~NH
cortisone acetate XXI
Curvularia lunata
Progesterone
1 1 p-Hydroxyprogesterone
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
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 , #
EcH
0
Norethindrone Finasteride
0vCH20H
OH
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
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
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
.
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
squalene
epoxidase
7-i-Y
0 -
2 NADPH NADPi
squalene + Hi
1squalene
cyclase
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.
Trophic
hormone2
Plasma membrane
GTP GDP
7
Mitochrondia CAMP + PP,
Lipid droplet
Other steroids
Endoplasmic reticulum
Figure 12.23. Trophic hormones and steroidogenesis.
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
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
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
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
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
Steroid (S)
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
--(
H ~ N AJB
- Nuclear localization
- Dimerization
Figure 12.32. General struc- -
ture of the steroid receptors. Transactivation
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CHAPTER THIRTEEN
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
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
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.
(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
3.3 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
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
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
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
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
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).
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
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.
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-
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
i
t
nized by other steroid receptors besides PR,
1 but generally not by ER.
Unlike the other steroidal progestins, (36)
i
of these might be available as new therapeutic can often be found in cited literature refer-
agents at a time coincident with the publica- ences proximate to the point in the text at
tion of this chapter. which these are described, in particular with
Because of their diminished reproductive regard to structure-activity relationships
tract hormonal effects, SERMs such as (89) (Section 7). The USP Dictionary of USAN and
may have therapeutic applications in men. Os- International Drug Names is a premier source
teoporosis is not a gender-specific disorder. A of generic names (including pronunciations),
significant proportion of elderly men exhibit structures, and CAS registry numbers of mar-
progressive bone density loss (233), which ev- keted drugs, and those in clinical trials.
idently is a consequence of diminished avail-
ability of (1)(234). In aged, intact male rats,
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
(235). A comparative reduction of serum cho- ER in tables of ER ligand aMinities
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)
pharmacologic profiling of (89)'seffects in this ERE estrogen response elements in DNA
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
ticipated that a variety of such steroidal and
nonsteroidal ER isoform-selective ligands will
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CHAPTER FOURTEEN
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
1 INTRODUCTION
2 HISTORICAL
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
(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
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.
0
&'&
0
Progesterone (8)
HO
17a-Hydroxypregnenolone(9)
Testosterone (1)
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
/
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,,,,@
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
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
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
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
H2N
COOH
1 559 624 919 a.a.
- Nuclear localization
- Dimerization
- Transactivation
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
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-
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
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
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
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
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
(54) methenolone
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.
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.
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
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.
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
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.
(120) aminoglutethimide
cl'
(121) ketoconazole
(122) liarozole
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
enous androgens are biosynthesized from cho- have also resulted in the abuse of these
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-
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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
1 INTRODUCTION
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
F
(8) Halobetasol propionate (9) Amcinonide (10) Desoximetasone
F F
(13) Triamcinolone acetonide (14) Clocortolone pivalate (15) Flurandrenolide
(13a) Triamcinolone 16, 17-diol
(13b) Triamcinolone hexacetonide
(21-pivalate ester)
F
(16)Fluticasone propionate (17)Mometasone furoate (18)Aclometasone diprorionate
0 &IH/
(°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
(42) pro-antedrug
colonic bacteria
OH
esterases
0
(44) active antedrug (45) inactive
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.
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
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
~- -
/
OCOEt
CH3
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
(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.
OAc
OAc
OCOEt
OCOPr
CI
OAc
CI
C1
F
C1
"Ref. 42.
bCroton oil ear edema assay at 5 h and (5days).
"Betamethasonevalerate, control.
Cortisone Cortisol
\ 6p-Hydroxycortisol
0
H
& $:
Dihydrocortisone Dihydrocortisol Allodihydrocortisol
&&::&&::
Tetrahydrocortisone Tetrahydrocortisol (13%) Allotetrahydrocortisol (7%)
HO'
H
HOP
H
HO - H
HO'
H
2Op-Cortolone 20a-Cortolone 20&Cortol 2Oa-Cortol
\ \ 1
COOH
b OH
HO' .@-o~
H
-
HO' -dPo H
20g-Cortolonic acid
.&
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
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)
Budesonide, (44)
16a-Hydroxyprednisolone,
16-butyrate ester, (45)
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
"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
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
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
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
(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
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.
"Ref. 153.
+, retention; -, excretion.
790 Anti-Inflammatory Steroids
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-
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)"
Inactive
Active
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
~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.
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
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
<OH
(163)
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
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
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 15.36 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
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
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
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
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.
Biological transformation
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
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
"Ref. 305.
bMcKenzieassay in human volunteers; BV = control.
'BV = betamethasone valerate (Sc).
(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
Mouse
Compound Z Y X R 16 Human Va AITb HPAc
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
Liver
___f
0
F (271) inactive
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).
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
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.
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-
"Ref. 332.
bDexamethasone = 100%.
'Progesterone = 100%.
842 Anti-Inflammatory Steroids
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-
"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
Compound
(328)
(329)
(330)
(331) (racemic)
(+)-(332)
(-14333)
(334)
(335)
(336)
Prednisolone
"Ref. 336.
C-5-aryl ring was undertaken. Compounds 10. G. K. McEvoy, AHFS Drug Information,
(334-336)established that GR activity could American Society o f Health-System Pharma-
be separated from unwanted PR, MR, AR, and cists, Bethesda, MD, 1995.
ER activities by simple modification to the 3'- 11. Y. Uoneda, D. Han, K. Ogita, a n d k Watanabe,
position. The candidate compounds (335)and Brain Res., 685, 105-116 (1995).
(336)were compared for functional repression 12. K. Iwasaki, E. Mishima, M. Miura, N. Sakai,
and activation vs. prednisolone in cotransfec- and S. Shimao, J. Dermatol. Sci., 10, 151-158
tion assays, with (336) appearing to be the (1995).
better lead. An oral dose of (336)was compa- 13. W. Rostene, A. Sarrieau, A. Nicot, V. Scarceri-
rable to prednisolone in a rat model of asthma. aux, C. Betancur, et al., J. Psychiatry Neuro-
sci., 20,349356 (1995).
Finally, the C-5 chiral center was examined in
a few cases, as shown in Table 15.57 for (+)- 14. G. Wang and A. T. Lim, Endocrinology, 137,
379-382 (1996).
332 and (-)-333.It was clear that the (-)-
enantiomer was far more potent, and the au- 15. H. Shibata, T. E. Spencer, S. A. Onate, G. Jen-
ster, S. Y. Tsai, et al., Recent Prog. Horm. Res.,
thors suggest that this stereocenter may have
52,141-165 (1997).
the S configuration on the basis of other sim-
16. P. Y. Sze and B. H. Yu, J. Steroid Biochem.
ilar nonsteroidal PR ligands.
Mol. Biol., 55, 185-192 (1995).
17. T. D. Long and R. G. Kathol, Ann. Clin. Psy-
chiatry, 5,259-270 (1993).
13 ACKNOWLEDGMENTS
18. K. M. Campbell
- and D. S. Schubert, Gen. Hosp.
Psychiatry, 13,270-272 (1991).
M.A.A. is grateful to Dr. Masato Tanabe, who
19. S. Teramoto and Y. Fukuchi, Am. J. Respir.
for many years freely offered general insights
Crit. Care Med., 153,879-880 (1996).
and specific discussions regarding steroid hor-
20. J. K. Saito, J. W. Davies, R. D. Wasnich, and
mone synthesis, SAR, biochemistry, clinical
P. D. Ross, Calcif. Tissue Znt., 57, 115-119
applications, and medical significance. (1996).
21. T. Olbricht and G. Benker, J. Intern. Med.,
234,237-244 (1993).
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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
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