Professional Documents
Culture Documents
CHEMISTRY AND
DRUG DISCOVERY
Sixth Edition
Volume 6: Nervous System Agents
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
CONTENTS
xiii
xiv Contents
INDEX, 895
CUMULATIVE INDEX, 923
BURGER'S
MEDICINAL CHEMISTRY
AND
DRUG DISCOVERY
CHAPTER ONE
Contents
1 Introduction, 2
2 Clinical Applications, 2
2.1 Current Drugs, 2
2.1.1 Applications of General Adrenergic
Agonists, 9
2.1.2 Applications of a,-Agonists, 12
2.1.3 Applications of a,-Agonists, 13
2.1.4 Applications of p-Agonists, 14
2.1.5 Applications of Antiadrenergics, 14
2.1.6 Applications of Nonselective a-
Antagonists, 15
2.1.7 Applications of Selective a,-
Antagonists, 15
2.1.8 Applications of p-Antagonists,16
2.1.9 Applications of alp-Antagonists, 16
2.1.10 Applications of Agonists/Antagonists,
16
2.2 Absorption, Distribution, Metabolism, and
Elimination, 16
2.2.1 Metabolism of Representative
Phenylethylamines, 16
2.2.2 Metabolism of Representative
Imidazolines and Guanidines, 18
2.2.3 Metabolism of Representative
Quinazolines, 19
2.2.4 Metabolism of Representative Aryl-
oxypropanolamines, 19
3 Physiology and Pharmacology, 21
3.1 Physiological Significance, 21
3.2 Biosynthesis, Storage, and Release
of Norepinephrine, 22
3.3 Effector Mechanisms
Burger's Medicinal Chemistry and Drug Discovery of Adrenergic Receptors, 25
Sixth Edition, Volume 6: Nervous System Agents 3.4 Characterization of Adrenergic
Edited by Donald J. Abraham Receptor Subtypes, 25
ISBN 0-471-27401-1 02003 John Wiley & Sons, Inc. 4 History, 26
1
Adrenergics and Adrenergic-Blocking Agents
"Not all trade names are listed, particularly for drugs no longer under patent.
bAU dose information from Drug Facts a n d Comparisons 2002(14).
"Not all doses and dosage forms are listed. For further information consult reference (14).
Table 1.2 Phenylethylamines (Structures 1-28)
R4
4' \
5'
Compound R1 R2 R3 R4 Receptor Activity"
(1) H H OH 3',4'-diOH a+@
(2) CH3 H OH 3',4'-diOH pza!
(3) H CH3 H H (a! + mb
(4) CH3 H OH 3',4'-di-02CC(CH3), (P 2 a!)"
(5) CH3 CH3 OH H (a! + P ) ~
(6) cH3 2,2-diCH3 OH H (a + PIb
m (7) H CH3 OH 3',4'-&OH a
(8) H CH3 OH 3'-OH a!
Y Q
P2
0CH3
(16) CH(CH3I2 CH2CH3 OH 3',4'-diOH P
(17) CH(CH& H OH 3',4'-&OH P
(18) C(CH& H OH 3',5'-diOH P2
(19) C(CH3I3 H OH 2'-aza, 3'-CH20H, 4'-OH PZ
(20) cH3 OH 4'-OH P2
OH
CH~
"Agonist activity unless indicated otherwise.
bIndirectactivity through release of norepinephrine and reuptake inhibition.
"Prodrug.
dMixeddirect and indirect activity.
'Norepinephrine biosynthesis inhibitor.
fNet sum of effects of enantiomers.
8 Adrenergics and Adrenergic-Blocking Agents
tic application; for example, p-blockers, a,- raises blood pressure, (1)is used to counteract
blockers, and a,-agonists are all employed to various hypotensive crises and as an adjunct
treat hypertension. treatment in cardiac arrest where its p-activ-
ity stimulates the heart. Although it also lacks
2.1.1 Applications of General Adrenergic oral activity because it is a catechol, epineph-
Agonists. The mixed a- and p-agonist norepi- rine (2) is far more widely used clinically than
nephrine (1)has limited clinical application (1).Epinephrine, like norepinephrine, is used
because of the nonselective nature of its action to treat hypotensive crises and, because of its
in stimulating the entire adrenergic system. greater p-activity, is used to stimulate the
In addition to nonselective activity, it is orally heart in cardiac arrest. When administered in-
inactive because of rapid first-pass metabo- travenously or by inhalation, epinephrine's
lism of the catechol hydroxyls by catechol-0- &activity makes it useful in relieving bron-
methyl-transferase (COMT) and must be ad- choconstriction in asthma. Because it has sig-
ministered intravenously. Rapid metabolism nificant a-activity, epinephrine is also used in
limits its duration of action to only 1 or 2 min, topical nasal decongestants. Constriction of
even when given by infusion. Because its a-ac- dilated blood vessels by a-agonists in mucous
tivity constricts blood vessels and thereby membranes shrinks the membranes and re-
10 Adrenergics and Adrenergic-Blocking Agents
duces nasal congestion. Dipivefrin (4) is a pro- mic mixture of R,R and S,S stereoisomers.
drug form of (2), in which the catechol hy- Ephedrine is a natural product isolated from
droxyls are esterified with pivalic acid. several species of ephedra plants, which were
Dipivefrin is used to treat open-angle glau- used for centuries in folk medicines in a vari-
coma through topical application to the eye ety of cultures worldwide (9). Ephedrine has
where the drug (4) is hydrolyzed to epineph- both direct activity on adrenoceptors and indi-
rine (2),which stimulates both a-and P-recep- rect activity, through causing release of nor-
tors, resulting in both decreased production epinephrine from adrenergic nerve terminals.
and increased outflow of aqueous humor, Ephedrine is widely used as a nonprescription
which in turn lowers intraocular pressure. bronchodilator. It has also been used as a va-
Amphetamine (3) is orally active and, sopressor and cardiac stimulant. Lacking phe-
through an indirect mechanism, causes a gen- nolic hydroxyls, ephedrine crosses the blood-
eral activation of the adrenergic nervous sys- brain barrier far better than does epinephrine.
tem. Unlike (1)and (2), amphetamine readily Because of its ability to penetrate the CNS,
crosses the blood-brain barrier to activate a ephedrine has been used as a stimulant and
number of adrenergic pathways in the central exhibits side effects related to its action in the
nervous system (CNS). Amphetamine's CNS brain such as insomnia, irritability, and anxi-
activity is the basis of its clinical utility in ety. It suppresses appetite and in high doses
treating attention-deficit disorder, narco- can cause euphoria or even hallucinations. In
lepsy, and use as an anorexiant. These thera- the United States the purified chemical ephed-
peutic areas are treated elsewhere in this rine is considered a drug and regulated by the
volume. FDA. However, the dried plant material ma
Ephedrine erythro-(5) and pseudoephed- huang is considered by law to be a dietary sup-
rine threo-(5) are diastereomers with ephed- plement, and not subject to FDA regulation.
rine, a racemic mixture of the R,S and S,R As a result there are a large number of ma
stereoisomers, and pseudoephedrine, a race- huang-containing herbal remedies and "nu-
2 Clinical Applications 11
Plt Pz
triceuticals" on the market whose active in- 2.1.2 Applications of a,-Agonists. All se-
gredient is the adrenergic agonist ephedrine. lective a,-agonists are vasoconstrictors, which
Pseudoephedrine, the threo diastereomer, has is the basis of their therapeutic activity. The
virtually no direct activity on adrenergic re- sole use of levonordefrin (7)is in formulations
ceptors but acts by causing the release of nor- with parenteral local anesthetics employed in
epinephrine from nerve terminals, which in dentistry. Vasoconstriction induced by the
turn constricts blood vessels. Although it too a-agonist activity of (7) helps retain the local
crosses the blood-brain barrier, pseudoephed- anesthetic near the site of injection and pro-
rine's lack of direct activity affords fewer CNS longs the duration of anesthetic activity. Met-
side effects than does ephedrine. Pseudo- araminol (8) and methoxamine (9) are both
ephedrine is widely used as a nasal deconges- parenteral vasopressors selective for a-recep-
tant and is an ingredient in many nonprescrip- tors and so have few cardiac stimulatory prop-
tion cold remedies. erties. Because they are not substrates for
Mephentermine (8) is another general ad- COMT, their duration of action is significantly
renergic agonist with both direct and indirect longer than that of norepinephrine, but their
activity. Mephentermine's therapeutic utility primary use is limited to treating hypotension
is as a parenteral vasopressor used to treat during surgery or shock. Methoxamine is also
hypotension induced by spinal anesthesia or used in treating supraventricular tachycardia.
other drugs. Midodrine (10) is an orally active glycine-
To%
2 Clinical Applications
H\\\'
0CH3
CH302C =-
OCH~
0CH3
0CH3
(61) N-N
H3C
amide prodrug, hydrolyzed in vivo to (63), an severe hypotension or shock but is much
analog of methoxamine, and a vasoconstrictor. more widely employed as a nonprescription
Midodrine is used to treat orthostatic hypo- nasal decongestant in both oral and topical
tension. preparations.
The imidazolines naphazoline (29), oxy-
metazoline (301,tetrahydozoline (31),and xy-
lometazoline (32) are all selective a,-agonists,
widely employed as vasoconstrictors in topical
nonprescription drugs for treating nasal con-
gestion or bloodshot eyes. Naphazoline and
oxymetazoline are employed in both nasal de-
congestants and ophthalmic preparations,
whereas tetrahydrozoline is currently mar-
keted only for ophthalmic use and xylometa-
zoline only as a nasal decongestant.
which oxidatively deaminates the side chain of shown. Any catechol-containing drug will also
either to form the same product DOPGAL likely be subject to metabolism by COMT.
(67), and for catechol-0-methyltransferase Ephedrine (51, a close structural analog of
(COMT), which methylates the 3'-phenolic (2), having no substituents on the phenyl ring,
OH of each to form (68). Metabolite (68) is is well absorbed after an oral dose and over
subsequently oxidized by MA0 to form alde- half the dose is eliminated unchanged in the
hyde (69), and aldehyde (68) may be methyl- urine. The remainder of the dose is largely
ated by COMT to also form (69). This alde- desmethylephedrine (72), deamination prod-
hyde may then be either oxidized by aldehyde uct (73), and small amounts of benzoic acid
dehydrogenase (AD) to (70) or reduced by al- and its conjugates (16). No aromatic ring-hy-
dehyde reductase to alcohol (71). Alternate droxylation products were detected. This is in
routes to (70) and (71) from (67) are also marked contrast to the case with amphet-
shown. Several of these metabolites are ex- amine (3), in which ring-hydroxylated prod-
creted in the urine as sulfate and glucuronide ucts are major metabolites.
conjugates (15).As previously mentioned, nei- Albuterol(13) is not subject to metabolism
ther (1)nor (2) is orally active because of ex- by COMT and is orally active but does have a
tensive first-pass metabolism by COMT, and 4'- OH group subject to conjugation. The ma-
both have short durations of action because of jor metabolite of albuterol (13) is the 4'-0-
rapid metabolic deactivation by the routes sulfate (74) (17). The sulfation reaction is ste-
HO \
Adrenergics and Adrenergic-Blocking Agents
CH20H
+ conjugates + Z-isomer of (36)
C1
20 Adrenergics and Adrenergic-Blocking Agents
3 Physiology and Pharmacology 21
A
OH 0/\f\cH20H
'OZH+ many conjugates
@ O"
/ / / /
(96) active
originally defined anatomically (38).Today of the axon near the junction with the effector
the terms adrenergic nervous system and sym- cell. The amino acid L-tyrosine(97)is actively
pathetic nervous system are generally used in- transported into the neuron cell (411,where
terchangeably. The sympathetic nervous sys- the cytoplasmic enzyme tyrosine hydroxylase
tem is a division of the autonomic nervous (tyrosine-3-monooxygenase) oxidizes the 3'-
system, which innervates organs such as the position to form the catechol-amino-acid L-
heart, lungs, blood vessels, glands, and smooth dopa (98) in the rate-limiting step in norepi-
muscle in various tissues and regulates func- . nephrine biosynthesis (42). L-Dopa is
tions not normally under voluntary control. decarboxylated to dopamine(99)by aromatic-
The effects of the sympathetic stimulation on L-amino acid decarboxylase, another cytoplas-
a few organs and tissues of particular rele- mic enzyme. Aromatic-L-aminoacid decarboxyl-
vance to current pharmaceutical interven- ase is more commonly known as dopa
tions are shown in Table 1.7 (39,40). Excellent decarboxylase. Doparnine is then taken up by
overviews of the adrenergic nervous system active transport into storage vesicles or granules
and its role in control of human physiology are located near the terminus of the adrenergic neu-
provided in Katzung (39) and Hoffman and ron. Within these vesicles, the enzyme dopa-
Palmer(40). mine P-hydroxylase stereospecifically intro-
duces a hydroxyl group in the R absolute
3.2 Biosynthesis, Storage, and Release
configuration on the carbon atom beta to the
of Norepinephrine
amino group to generate the neurotransmitter
Biosynthesis of norepinephrine takes place norepinephrine (1).Norepinephrine is stored in
within adrenergic neurons near the terminus the vesicles in a 4:l complex, with adenosine
3 Physiology and Pharmacology 23
triphosphate (ATP)in such quantities that each nephrine biosynthesis, release, and fate is
vesicle in a peripheral adrenergic neuron con- given in Fig. 1.1.After release, norepinephrine
tains between 6000 and 15,000 molecules of nor- diffuses through the intercellular space to
epinephrine (43). The pathway for epinephrine bind reversibly to adrenergic receptors (alpha
(2) biosynthesis in the adrenal medulla is the or beta) on the effector cell, triggering a bio-
same, with the additional step of conversion of chemical cascade that results in a physiologic
(1)to (2) by phenylethanolamine-N-methyl- response by the effector cell. In addition to the
transferase. receptors on effector cells, there are also adre-
Norepinephrine remains in the vesicles un- noreceptors that respond to norepinephrine
til it is released into the synapse during signal (a2-receptors) or epinephrine (P2-receptors)
transduction. A wave of depolarization reach- on the presynaptic neuron, which modulate
ing the terminus of an adrenergic neuron trig- the release of additional neurotransmitter
gers the transient opening of voltage-depen- into the synapse. Activation of presynaptic a,-
dent calcium channels, causing an influx of adrenoceptors by (1)inhibits the release of ad-
calcium ions. This influx of calcium ions trig- ditional (I),whereas stimulation of presynap-
gers fusion of the storage vesicles with the tic P,-adrenoceptors by (2) enhances the
neuronal cell membrane, spilling the norepi- release of (I),thus increasing overall sympa-
nephrine and other contents of the vesicles thetic activation. Removal of norepinephrine
into the synapse through exocytosis. A sum- from the synapse is accomplished by two
mary view of the events involved in norepi- mechanisms, reuptake and metabolism, to in-
Epinephrine
enhancement of
NE release
Storage vesicle
Dopamine-
depolarization
and Ca++influx
rn - biochemical
cascade
t 2 NE
I
Tyrosine
\\
recycling
Mitochondria1
DOPGAL, 67 4 MA0
t Uotake-1
I
I
Tyrosine metabolism
Figure 1.1. Diagram of synapse between an adrenergic neuron and its effector cell. NE, norepineph-
rine; aR, a-adrenoceptor; pR, P-adrenoceptor.
3 Physiology and Pharmacology
active compounds. The most important of ceptors, except that linkage through a G-pro-
these mechanisms is transmitter recycling tein (G,) leads to inhibition of adenylyl cyclase
through active transport uptake into the pre- instead of activation.
synaptic neuron. This process, called up- The a,-adrenoreceptor, on the other hand,
take-1, is efficient and, in some tissues, up to is linked through yet another G-protein to a
95% of released norepinephrine is removed complex series of events involving hydrolysis
from the synapse by this mechanism (44).Part of polyphosphatidylinositol (46). The first
of the norepinephrine taken into the presyn- event set in motion by activation of the a,-
aptic neuron by uptake-1 is metabolized by receptor is activation of the enzyme phospho-
MA0 through the same processes discussed lipase C, which catalyzes the hydrolysis of
earlier under norepinephrine metabolism, but phosphatidylinositol-4,5-biphosphate(PIP,).
most is sequestered in the storage vesicles to This hydrolysis yields two products, each of
be used again as neurotransmitter. This up- which has biologic activity as second messen-
take mechanism is not specific for (1)and a gers of the a,-receptor. These are 1,2-diacyl-
number of drugs are substrates for the uptake glycerol (DAG) and inositol-1,4,5-triphos-
mechanism and others inhibit reuptake, lead- phate (IP,). IP, causes the release of calcium
ing to increased adrenergic stimulation. A less ions from intracellular storage sites in the en-
efficient uptake process, uptakeS, operates in doplasmic reticulum, resulting in an increase
a variety of other cell types but only in the in free intracellular calcium levels. Increased
presence of high concentrations of norepi- free intracellular calcium is correlated with
nephrine. That portion of released norepi- smooth muscle contraction. DAG activates cy-
nephrine that escapes uptake-1 diffuses out of tosolic protein kinase C, which may induce
the synapse and is metabolized in extraneuro- slowly developing contractions of vascular
nal sites by COMT. MA0 present at extraneu- smooth muscle. The end result of a complex
ronal sites, principally the liver and blood series of protein interactions triggered by ag-
platelets, also metabolizes norepinephrine. onist binding to the a,-adrenoceptor includes
increased intracellular free calcium, which
3.3 Effector Mechanisms
leads to smooth muscle contraction. Because
of Adrenergic Receptors
smooth muscles of the wall of the peripheral
Adrenoceptors are proteins embedded in the vascular bed are innervated by a,-receptors,
cell membrane that are coupled through a G- stimulation leads to vascular constriction and
protein to effector mechanisms that translate
.
an increase in blood pressure.
conformational changes caused by activation 3.4 Characterization of Adrenergic
of the receptor into a biochemical event within
Receptor Subtypes
the cell. All of the P-adrenoceptors are coupled
through specific G-proteins (G,) to the activa- The discovery of subclasses of adrenergic re-
tion of adenylyl cyclase (45). When the recep- ceptors and the ability of relatively small mol-
tor is stimulated by an agonist, adenylyl cy- ecule drugs to stimulate differentially or block
clase is activated to catalyze conversion of these receptors represented a major advance
ATP to cyclic-adenosine monophosphate in several areas of pharmacotherapeutics. An
(CAMP),which diffuses through the cell for at excellent review of the development of adreno-
least short distances to modulate biochemical ceptor classifications is available in Hiebel et
events remote from the synaptic cleft. Modu- al. (47).
lation of biochemical events by CAMPincludes The adrenoceptors, both alpha and beta,
a phosphorylation cascade of other proteins. are members of a receptor superfamily of
CAMPis rapidly deactivated by hydrolysis of membrane-spanning proteins, including mus-
the phosphodiester bond by the enzyme phos- carine, serotonin, and dopamine receptors,
phodiesterase. The a,-receptor may use more that are coupled to intracellular GTP-binding
than one effector system, depending on the proteins (G-proteins), which determine the
location of the receptor; however, to date the cellular response to receptor activation (48).
-
best understood effector system of the a,-re- All G-protein-coupled receptors exhibit a
ceptor appears to be similar to that of the p-re- common motif of a single polypeptide chain
Adrenergics and Adrenergic-Blocking Agents
that is looped back and forth through the cell including the actual peptide sequence and
membrane seven times, with an extracellular length. Each of the adrenoceptors is encoded
N-terminus and intracellular C-terminus. on a distinct gene, and this information was
One of the most thoroughly studied of these considered crucial to the proof that each adre-
receptors is the human P,-adrenoreceptor noreceptor is indeed distinct, although re-
(49). The seven transmembrane domains, lated. The amino acids that make up the seven
TMD1-TMD7, are composed primarily of li- transmembrane regions are highly conserved
pophilic amino acids arranged in a-helices among the various adrenoreceptors, but the
connected by regions of hydrophilic amino ac- hydrophilic portions are quite variable. The
ids. The hydrophilic regions form loops on the largest differences occur in the third intracel-
intracellular and extracellular faces of the lular loop connecting TMD5 and TMD6, which
membrane. In all of the adrenoceptors the ag- is the site of linkage between the receptor and
onistlantagonist recognition site is located its associated G-protein. Sequences and bind-
within the membrane-bound portion of the re- ing specificities have been reported for numer-
ceptor. This binding site is within a pocket ous a- and P-adrenoceptor subtypes (47, 53-
formed by the membrane-spanning regions of 56). For purposes of drug design and
the peptide. All of the adrenoceptors are cou- therapeutic targeting, the most critical recep-
pled to their G-protein through reversible tors are the a,, on prostate smooth muscle,
binding interactions with the third intracellu- a,, on vascular smooth muscle and in the kid-
lar loop of the receptor protein. ney, a, in the CNS, P, in heart, P, in bronchial
Binding studies with selectively mutated smooth muscle, and p, in adipose tissue.
P,-receptors have provided strong evidence
for binding interactions between agonist func-
tional groups and specific residues in the 4 HISTORY
transmembrane domains of adrenoceptors
(50-52). Such studies indicate that Asp,,, in In 1895 Oliver and Schafer reported (57) that
transmembrane domain 3 (TMD3) of the P,- adrenal gland extracts caused vasoconstric-
receptor is the acidic residue that forms a tion and dramatic increases in blood pressure.
bond, presumably ionic or a salt bridge, with Shortly thereafter various preparations of
the positively charged amino group of cate- crude adrenal extracts were being marketed
cholamine agonists. An aspartic acid residue is largely to staunch bleeding from cuts and
also found in a comparable position in all of abrasions. In 1899 Abel reported (58) isolation
the other adrenoceptors as well as other of a partially purified sample of the active con-
known G-protein-coupled receptors that bind stituent (2), which he named epinephrine.
substrates having positively charged nitro- Shortly thereafter von Fiirth (59)employed an
gens in their structures. Elegant studies with alternative procedure to isolate another im-
mutated receptors and analogs of isoprotere- pure sample of (2), which he named suprare-
no1 demonstrated that Ser,,, and Ser,,, of nin, claiming it to be a different substance
TMD5 are the residues that form hydrogen than that isolated by Abel. The pure hormone
bonds with the catechol hydroxyls of &-ago- (2) was finally obtained in 1901 by both Taka-
nists. Furthermore, the evidence indicates mine (60) and Aldrich (61). Takamine gave (2)
that Ser,,, interacts with the meta hydroxyl yet a third name, adrenalin. Although the
group of the ligand, whereas Ser,,, interacts chemical structure was still not definitively
specifically with the para hydroxyl group. known, a pure preparation of (2) was first
Serine residues are found in corresponding po- marketed by Parke, Davis & Co. under the
sitions in the fifth transmembrane domain of trade name Adrenaline (62, 63). Adrenaline
the other known adrenoceptors. Evidence in- eventually became the generic name employed
dicates that the phenylalanine residue of outside the United States, whereas epineph-
TMD6 is also involved in ligand-receptor rine became the U.S. approved name. By 1903
bonding with the catechol ring. Structural dif- Pauly (64) had demonstrated that "adrena-
ferences exist among the various adrenocep- line" was levorotatory and proposed two pos-
tors with regard to their primary structure, sible structures consistent with the available
4 History
data. The structure of racemic (2) was conclu- chlorines, was discovered to be a P-antagonist
sively proved through nearly simultaneous that blocked the effects of sympathomimetic
synthesis by Stolz at Farbwerke Hoechst (65) amines on bronchodilation, uterine relax-
and Dakin at the University of Leeds (66), but ation, and heart stimulation (75). Although
it had only one half the activity of the natural DCI had no clinical utility, replacement of the
levorotatory isomer (67). The racemate was 3,4-dichlorosubstituents with a carbon bridge
resolved by Flacher in 1908 (68). to form a naphthylethanolamine derivative
The earliest major clinical application of (2) did afford a clinical candidate, pronethalol
was the report in 1900 (69) of the utility of (1011, introduced in 1962 only to be with-
injected adrenal extracts in treating asthma drawn in 1963 because of tumor induction in
attacks, followed in 1903 by a report (70)of the animal tests.
use of purified (2) for the same purpose. In-
jected epinephrine rapidly became the stan-
dard therapy for treatment of acute asthma
attacks. A nasal spray containing epinephrine
was available by 1911 and administration
through an inhaler was reported in 1929. Also,
early in the 1900s Hoechst employed the vaso-
constrictor properties of epinephrine to pro-
long the duration of action of their newly de-
veloped local anesthetic procaine (63).
It had been recognized early on (71) that
there were similarities between the effects of
administration adrenal gland- extracts and
stimulation of the sympathetic nervous sys-
tem. Elliot (72) suggested that adrenaline
might be released by sympathetic nerve stim-
ulation and over the years the term adrenergic
nerves became effectively synonymous with
sympathetic nerves. In 1910 Barger and Dale Shortly thereafter, a major innovation was
(73) reported a detailed structure-activity re- introduced when it was discovered that an
lationship study of epinephrine analogs and oxymethylene bridge, OCH,, could be intro-
introduced the term sympathomimetic for duced into the arylethanolamine structure of
chemicals that mimicked the effects of sympa- pronethalol to afford propranolol(57), an ary-
thetic nerve stimulation, but they also noted loxypropanolamine and the first clinically suc-
some important differences between the ef- cessful P-blocker.
fects of administered adrenaline and stimula- To clarify some of the puzzling differential
tion of sympathetic nerves. It was not until effects of sympathomimetic drugs on various
1946 that von Euler demonstrated that the tissues, in 1948 Ahlquist (76) introduced the
actual neurotransmitter released at the termi- concept of two distinct types of adrenergic re-
nus of sympathetic neurons was norepineph- ceptors as defined by their responses to (11,
rine (1)rather than epinephrine (2) (74). In (2), and (17), which he called alpha receptors
1947 compound (17), the N-isopropyl analog and beta receptors. Alpha receptors were de-
of (1)and (21, was reported to possess bron- fined as those that responded in rank order of
chodilating effects similar to those of (2) but agonist potency as (2) > (1)>> (17). Beta re-
lacking its dangerous pressor effects. In 1951 ceptors were defined as those responding in
(17) was introduced into clinical use as isopro- potency order of (17) > (2) > (1).Subse-
terenol (isoprenaline) and became the drug of quently, P-receptors were further divided into
choice for treating asthma for two decades. PI-receptors, located primarily in cardiac tis-
In the 1950s, dichloroisoprotereno1 (DCI, sue, and &-adrenoceptors, located in smooth
loo), a derivative of isoproterenol, in which muscle and other tissues, given that (1)and
the catechol hydroxyls had been replaced by (2) are approximately equipotent at cardiac
Adrenergics and Adrenergic-Blocking Agents
P-receptors, although (2) is 10 to 50 times ture of the other substituents determines re-
more potent than (1)at most smooth muscle ceptor selectivity and duration of action.
P-receptors (77). Alpha receptors were also
subdivided into a, (postsynaptic) and a, (pre- 5.1.1 R' Substitution on the Amino Nitrogen.
synaptic) adrenoceptors (78). Development of As R1 is increased in size from hydrogen in
selective agonists and antagonists for these norepinephrine to methyl in epinephrine to
various adrenoceptors has been thoroughly re- isopropyl in isoproterenol, activity at a-recep-
viewed in Ruff010 et al. (79). tors decreases and activity at P-receptors in-
creases. Activity at both a- and P-receptors is
maximal when R1 is methyl as in epinephrine,
5 STRUCTURE-ACTIVITY RELATIONSHIPS
but a-agonist activity is dramatically de-
creased when R1 is larger than methyl and is
Comprehensive reviews of the structure-activ-
negligible when R1 is isopropyl as in (17), leav-
ity relationships (SAR) of agonists and antag-
ing only P-activity. Presumably, the P-recep-
onists of a-adrenoceptors (80) and P-adreno-
tor has a large lipophilic binding pocket adja-
ceptors (81) are available, which thoroughly
cent to the amine-binding aspartic acid
cover developments through the late 1980s.
residue, which is absent in the a-receptor. As
Only summaries of these structure-activity re-
R1 becomes larger than butyl, affinity for a,-
lationships are provided here.
receptors returns, but not intrinsic activity,
which means large lipophilic groups can afford
5.1 Phenylethylamine Agonists
compounds with a,-blocking activity [e.g.,
The structures of the phenylethylamine ad- tamsulosin (24) and labetalol (26)l. Tarnsulo-
renergic agonists were summarized in Table sin (24) is more selective for a,,, the a,-adre-
1.2. Agents of this type have been extensively noceptor subtype found in the prostate gland,
over those found in vascular tissue. In addi-
tion, the N-substituent can also provide selec-
tivity for different P-receptors, with a t-butyl
group affording selectivity for &-receptors.
For example, with all other features of the
molecules being constant, (66) [the active
metabolite of prodrug bitolterol (1411 is a se-
lective P,-agonist, whereas (17) is a general
P-agonist. When considering its use as a bron-
studied over the years since the discovery of chodilator, it must be recognized that a gen-
the naturally occurring prototypes, epineph- eral P-agonist such as (17) has undesirable
rine and norepinephrine, and the structural cardiac stimulatory properties (because of its
requirements, and tolerances for substitu- &-activity) that are greatly diminished in a
tions at each of the indicated positions have selective P,-agonist.
been well established and reviewed (79,82).In
5.1.2 RZ Substitution a to the Basic Nitro-
general, a primary or secondary aliphatic
gen, Carbon-2. Small alkyl groups, methyl or
amine separated by two carbons from a substi-
ethyl, may be present on the carbon adjacent
tuted benzene ring is minimally required for to the amino nitrogen. Such substitution
high agonist activity in this class. Tertiary or slows metabolism by MA0 but has little over-
quaternary amines have little activity. Be- all effect on duration of action of catechols be-
cause of the basic amino groups, pK, values cause they remain substrates for COMT. Re-
range from about 8.5 to 10, and all of these sistance to MA0 activity is more important
agents are highly positively charged at physi- in noncatechol indirect-acting phenylethyl-
ologic pH. Most agents in this class have a amines. An ethyl group in this position dimin-
hydroxyl group on C-1 of the side chain, P to ishes a-activity far more than P-activity, and is
the amine, as in epinephrine and norepineph- present in isoetharine (16). Substitution on
rine. Given these features in common, the na- this carbon introduces an asymmetric center,
5 Structure-Activity Relationships
producing pairs of diastereomers when an OH substituent does not affect the ability of the
group is present on C-1. These stereoisomers drug to bind to the a,-receptor but does affect
can have significantly different biologic and the ability of the molecule to activate the re-
chemical properties. For example, maximal ceptor; that is, the stereochemistry of the
direct activity in the stereoisomers of a-meth- methyl group affects intrinsic activity but not
ylnorepinephrine resides in the erythro ste- affinity. Because both stereoisomers are
reoisomer (65), with the (1R,2S) absolute con- p-agonists, with the (+)-isomer about 10
figuration (83), which is the active metabolite times as potent as the (-)-isomer, the net ef-
of the prodrug methyldopa (12) (84). The con- fect is p-stimulation. Dobutamine is used as a
figuration of C-2 has a great influence on re- cardiac stimulant after surgery or congestive
ceptor binding because the (1R,2R) diaste- heart failure. As a catechol, dobutamine is
reomer of a-methylnorepinephrine has readily metabolized by COMT and has a short
primarily indirect activity, even though the ab- duration of action with no oral activity.
solute configuration of the hydroxyl-bearing C-1
is the same as that in norepinephrine. In 5.1.4 R4 Substitution on the Aromatic
addition, with respect to a-activity, this addi- Ring. The natural 3',4'-dihydroxy substituted
tional methyl group also makes the direct-acting benzene ring present in norepinephrine pro-
(1R,2S) isomer of a-methylnorepinephrine se- vides excellent receptor activity for both a-
lective for a,-adrenoceptors over a,-adrenocep- and p-sites, but such catechol-containing com-
tors, affording the central antihypertensive pounds have poor oral bioavailability and
properties of methyldopa. short durations of action, even when adminis-
tered intravenously, because they are rapidly
5.1.3 R3 Substitution on Carbon-1. In the metabolized by COMT. Alternative substitu-
phenylethyamine series, a hydroxyl group at tions have been found that retain good activity
this position in the R absolute configuration is but are more resistant to COMT metabolism.
preferred for maximum direct agonist activity For example, 3'3'-dihydroxy compounds are
on both a- and p-adrenoceptors. If a hydroxyl not good substrates for COMT and, in addi-
is present in the S absolute configuration, the tion, provide selectivity for p,-receptors.
activity is generally the same as that of the Thus, because of its ring-substitution pattern,
corresponding chemical with no substituent. metaproterenol (18) is an orally active bron-
This is the basis for the well-known Easson- chodilator having little of the cardiac stimula-
Stedman hypothesis of three-point attachment tory properties possessed by isoproterenol
of phenylethanolamines to adrencoceptors (17).
through stereospecific bonding interactions Other substitutions are possible that en-
with the basic amine, hydroxyl group, and ar- hance oral activity and provide selective p,-
omatic substituents (85). A comprehesive and activity, such as the 3'-hydroxymethyl, 4'-hy-
excellent review of the stereochemistry of ad- droxy substitution pattern of albuterol (131,
renergic drug-receptor interactions was writ- which is also not a substrate for COMT. A re-
ten by Ruff010 (86). cently developed selective p,-agonist with an
An example of a phenethylamine agonist extended duration of action is salmeterol(21),
lacking an OH group on C-1 is dobutamine which has the same phenyl ring substitution
(27), which has activity on both a- and p-re- R4 as that of (13)but an unusually long and
ceptors but, because of some unusual proper- lipophilic group R1 on the nitrogen. The octa-
ties of the c h i d center on R1, the bulky nitro- noltwater partition coefficient log P for salme-
gen substituent, the overall pharmacologic terol is 3.88 vs. 0.66 for albuterol and the du-
response is that of a selective PI-agonist (87). ration of action of salmeterol is 12 vs. 4 h for
he (-)-isomerof dobutamine is an a,-agonist albuterol (88).
. . There is substantial evidence
and vasopressor. The (+)-isomer is a n a,- that the extended duration of action is attrib-
antagonist; thus, when the racemate is used uted to a specific binding interaction of the
clinically, the a-effects of the enantiomers ef- extended lipophilic side chain with a specific
fectively cancel, leaving the p-effects to pre- region of the P,-receptor, affording salmeterol
dominate. The stereochemistry of the methyl a unique binding mechanism (89). The long
Adrenergics and Adrenergic-Blocking Agents
ter soluble (94), as would be expected, given Labetalol(26) and carvedilol(59) have un-
tetrahydrofuran's greater water solubility usual activity, in that they are antihyperten-
than that of furan. sives with a,-, PI-, and Pz-blockingactivity. In
terms of SAR, you will recall from the earlier
5.1.7 Aryloxypropanolamines. In general, discussion of phenylethanolamine agonists
the aryloxypropanolamines are more potent that, although groups such as isopropyl and
P-blockers than the corresponding aryletha- t-butyl eliminated a-receptor activity, still
nolamines, and most of the p-blockerscurrently larger groups could bring back a,-affinity but
used clinically are aryloxypropanolamines. not intrinsic activity. Thus these two drugs
Beta-blockers have found wide use in treating have structural features permitting binding to
hypertension and certain types of glaucoma. both the a,- and both P-receptors. The
At approximately this same time, a new se- P-blocking activity of labetalol is approxi-
ries of Csubstituted phenyloxypropanolo- mately 1.5 times that of its a-blocking activity.
lamines emerged, such as practolol, which The more recently developed carvedilol has an
selectively inhibited sympathetic cardiac stim- estimated P-blocking activity 10 to 100 times
ulation. These observations led to the recogni- its a-blocking activity.
tion that not all preceptors were the same, A physicochemical parameter that has clin-
which led to the introduction of P, and P, no- ical correlation is relative lipophilicity of dif-
menclature to differentiate cardiac P-recep- ferent agents. Propranolol is by far the most
tors from others. lipophilic of the available P-blockers and en-
6 Recent Developments
ters the CNS far better than less lipophilic on efforts to discover new selective a,,-antag-
agents, such as atenolol or nadolol. Lipophilic- onists for treatment of prostatic hypertrophy
ity as measured by octanollwater partitioning and to develop selective P,-agonists for use in
also correlates with primary site of clearance. treating obesity and type 2 diabetes.
The more lipophilic drugs are primarily
cleared by the liver, whereas the more hydro- 6.1 Selective a!,,-Adrenoceptor Antagonists
philic agents are cleared by the kidney. This
The successful application of tamsulosin (24)
could have an influence on choice of agents in
to the treatment of BPH with minimal cardio-
cases of renal failure or liver disease (27).
vascular effects has led to an extensive effort
to develop additional antagonists selective for
6 RECENT DEVELOPMENTS the a,,-receptor. Phenoxyethylamine (102,
KMD-3213), a tamsulosin analog, has been re-
Recently, major research efforts in develop- ported to be in clinical trial in Japan, as has
ment of adrenergic drugs have focused largely (103) (95).
Adrenergics and Adrenergic-Blocking Agents
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CHAPTER TWO
Cholinergics
JOSEPH G. CANNON
The University of Iowa
Iowa City, Iowa
Contents
1 Introduction, 40
2 Cholinergic (Acetylcholine) Receptors, 41
3 Acetylcholine and Analogs, 43
3.1 Variations of the Quaternary
Ammonium Group, 43
3.2 Variations of the Acyl Group, 45
3.3 Variations of the Ethylene Bridge, 46
3.4 Substitution of the Ester Group
by Other Groups, 47
4 Cholinergics Not Closely Related Structurally to
Acetylcholine, 49
4.1 Nicotine, Its Analogs and Congeners, and
Other Nicotinic Receptor Stimulants, 49
4.2 Muscarine, Muscarone, and Related
Compounds, 57
4.3 Pilocarpine and Analogs and Congeners, 61 .
4.4 Arecoline and Analogs and Congeners, 62
4.5 Oxotremorine and Analogs and Congeners,
71
4.6 Miscellaneous, Structurally Unique
Muscarinic Agonists, 78
5 Conformation-Activity Relationships of Some
Cholinergic Agonists, 80
6 Anticholinesterases, 84
6.1 Quaternary Ammonium Reversible
Inhibitors, 85
6.2 Reversible, Noncovalent Inhibitors Related
to 1,2,3,4-Tetrahydro-
9-Aminoacridine, 86
6.3 Carbamate-Derived Inhibitors, 88
6.4 Phosphorus-Derived Inhibitors, 90
6.5 Miscellaneous Inhibitors, 93
7 Acetylcholine-Release Modulators, 97
ceptors are present in the cerebellum, heart, have a similar pentameric structure, but they
smooth muscle, and at some potassium chan- vary considerably in the nature of the subunit
nels (15); M, receptors are found in secretory combinations (29).
. .
glands and smooth muscle. M, receptors are At least some neuronal nicotinic subunits
located in the basal forebrain and the stria- are homologous to those found in muscle.
tum. M, receptors are found in the substantia These have been designated as a if they con-
nigra. MI, M,, and M, receptors modulate tain vicinal cysteine residues analogous to
some potassium, chloride, and calcium chan- Cys192-Cys193of the Torpedo receptor, or P if
nels, and M, and M, receptors modulate some they do not. Stimulation of these receptors
calcium channels (19). All of these ion channel produces depolarization (a result of cation
effects are indirect and complex. The central channel opening) and firing of the postgangli-
nervous system contains all known subtypes onic neuron. Nine homologous nicotinic recep-
of muscarinic receptors (18).Both muscarinic tor subunits have been identified thus far in
M, (20) and nicotinic (21) receptors decrease mammalian nervous systems (30). Two main
in numbers with the progression of Alzhei- categories of nicotinic receptor pentamers
mer's syndrome, whereas the numbers of have been identified in the brain, based on
M, receptors do not decrease (22). Alzheimer their high affinity for either nicotine or for the
patients also show reduced activity of acetyl- nicotinic receptor blocker a-bungarotoxin.
cholinesterase; of high affinity, sodium-depen- The former are considered to be formed by a4-
dent choline uptake; and of choline acetyl- and P2-subunit receptors, and the latter are
transferase (23). Contemporary and projected thought to be a7 or a7* nicotinic receptors
therapeutic roles for muscarinic receptors in (30). Nicotinic receptor glycoprotein has been
the central nervous system have been re- isolated and extensively " studied (31-33). Re-
viewed (24). views of the classification, recommended no-
Muscarinic receptors are glycoproteins menclature, and function of nicotinic receptor
with molecular weights of approximately subtypes are available (29,30,34-36).
80,000. They are located on the outer surface A family of presynaptic ion channel-type
of the cell membrane, and they are of the G- acetylcholine receptors in the brain modulates
protein-linked type; M,, M,, and M, receptors the release of acetylcholine, dopamine, and
couple with G, proteins to stimulate phospho- other neurotransmitters implicated in learn-
lipase-C, whereas M, and M, receptors couple ing and memory processes (37). There is con-
with Gi proteins to inhibit adenylate cyclase vincing evidence to implicate a deficit in nico-
(25). The molecular basis of muscarinic recep- tinic receptors in the symptomatology of
tor function has been reviewed (26). Nordvall Alzheimer's disease. Furthermore, the neuro-
and Hacksell (27) proposed a molecular model toxin P-amyloid attenuates nicotine-induced
of the transmembrane domains of the M, re- release of acetylcholine and dopamine (37).
ceptor, to explain the three-dimensional inter- Although acetylcholine is optically inac-
action of the receptor with its ligands. How- tive, its in vivo receptors exhibit discrimina-
ever, the authors specified that the model is tion between enantiomers of synthetic and
"primarily of qualitative value". naturally occurring cholinergic stimulants.
Postsynaptic nicotinic receptors in the pe- Both central and peripheral muscarinic recep-
ripheral nervous system are designated as N, tors are highly stereospecific; peripheral nico-
(in autonomic ganglia) and N, (at myoneural tinic receptors seem to be less so, although
junctions). Nicotinic receptors are of the ion these usually show preference for one or the
channel type. They are pentameric proteins other member of enantiomeric pairs. Central
that are composed of one, two, or more distinct nicotinic receptors frequently demonstrate a
subunits, each of which contains multiple higher degree of stereoselective binding char-
membrane-spanning regions, and the individ- acter than is noted with the peripheral recep-
ual subunits surround an internal channel tors. Understanding of nicotinic receptor ste-
(28). Nicotinic receptors are highly heteroge- reoselectivity and specificity is complicated by
neous, and subcategorization has been diffi- the liklihood that, as was mentioned previ-
cult; those in neuronal tissue are believed to ously, at some in vivo sites, some nicotinic
3 Acetylcholine and Analogs
stimulant agents function indirectly by pro- these older studies were performed before the
moting presynaptic release of acetylcholine. exquisite heterogeneity of both muscarinic
and nicotinic receptors was recognized; thus,
3 ACETYLCHOLINE AND ANALOGS many compounds were classified merely as
"muscarinic" or "nicotinic." The observed ef-
Acetylcholine has virtually no clinical uses. Its fectiveness of a cholinergic agent in producing
rapid rate of hydrolysis in the gastrointestinal a biological response depends, inter alia, on its
tract precludes oral administration, and a sim- inherent potency and intrinsic activity as well
ilarly rapid hydrolysis by esterases in the as on the rate at which it is metabolically in-
blood and by acetylcholinesterase in the ner- activated and/or excreted. Frequently, these
vous tissue limits its usefulness. individual factors were not separately and in-
The need for therapeutically satisfactory dividually assessed. This problem has been
cholinergic agents coupled with the simple cited (40) with regard to the lack of consis-
and easily synthesized structures necessary tency among laboratories in the methods used
for cholinergic activity have stimulated prep- to determine cholinergic receptor subtype se-
aration and study of a great number of analogs lectivity. Therefore, in the following discus-
of acetylcholine. The following structural vari- sion of the relationship of chemical structure
ations have been addressed: to cholinergic activity, frequently only gener-
alized (and tentative) conclusions can be
1. Alteration of the quaternary ammonium made, and these may have been based on a
head. composite of the cholinergic activities for
2. Replacement of the acetyl group by other which the compound was tested.
acyl moieties.
3.1 Variations of the Quaternary
3. Alteration of the ethylene bridge connect- Ammonium Croup
ing the quaternary ammonium and the es-
ter groups. Two types of alterations of the quaternary
4. Substitution of another group for, or elim- head have been studied: replacement of the
ination of, the ester moiety. nitrogen by other atoms and replacement of
the N-methyl groups by hydrogen, alkyl, ni-
trogen, or oxygen. Acetyl phosphonocholine
The "five atom rule," first suggested by
( 5 ) (391, acetylarsenocholine (6) (391, and ace-
Alles and Knoefel (38) and stated more for-
mally by Ing (39), proposes that, for maximum
muscarinic activity, there should be attached
to the quaternary nitrogen atom, in addition
to three methyl groups, a fourth group with a
chain of five atoms, as illustrated for acetyl-
choline: C-C-0-C-C-N. This empirical obser-
vation has been found to be valid for a large
number of molecules, regardless of the precise tylsulfonocholine (7) (41) exhibit muscarinic
nature of the five atoms involved. effects, but they are considerably less potent
Studies of a large number of compounds than acetylcholine.
have supplied considerable information on Ing (39) noted that the potencies of acetyl-
structural requirements for cholinergic activ- choline analogs containing charged atoms
ity; however (especiallyin the older literature) other than nitrogen (phosphorus, arsenic, sul-
these data must be interpreted with caution. fur) are in inverse order to the volumes occu-
They have been obtained using a variety of in pied by these atoms. The carbon isostere (8) of
vivo and in vitro testing procedures and bio- acetylcholine exhibits no cholinergic activity,
logical preparations in a variety of animal spe- but it is an excellent substrate for acetylcho-
cies, and different biological responses associ- linesterase (42). Studies of the role of nitrogen
ated with stimulation of the cholinergic substituents in the acetylcholine molecule in-
nervous system were measured. Additionally, dicate that the N,N,N-trimethyl quaternary
Cholinergics
Number R Reference
HCO
BrCH2C0
C2H5C0
N-methyl,N,N-diethylcholine,and it is decid-
H,NCH2C0 edly more potent than the diethylcholine es-
n-C3H7C0 ter.
i-C3H7C0 However, in general, incorporation of the
n-C4H,C0 choline nitrogen into a heterocyclic ring mark-
C6H5C0 edly lowers the potency compared with acetyl-
C6H5CH2C0 choline (47,48).
C6H5CH=CHC0 The report (49) that the tertiary amine (12)
(C$,)&XOH)CO is a full and nonselective muscarinic agonist
CH,(CH2),,CO
CH3(CH2),4C0
HOCH,CO
CHflHCO
CH,COCO
CH,CHOHCO
O2N
H2NC0
(CH,O),PO
thoxyl permits retention of some cholinergic 3.2 Variations of the Acyl Croup
effects, and in certain compounds, nicotinic or
Qualitatively, choline has the same pharmaco-
muscarinic activities are enhanced over the
parent N,N,N-trimethyl system (53). logical actions as acetylcholine, but it is far
The reverse N-alkoxy systems (17) demon- less active at most sites (58). However, choline
strated only extremely weak muscarinic activ- has been reported (34) to be a full agonist at
one nicotinic receptor subtype, and at some
other nicotinic subtypes it can act as a partial
agonist or a coagonist.
Acetylation of the alcohol function of cho-
line greatly increases the potency. However,
(17) R = CH3 or NH2 formylcholine is less potent than acetylcho-
line, and homologation of the acetate methyl
-
R', R = combinations of H, CH3
group of acetylcholine generally produces
compounds that are much less potent than
ity (54). These compounds violate the five acetylcholine (see Table 2.1). Polar groups
atom rule. such as OH (Table 2.1,17) and NH, (4) mark-
Amine oxide analogs of the cholinergic ago- edly decrease muscarinic potency, but a
nists (18-21) exhibit little or no cholinergic group (16) permits retention of considerable
effect, and they are not substrates for cho- activity. Bromoacetylcholine (number 2) is a
linesterases (55). muscarinic and nicotinic agonist (59) and, un-
der reducing conditions, it binds covalently to
nicotinic receptors but not to muscarinic re-
ceptors (60).
Acrylylcholine (Table 2.1, 151, which has
been isolated from tissues of a marine gastro-
pod (72), has relatively high cholinergic activ-
ity. Higher fatty acid esters (12 and 13) were
prepared for testing as hemolytic agents, but
apparently they have never been evaluated for
cholinergic activity. A study of acetylcholine
congeners derived from relatively high molec-
ular weight acids (73), most of which con-
The observed biological effects of several tained a benzene ring, revealed that as the
variations of the quaternary head of acetyl- molecular weight of the acid increases para-
choline and its congeners may be rationalized sympathetic stimulant activity dimninishes,
by invoking results of molecular orbital calcu- and there is a gradual change to atropine-like
lations (56), which indicate that in both mus- (muscarinic blocking) activity.
carine and acetylcholine the nitrogen atom is In general, carbamic acid esters of choline
nearly neutral and a large part (70%) of the and its congeners are more potent and more
formal charge is distributed among the three toxic than the corresponding acetates. Car-
attached methyl groups, which form a large bamyl choline (19 in Table 2.1) is a potent
ball of spreading positive charge. muscarinic agent, and it demonstrates pro-
Furthermore, Kimura and coworkers (57) nounced nicotinic stimulant effects a t auto-
determined that chain extension of one alkyl nomic ganglia. It is likely that these ganglionic
group of the tetramethylammonium cation actions are due, at least in part, to release of
produces a great decrease in the charge den- endogenous acetylcholine from the terminals
sity on the nitrogen, and they proposed that of cholinergic fibers (74).
cholinergic agonist activity for a quaternary Carbamyl choline is is a poor substrate for
ammonium compound requires a minimum acetylcholinesterase and nonspecific cholines-
level of charge density on the nitrogen. terases (74). The nitrate ester (18, Table 2.1)
Cholinergics
has marked nicotinic and muscarinic agonist 3.3 Variations of the Ethylene Bridge
effects, and it also displays an intense paralyz- The distance between the ester moiety and the
ing nicotine action. The dimethylphosphate
cationic head of acetylcholine seems to be crit-
ester (20,Table 2.1) has powerful nicotinic ac- ical. Acetoxytrimethylammonium (27), com-
tion but little muscarinic effect.
The "reversed ester" congener (22)of ace-
tylcholine exhibits weak muscarinic and no
permits retention of potent nicotinic effects. It was concluded that the nicotinic activity
p-Methyl substitution (compounds 37 and 39) of choline phenyl ether and of choline o-tolyl
permits retention of some degree of musca- ether is a reflection of the ability of the mole-
rinic effect, but nicotinic effects are com- cule to assume a "planar" conformation when
pletely abolished (92). A series of open-chain interacting with the ganglionic nicotinic re-
congeners of muscarine, typified by structure ceptor. In contrast, the inactive 2,6-xylylether
(41), exhibited low muscarinic potency, which of choline cannot assume this planar disposi-
tion. Evaluation of additional conformation-
ally restricted aryl choline ethers (44-47) re-
vealed that only the piperidine derivative (47)
is a ganglionic stimulant (98).
However, the alkaloid is nonselective in its ac- positions 5 or 6 in the 3-pyridyl fragment are
tions on nicotinic receptors, including the gan- potent nicotinic receptor ligands (126).
glionic (a3&) and neuromuscular (a1P18y~)
subtypes, as a consequence of which there is a
very narrow therapeutic index between bene-
ficial analgesic actions and toxic and ulti-
mately lethal actions on the cardiorespiratory
system. Replacement of the chlorine atom of
(+)-epibatidinewith hydrogen resulted in re-
tention of comparable affinity for nicotinic
sites, whereas replacement with methyl or io-
dine lowered affinity (118).
ABT-594 (721, described as an azetidine
bioisostere of nicotine with the (Rbabsolute
H-C
II
C-H
Beckett and coworkers (147) reported the (C4 = R ) is more than 100 times more potent
approximate equivalence of muscarinic action than the D-(-) enantiomer (C4 = S), and is
shown by enantiomers of 4,5-dehydromusca- approximately six times more potent than ace-
rone (104). tylcholine in a guinea pig ileum assay. The
more active L-(+) compound is related config-
urationally to the most potent muscarine ste-
reoisomer (3),although it should be noted that
several authors in the older literature incor-
rectly assigned the S absolute configuration to
position 4 of the L-(+)-cis compound (1071,
apparently through misapplication of priority
rules. 2,2-Dialkyl analogs of these dioxolanes
are much weaker muscarinic agonists than
dl-Dehydromuscarine (105)retains consid- the parent systems (107), and the difference
erable muscarinic agonist activity, but it in potency between the C4 R and S enan-
shows no effects at nicotinic receptors (141). tiomers diminishes sharply with increasing
Incorporation of the elements of the musca- size of substituents at C2 (150). Both enantio-
rine structure into an aromatic ring has pro- mers of the cis- and trans-oxathiolane system
duced some systems, such as compound (1061, (lOS), bioisosteres of the dioxolanes, were
which approach acetylcholine in muscarinic evaluated for nicotinic and muscarinic effects
potency (41). Activity is lowered by changing (151).
4 Cholinergics Not Closely Related Structurally to Acetylcholine 59
0
II
C-OR
(136)of dihydroarecoline is approximately 1% I
as potent as arecoline in this assay (175). The
sulfur bioisostere (137)of arecoline (R = CHJ
is more potent and active than its N,N-di-
methyl quaternary ammonium congener, be-
ing approximately equipotent to arecoline it-
self (176).The ester group positional isomer of
the sulfur bioisostere (138)(R = CH,) retains
muscarinic effects, but it is somewhat less po-
tent and less active than the &substituted
compound (137). In both (137) and (138)the
ethyl and n-propyl esters are inferior to the It was proposed that the amidine moiety of
methyl. the tetrahydropyrimidine ring would be a suit-
able ammonium bioisostere, lacking the per-
manent cationic head present in a quaternary
ammonium system. This might facilitate pen-
etration of the blood-brain barrier. Of this se-
ries, the methyl ester (139a)shows high a n -
ity for muscarinic receptors in rat brain, and it
stimulates phosphoinositide metabolism in
the rat hippocampus. It ameliorated memory
deficits associated with lesions in the septohip-
pocampal cholinergic system in rats. In a sub-
sequent communication (178) it was reported
that compounds (139b), (139e), (1401, and
(141) exhibited marked functional selectivity
for MI vs. M, receptors.
The potential utility of arecoline in produc-
ing significant cognitive improvement in Alz-
heimer patients (179) and in enhancing learn-
ing in normal young humans and in aged
nonhuman primates (180, 181) is largely ne-
gated by its short duration of action, which has
Further application of the bioisosterism been ascribed (182) to rapid in vivo hydrolysis
strategy replaced the tetrahydropyridine ring of the ester group. This metabolic lability
of arecoline with tetrahydropyrimidine stimulated preparation of metabolically stable
(139a-e) (177). aldoxime derivatives (142) (183); compounds
Cholinergics
N-OR'
II
N-OR'
II
E Rotamer Z Rotamer
4 Cholinergics Not Closely Related Structurally to Acetylcholine 67
and also included some branched chain C, al- antinociceptive effects from undesirable mus-
kyl groups, demonstrated high potency in dis- carinic side effects (salivation, tremors).
placing tritiated oxotremorine-M (a nonse- These efforts were unsuccessful. As a result of
lective muscarinic agonist) and tritiated a concurrent study of rigid, conformationally
pirenzepine (a selective M, antagonist) from restricted analogs of (156), it was proposed
rat brain membrane tissue. The n-butyloxy that the biologically active conformation of
and n-pentyloxy substituents provided maxi- (156) has a torsion angle T C3-C4-C3'-N2'
mal pharmacological effects. A subsequent "close to 180°."
communication (191) reported that a deriva- Study of a series of arecoline congeners
tive of (155) in which R = n-hexyl (xanome- (158)in which the carbomethoxy group is re-
line) is an M, agonist with potential value in placed by a pyrazine moiety (R = CH,-
treatment of Alzheimer's syndrome. The pri- C,H,,) revealed that M, agonist activity is re-
mary site of oxidative metabolism of xanome- lated to chain length, with n-hexyl providing
line is the hexyloxy side chain (192), and a maximum activity (195). A comparison of MI
secondary metabolic process involves N-de- agonist efficacy of these pyrazines and related
methylation. To prevent this oxidative N-de- 1,2,5-thiadiazoles (155)and 1,2,5-oxadiazoles
methylation, the N-methyl group was incorpo- (154) suggested that M, efficacy may be re-
rated into a series of azabicyclic and tricyclic lated to the magnitude of electrostatic poten-
systems, illustrated by (159)and (160). tial located over the nitrogens of the respec-
Some members of the series exhibited se- tive heterocycles. The heteroatom directly
lectivity for M, receptor binding. The alkyl- attached to the 3 position of the pyrazine or of
thio analogs (156) demonstrated a similar the 1,2,5-thiadiazole markedly influences the
structure-activity relationship to the alkoxy M, efficacy of the compounds by determining
series (155); however, the thio ethers have the energetically favorable conformers for ro-
higher receptor affinity and higher potency. tation about the bond connecting the tetrahy-
Thus, these systems (155) and (156) show a dropyridyl ring and the heterocycle. A three-
higher degree of selectivity for M, receptors dimensional model for the M, agonist
than for M,. The unsubstituted system (157) pharmacophore was proposed as a result of
(R = H) is a potent but nonselective musca- these studies.
rinic agonist. Derivatives of (1571, where R =
n-propyl, n-pentyl, n-heptyl, or n-octyl, have
10 to 100 times less affinity for central musca-
rink receptors than the corresponding alkoxy
and alkylthio derivatives.
&T OR'
'2-
(1) 1.74 1.48
Q 0
0.001
(4)
(5)
(6)
(7)
06
CH,CON(CH,)-
(CH,),NCON(CH,)-
(CH3)2NCOO-
CH3COCH2-
0.87
0.03
0.15
2.10
1.15
0.04
0.004
0.005
(8) CH,COO- 0.47 0.005
4 Cholinergics Not Closely Related Structurally to Ac
I" R
N-CH2-C-C-CH2-N
(l83a) R = R' = H
(183b) R = H; R' = CH3
,yR'
b N
.-+ R'
N-CH2-CrC-CH2-R
CH3
(190a) R = N(CH3)2
(190b) R = N(CH3)3
+
a
CH3-C-N-CH-C=C-
CH3
I
the Qbutyne chain; and variation of the C4 ter- to form a six- or seven-membered ring pre-
tiary amino group or quaternary group (230). served affinity for muscarinic receptor(s), but
Replacement of the acetyl group or the N- abolished efficacy (232).
methyl group in (191) and its analogs by a In a series of oxotremorine and oxotremo-
methansulfonyl group abolishes efficacy and n e (196) analogs (197, 198), all com-
decreases affinity at guinea pig ileal receptors. pounds studied (tertiary and quaternary
The trifluoroacetamide analogs of (191) also amines) demonstrated analgesic effects in
exhibited diminished affinity and efficacy. neurogenic and inflammatory pain models
Substitution of an acetyl group for the (233). It was concluded that these compounds
N-methyl group of (191) decreases efficacy, were acting as muscarinic analgesics. The an-
but has little effect on affinity for the recep- tinociceptive effects were blocked by atropine
tor(~). Most of the tertiary amines showed cen- but not by naloxone or mecamylamine. Mem-
tral antimuscarinic effects. Bioisosteres of bers of the series varied as to classic musca-
(191)bearing a urea moiety (194) in which the rinic agonist effects (e.g., salivation).
R, R', and R"groups were combinations of CH,
and H, displayed muscarinic agonism, partial
agonism, or antagonism (218); a structure-ac-
tivity relationship is not apparent in this se-
ries.
3-5 in Table 2.4) and the pyrrolidinium prod- of compound (203) (239). The (5')-enan-
uct of the 4-halobutylamine moiety of com- tiomers of (204) and (205) exhibit low eudis-
pound (6) in Table 2.4 should be much less mic ratios (1.5 and 4.9, respectively). Com-
susceptible to nucleophilic attack in vivo, and pound (205) has been described (240)as one of
hence these quaternary systems, unlike the the most potent MI-selective agonists (sic)
aziridinium moiety, should have little or no known.
tendency to bond covalently with the musca- Dimethylsulfonium (206a and 206b) and
rinic receptor(s) to produce blockade. Central thiolanium (206c and 206d) analogs of ox-
muscarinic effects (tremors, analgesia) of this otremorine demonstrate higher affinities for
series of compounds correlated well with the peripheral muscarinic receptors than the cor-
k, and t,,, data. The slow cyclization rate of responding trimethylammonium and N-meth-
compound (3)and the extremely rapid cycliza- ylpyrrolidinium compounds (241).
tion rate of compound (6) were reflected in
weak or no CNS-related activities. As might be
predicted, compound (3) showed relatively
weak peripheral muscarinic activity (saliva-
tion) whereas compound (6)was potent. Com-
pounds (4) and (5)were cited as meriting fur-
ther study.
McN-A-343 (203) is a selective M, agonist (206a) X = CH2; Y = +S(CH&
and it has been reported to exhibit antinoci- (206b) X = CO; Y = +S(CH&
ceptive effects (238). It was suggested that (206c) X = CH2; Y = c+SC4H8
postsynaptic M, receptors are involved in an- (206d) X = CO; Y = c+SC4H8
tinociception, and that presynaptic M, recep-
tors may also be involved, on the basis that
they participate in modulation of acetylcho- However, the sulfur compounds have lower
line release. intrinsic activities than their nitrogen ana-
Of a series of congeners of compound (2031, logs. The sulfur compounds also demonstrate
(2)-204 and (2)-205 were concluded to be potent affinity for rat cerebrocortical tissue in
muscarinic partial agonists, showing five- and a (-)-[3H]-N-methylscopolaminedisplace-
16-fold higher potency, respectively, than that ment assay. Sulfonium congeners (207) bear-
Cholinergics
5 CONFORMATION-ACTIVITY
RELATIONSHIPS OF SOME
CHOLINERCIC ACONISTS
+
N(CH3)3
0-C-CH3
II
0
(231) All stereoisomers of (221) and (233) are
feeble nicotinics. X-ray analysis of (+)-trans-
bilize the 1,2-trans-diaxial geometry did not ACTM (221) (282) established the 7, angle as
lead to greatly increased muscarinic effect 137" (which is within the anticlinal range),
and because of the rigidity of the cyclopropane
The cis- and trans-cyclopentane systems ring, this value probably closely approaches
(232)have been described (256)as feeble spas- the solution conformation. The (IS, 2s) abso-
mogenics with a T~ angle near anticlinal. lute configuration of (+)-trans-ACTM super-
imposes on the equivalent centers in the po-
tent muscarinic agonists (S)-(+)-acetyl-p-
methylcholine (31) (see Table 2.2) and
(2S,4R,5S)-( + )-muscarine (3) (283). A race-
mic cyclobutane analog (234) of trans-ACTM
is much less potent than (2)-trans-ACTM
0-C-CH3 (284).
II
0
(232)
ACTM (221): r, = 180"; 7, = +73" to +1370; has increased greatly over the past several
r3 = 180 + 3 9 ; r4 = 180' or -1370. Inter- years, as a result of recognition of the poten-
atomic distances were defined as: N+-0' = tial value in therapy of Alzheimer's disease as
360; N+-C6= 450; Nt-C7 = 540 pm. Low po- well as of other defects in memory and learn-
tency or inactivity of certain acetylcholine de- ing. Giacobini (286) presented data confirm-
rivatives was attributed to deviation from one ing that a steady-state increase in acetylcho-
or more of these parameters. However, Casy line resulting from cholinesterase (sic)
(256) cited examples of deviations from these inhibition in the brain results in improvement
values in which high agonist activity is mani- of cognitive function and mild-to-moderate
fested. cases of Alzheimer's syndrome. Sussman and
It has not been possible to demonstrate un- coworkers (287) showed that the catalytic site
equivocally that acetylcholine assumes differ- of acetylcholinesterase is located at the bot-
ent conformations for interaction at nicotinic tom of a deep and narrow gorge surrounded by
and muscarinic receptors and/or at the sub- 14 aromatic amino acids. Moreover, these
populations of each major receptor subtype; workers presented evidence that the quater-
neither has this theory been disproved by the nary ammonium moiety of acetylcholine does
body of chemical and biological data. It must not interact with an anionic site on acetylcho-
be concluded that the relationship of acetyl- linesterase, but rather it binds with the ~ e l e c -
choline's molecular geometry to its physiolog- trons of Trp-84 (tryptophan). QSAR studies
ical roles is still not understood. (288) of a series of nicotine analogs and conge-
ners indicated that the cationic moieties of
various nicotinic receptor ligands interact
with aromatic groups on a,& and a, nicotinic
Symptoms resulting from an inadequate sup- receptors to an extent proportional to their
ply of acetylcholine may be relieved by block- receptor binding coefficients.
ing the body's acetylcholine-deactivating Figure 2.1 is a simplified representation of
mechanism. Interest in this category of agents the catalytic region of acetylcholinesterase
Figure 2.1.
6 Anticholinesterases
(289).Significant features are the Trp-84 aro- some or all of the methyl groups on the tetra-
matic center that anchors the quaternary methylammonium molecule tends to increase
head of the substrate; a serine residue, the potency (292). However, attempts to correlate
primary alcohol moiety of which participates biological test data with the calculated diame-
in a transesterification reaction with acetyl- ter of the unhydrated quaternary head led to
choline, resulting in acetylation of the en- inconclusive results.
zyme; and an imidazole ring (part of a histi- Belleau (293) calculated entropies and en-
dine residue) that, as a neighboring group, thalpies of binding to acetylcholinesterase for
participates in and facilitates the acetyl group a homologous series of alkyltrimethylammo-
transfer. The resulting acetylated serine moi- nium compounds RN+(CH,),, where R = CH,
ety is extremely labile and rapidly undergoes through n-C,,H,,. The observed relative po-
spontaneous hydrolytic cleavage to liberate tencies in the series were rationalized on the
acetate anion and to regenerate the active cat- basis of hydrophobic bonding phenomena cou-
alytic surface. pled with the ability of the alkyl chains to dis-
Taylor (290) described three classes of ace- place ordered water from the acetylcholines-
tylcholinesterase inhibitors, based on their terase surface.
mechanism of action: Edrophonium (236) combines a quater-
nary head for binding to the complimentary
1. Reversible inhibitors. site of the enzyme with a phenolic OH, which
2. Agents having a carbamate ester moiety presumably hydrogen bonds to a portion of the
that is hydrolyzed by acetylcholinesterase, esteratic area.
but much more slowly than acetylcholine.
3. Phosphoric acid- or phosphonic acid-de-
rived inhibitors, which are true hemisub-
strates for acetylcholinesterase.
reported by Fulton and Mogey (295) and by tion. Compound (240) is a somewhat less po-
Cavallito and Sandy (296), who noted that tent acetylcholinesterase inhibitor in vitro
there is a gradual increase in antiacetylcho- than THA. However, the two compounds are
linesterase activity as the chain joining the approximately equipotent in reversal of sco-
two quaternary heads increases. The optimum polamine-induced memory impairment in
connecting chain length was stated to be five mice, a putative predictive model of activity in
or six carbons. In addition, enzyme inhibitory Alzheimer's disease. These data suggest that,
activity was maximal in those molecules in in addition to acetylcholinesterase inhibition,
which the substituent(s) on the quaternary ni- there may be other biochemical components to
trogen~were decidedly lipophilic. Later stud- the mechanism of action of (240). This specu-
ies (297) demonstrated efficient bonding of
decamethonium (238) to Torpedo acetylcho-
linesterase, contradicting earlier (296) reports.
Comparison of calculated desolvation free (310) to have memory enhancing effects in pa-
energies with IC,, values suggested the im- tients with Alzheimer's disease. However, for
portance of ligand hydrophobicity (low desol- clinical use it has a relatively short half- life,
vation free energy) for effective cation-n variable bioavailability, a low therapeutic in-
interaction of the homodimer (245) with pe- dex, and a multiplicity of unwanted cholin-
ripheral site(s). ergically related side effects.
(+)-Physostigmine, the enantiomer of the
6.3 Carbarnate-Derived Inhibitors naturally occurring alkaloid, has little effect
on acetylcholinesterase in vitro (311,312);it is
The prototype carbarnate-derived acetylcho-
linesterase inhibitor is physostigmine (247),
a weak centrally acting cholinergic agonist.
Eseroline (248), the ester cleavage product of
an alkaloid isolated from the seeds of the Cala-
bar bean, Physostigma venenosum. Physostig-
mine exhibits equal inhibitory activity against
acetylcholinesterase and butyrylcholinester-
ase (308).
wide for this purpose. The reaction between potency of the organophosphorus compounds
acetylcholinesterase and most organophos- parallels the ease of nucleophilic attack on the
phorus inhibitors has been presumed to occur phosphorus atom. Compounds in this category
only at the esteratic site of the enzyme, and include ester and amide derivatives of or-
the reaction here is a transesterification, com- thophosphoryl halides, pyrophosphate esters
parable to that involving the carbamate esters and amides, alkyl and aryl phosphonic acid de-
and acetylcholine itself. The reaction at the rivatives, and thiophosphoric acid derivatives.
esteratic site of acetylcholinesterase is en- Representative compounds are shown in Ta-
hanced by the geometry of the tetrahedral ble 2.5.
phosphates, which resemble the transition Holmsted (294, 325) and Hayes (326) have
state for acetate ester hydrolysis. The result- tabulated and discussed a large number of or-
ing serine-phosphorylated or -phosphonylated ganophosphorus acetylcholinesterase inhibi-
enzyme is extremely stable; if the R groups tors. Tabun, sarin, and soman (2, 3, and 4 in
(structure 259) are methyl or ethyl, regenera- Table 2.5) are among the most toxic "nerve
gases" known. OMPA (6) is inert as such, but
it is metabolized to an N-oxide derivative that
is the biologically active entity (327). Para:
thion (8)is inactive in inhibition of acetylcho-
linesterase in vitro; mixed-function oxidases
(in human liver) convert parathion into its ox-
I I ygen bioisostere paraoxon (9), the pharmaco-
serine residue
logically active metabolite (328). Echothio-
phate (7) is representative of inhibitors that
bind initially to the agonist cation binding site
tion of the enzyme by hydrolytic cleavage re- of acetvlcholinesterase
" as well as to the ester-
quires several hours. atic area. This compound is used clinically in
If the R groups are isopropyl, essentially no the treatment of certain types of glaucoma.
hydrolysis occurs and reestablishment of en- The 1,3,2-dioxaphosphorinane(260) is rep-
zyme activity can occur only after de novo resentative of organophosphorus acetylcho-
synthesis of the enzyme. A characteristic
structural feature of the anticholinesterase
phosphorus compounds is the grouping P-Z,
where Z is an electronegative moiety, a good
leaving group, and the cleavage of the P-Z
bond is accompanied by liberation of a large
amount of energy. The P-Z bond is eminently
susceptible to attack by nucleophiles, such as
the serine OH of the esteratic site of acetylcho-
linesterase. In general, the enzyme inhibitory
92 Cholinergics
Sarin (GB),isopropyl
methylphosphonofluoridate
Octamethylpyrophosphoramide
6 Anticholinesterases 93
S Parathion
Paraoxon
zene ring position isomers of the carbamate value in treatment of Alzheimer's syndrome
moiety were much less potent. The two enan- was suggested. Donepezil(276) is a reversible,
tiomers of (272) exhibited approximately the noncompetitive inhibitor of acetylcholinester-
same potency as the racemic material. ase (344). Its affinity for this enzyme is ap-
Of an extensive series of l-benzyl-442- proximately 1250 times greater than that for
(N-benzoylarnino)ethyl]piperidine derivatives, butyrylcholinesterase. The compound pro-
designed and evaluated as inhibitors of acetyl- -
duced a marked increase in the acetylcholine
cholinesterase, the sulfone derivative (273) content of rat; cerebral cortex. Introduction of
was the most potent (342). a fluorine atom at the 2-position of the in-
This compound showed an 18,000-fold danone ring of (276) resulted in a compound
preference for acetylcholinesterase over bu- with increased potency (345). Additionally,
tyrylcholinesterase. It was a reversible inhibi- there was a significant difference in the anti-
tor in a concentration-dependent manner. cholinesterase activity between the enantio-
Benzisoxazole derivatives (274) and (275) mers of 2-fluoro-(276) compared to that of the
showed threefold inhibitory selectivity for ace- nonfluorinated molecule. Docking simulations
tylcholinesterase over butyrylcholinesterase of fluorodonepezil with acetylcholinesterase
(343). have been reported (346). A "hypothetical
Compound (274) produced dose-dependent binding site" for (276) a t the acetylcholines-
elevation of acetylcholine in mouse forebrain terase catalytic region was proposed (344). Of
after oral administration. Possible palliative a series of aminopyridazines, (277) was the
7 Acetylcholine-Release Modulators
(27%
R1 = n- and branched chain alkyl, phenyl
Rz, R3 = H, F, CH3
dine) (280); which enhances potassium- This newer compound is effective both in
evoked acetylcholine release in rat cortex, vivo and in vitro. Its mechanism of acetylcho-
hippocampus, and caudate nucleus, in vitro line release was not established, but it was
(353,354). suggested that the blockade of certain K'
This enhancement of acetylcholine release channels may be involved.
from nerve terminals occurs only when the Compound (282) stimulates the release of
release has been triggered (355). Compound acetylcholine (and also of dopamine and nor-
(280) is reported (355) to exert significant ef- epinephrine) in brain regions involved in
fects on the human central nervous system.
Dopamine and serotonin release are also en-
hanced by this agent, but release of glutamate,
GABA, and norepinephrine is unaffected. Fur-
ther structure-activity studies of 3,3-disubsti-
tuted oxindoles have been reported (3561, and
the potential utility of this category of com-
pounds in treatment of cognitive and neuro-
logical deficiencies was stressed. Compound
(281) was reported to be superior to (280) as
an acetylcholine-releasing agent (23).
memory and learning (37). This compound
was resolved, and it was reported that neither
the (R)nor the (5')enantiomer alone was as
active as the racemate in any of a series of
whole animal behavioral experiments.
One of the possible advantages to a thera-
peutic strategy for Alzheimer's disease or
other deficits in memory and learning using
acetylcholine-releasing agents is that such a
process would seem to permit stimulation of
both nicotinic and muscarinic receptors in the.
brain and to permit stimulation of all sub-
groups of acetylcholine receptors.
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279. D. J. Triggle, Chemical Aspects of the Auto- K. Tachiki, and A. Kling, N. Engl. J. Med., 315,
nomic Nervous System, Academic Press, Lon- 1241-1245 (1986).
don, 1965, p. 83. 301. P. Taylor in Ref. 28, p. 174.
280. P. D. Armstrong, J. G. Cannon, and J. P. Long, 302. G. M. Shutske, F. A. Pierrat, M. L. Cornfeldt,
Nature, 220, 65-66 (1968). M. R. Szewczak, F. P. Huger, G. M. Bores, V.
281. C. Y. Chiou, J. P. Long, J. G. Cannon, and P. D. Haroutunian, and E. L. Davis, J. Med. Chem.,
Armstrong, J. Pharmacol. Exp. Ther., 166, 31, 1278-1279 (1988).
243-248 (1969). 303. M. T. McKenna, G. R. Proctor, L. C. Young,
282. C. Chothia and P. Pauling, Nature, 226, 541- and A. L. Harvey, J. Med. Chem., 40, 3516-
542 (1970). 3523 (1997).
References
Anticholinergic Drugs
B.V. RAMA SASTRY
School of Medicine
Vanderbilt University
Nashville, Tennessee
Contents
1 Introduction, 110
1.1Types and Selectivity of Antispasmodics, 111
1.2 Gastric Secretion, Peptic Ulcer, and
Anticholinergics as Antiulcer Agents, 111
1.3 Anticholinergics as Mydriatics
and Cycloplegics, 114
1.4 Anticholinergic Drugs in Prernedication
during Anesthesia, 115
1.5 Anticholinergic Activity as a Side Effect of
Drugs and Anticholinergic Syndrome, 115
1.6 Classification of Anticholinergic Agents
Based on Subtypes of Muscarinic Receptors,
115
2 Biocomparative Assay of Anticholinergics, 116
2.1 Antispasmodic Activity, 116
2.2 Antiulcer Activity, 118
2.3 Mydriatic and Cycloplegic Activities, 119
2.4 Miscellaneous Anticholinergic Activities, 120
3 Solanaceous Alkaloids, 120
3.1 History, 120
3.2 Chemical Structure, 120
3.3 Preparative Methods, 122
3.4 Molecular Factors in the Absorption, Fate,
and Excretion of Atropine and
Related Compounds, 122
3.5 Semisynthetic Derivatives
of Solanaceous Alkaloids, 124
4 Synthetic Anticholinergics, 126
4.1 Analogs of Atropine, 126
4.2 Receptor-Subtype-Selective Anticholinergics,
128
4.2.1 Tricyclic Benzodiazepines, 128
4.2.2 Benzothiazepines, 129
4.2.3 Quinuclidine-Based Antagonists, 129
4.2.4 Polymethylene Tetramines, 131
4.2.5 Indene Derivatives, 131
Burger's Medicinal Chemistry and Drug Discovery 4.2.6 Sila-difenidols, 132'
Sixth Edition, Volume 6: Nervous System Agents 4.2.7 Diphenylacetyloxy Derivatives, 132
Edited by Donald J. Abraham 4.2.8 Himbacine, 133
ISBN 0-471-27401-1 O 2003 John Wiley & Sons, Inc. 5 Structure-Activity Relationships, 133
109
Anticholinergic Drugs
by acid is instantaneous at pH 2.0. In humans, vous component, the intestinal phase includes
gastric juice contains hydrocholoric acid dur- humoral stimulation of secretion by unknown
ing the period of interdigestive secretion as agents. Gastrin released from the small intes-
well as during the period of digestive secre- tine may be involved. The response to what-
tion. Although the mechanisms of interdiges- ever humoral agent comes from the intestine
tive secretion are not known, they depend is greatly increased when subthreshold doses
partly on the tonic activity of the vagus. The of cholinergic drugs are given.
gastric secretory activity during the period of A number of humoral inhibitors of gastric
digestive secretion may be divided into three secretion arise in the small intestine. They are
phases, cephalic, gastric, and intestinal. Each termed enterogastrones. An enterogastrone is
phase is named to denote the region in which present in the jejunum and duodenal mucosa.
the stimuli act to induce gastric secretion. It is released in the presence of fat and inhibits
In the cephalic phase the stimuli are initi- gastric secretion and motility. The hormone
ated in the central nervous system. The stim- secretin, which stimulates pancreatic secre-
uli are the sight, smell, taste, and thought of tion, is an enterogastrone. It is produced in the
food, which act through conditioned and un- proximal duodenum and inhibits gastric se-
conditioned reflexes. The final efferent path is cretion in the presence of acids. Cholecystoki-
the vagus nerve. The impulses in the vagus nin, which is the same as pancreozymin, and
nerve stimulate the secretory cells in the gas- gastrin share the same terminal tetrapeptide.
tric glands. Acetylcholine, which is released Given alone, cholecystokinin is only a mild
from the postganglionic nerve endings, exerts stimulant of gastric acid secretion. It is a com-
a direct action on the secretory cells. Adminis- petitive inhibitor of the receptor for gastrin,
tration of atropine abolishes this phase. The which is a powerful stimulant of gastric acid
secretion is high in acid and pepsinogens, and secretion. Therefore, in the presence of gas-
its concentration of mucus is lower than that trin, cholecystokinin decreases the total out-
of the basal secretion; mucus output rises put of acid. Glucagon (and possibly enteroglu-
8-10 times as the volume increases. cagon) reduces the gastrin-induced acid
The gastric phase of secretion begins copi- secretion by noncompetitive inhibition of the
ously as soon as the food enters the stomach, receptors to gastrin. A gastric inhibitory
and it may continue 3-4 h, with a total volume polypeptide (GIP) that is present in duodenal
of 600 mL or more of strongly acid juice con- mucosa inhibits both histamine- and gastrin-
tain ng a high concentration of pepsinogens. induced acid secretion. A vasoactive intestinal
The gastric phase of secretion is caused by lo- peptide (VIP), which has been isolated from
cal and vagal responses to distension and by small intestinal mucosa, inhibits histamine-
the hormone gastrin, released by the musosa induced acid secretion. GIP and VIP are two
of the pyloric gland area. The local nerves of possible enterogastrones whose significance
the pyloric area are confined to the mucosa has yet to be established.
and are cholinergic. Irrigation of the pyloric Histamine, the exact role of which is not
gland area with acetylcholine releases gastrin, clearly understood, stimulates secretion of
and this liberation of gastrin is abolished by gastric juice that is rich in hydrochloric acid.
atropinization. There is a synergism between Recently, histamine receptors have been di-
gastrin and acetylcholine at the target cells; vided into three types, HI, H2, and H3. Stim-
the effect of injected gastrin on both acid and ulation of H2 receptors by histamine results in
pepsinogen secretion is increased two- to increased gastric acid secretion. H2 receptor
eightfold by subthreshold parasympathomec- antagonists (burinamide, metiamide, cimeti-
tic stimuli, and it is strongly inhibited by atro- dine) inhibit histamine-induced gastric acid
pinization. secretion in both humans and animals. In
The intestinal phase that begins when humans, H2 antagonists inhibit not only his-
chyme passes from the stomach to intestine, tamine- but also pentagastrin (a synthetic an-
contributes about 10% of the total response to alog of gastrin)-stimulated gastric acid secre-
a test meal. Protein and its digestion products, tion. This suggests that, at least in humans,
milk, dilute alcohol, and acid itself are effec- gastrin acts partially by histamine. Blockage
tive stimulants. Although there may be a ner- of acetylcholine receptors by atropine and his-
1 Introduction
Anticholinergics HPreceptor
(ACh) antagonists (H)
-Vagus nv
er, CP I Histamine
t
Antrum - Gastrin -
(-) Local hormones:
prostaglandins (G, H)
PGE, , PGA,
.- ,
Small
mtestine
Gastrin - (+) Parietal cell
- Mucus secretants
(carbenoxolone)
Enterogastrones 4 \ I
Secretin (G) I
~ntero~luca~on (G)
Cholicystokinin (G)
GIP (G, H)
VIP (H)
Fat (G, H)
Pepsinogen - Pepsin Pepsin inhibitors
(sulfated polysaccharides)
Figure 3.1. Interactions among neuronal and hormonal factors and pharmacological agents during
cephalic (Cp),gastric (Gp),and intestinal (Ip) phases of gastric acid secretion by parietal cell. ACh,
acetylcholine; GIP, gastric inhibitory peptide; VIP, vasoactive intestinal peptide; MI, muscarinic
receptor; HZ,histamine HZ receptor; GR, gastrin receptor; (+), stimulation of acid secretion; (-), ,
inhibition of acid secretion. In parentheses, next to the inhibitory agents, is indicated the blocked
stimulant agent (ACh, acetylcholine; H, histamine; G, gastrin).
influence on the gastric wall itself. The extent completely for many hours (6). Its effect on
of this insult is determined by the number of secretion during the feeding of milk and cream
acid- and pepsinogen-producingcells, their ir- is significant (7, 8). However, anticholinergic
ritability, and/or the magnitude of the stimuli drugs do not effectively perform a "medical
that reach them. These stimuli are partly ner- vagotomy" and they do not effectively reduce
vous (vagal) and partly hormonal (gastrin, gastric acidity to the extent of achlorhydria
corticosteroids). when patients are fed (8, 9). The effective an-
The healthy stomach does not digest itself. ticholinergic agent as an antiulcer drug should
Counteracting the aggression are defensive be selective for the subtype of muscarinic re-
factors such as buffering and dilution by food, ceptors localized on the secretory cells of gas-
inhibition of the secretion of gastric juice, and tric glands as well as mucosa of the pyloric
drainage of gastric contents. In addition, how- gland area. Anticholinergics selective for mus-
ever, the local condition of the mucosa (the carinic receptors of M1 subtype are useful in
mucosal resistance) is also of importance. decreasing gastric acid secretion.
Some of the determinants of mucosal resis-
tance are the mucous barrier, the local circu-
1.3 Anticholinergics as Mydriatics
lation, and the healing capacity of the mucosa.
and Cycloplegics
A peptic ulcer forms when the insult is more
powerful than the defense. In the case of duo- The size of the pupil is determined by the bal-
denal ulcers, the powerful irritation is often ance of forces exerted by the dilator muscles
the important factor; in gastric ulcers it is the fibers (sympathetically innervated and radi-
insufficient defense. ally arranged) and the constrictor muscle fi-
The ideal agent for the treatment of the bers (parasympathetically innervated and cir-
peptic ulcer would be one that selectively inac- cularly arranged) of the iris. Normally both
tivates pepsin or inhibits the output of hydro- sets of muscle fibers have a constant degree of
chloric acid so as to maintain the pH of the tonus and act reciprocally to dilate or constrict
gastric contents at about 4.5 for long periods the pupil. Any substance that paralyzes the
after its oral ingestion. It should produce no, constrictor muscle fibers (parasympatholytic)
or only minimal, side effects, induce no toler- allows the unopposed tone of dilator muscle
ance, and be inexpensive. It should be effective fibers to widen the pupil.
during all periods and phases of gastric secre- Acetylcholine is the transmitter between
tion and prevent the formation of ulcers. the constrictor muscle fibers and the parasym-
Atropine-like anticholinergics do not sat- pathetic nerve that innervates them. There-
isfy all requirements of an antiulcer agent. fore, acetylcholine and its congeners stimulate
They block acetylcholine action at the neu- the constrictor muscle fibers of the iris and
roeffector junction of the vagus. They give re- constrict the pupil. Atropine and related com-
lief to patients with a peptic ulcer by their an- pounds paralyze the constrictor muscle fibers
tisecretory and antispasmodic effects. They and cause widening or dilatation of the pupil.
decrease the basal hydrochloric acid and pep- The ciliary muscle is innervated by the
sin secretion, thereby allowing the healing of parasympathetic nerve, and acts to decrease
ulcers. The antispasmodic effects of atropine- the tone on the supporting muscle fibers of the
like agents are as consistent as their antisecre- lens, and thus increases the accommodative
tory effects. Motor activity is closely related to power of the eye. Acetylcholine and its conge-
ulcer pain, and the pain-relieving action of an- ners constrict the ciliary muscle fibers, and
ticholinergic agents seems to be related to atropine and related compounds paralyze the
their effect on depressing motor activity (anti- ciliary muscle.
spasmodic effect). Mydriatics are drugs that dilate the pupil,
An "effective" atropine-like anticholinergic but have minimal effect on the ciliary muscle
drug is capable of favorably influencing the and thus on accommodation. Cycloplegics are
excessive gastric secretion under certain con- drugs that partially or completely paralyze ac-
ditions. It exerts a significant effect on acid commodation. Most of the anticholinergics
secretion during the basal and interdigestive have both properties to varying degrees. For
night secretion to the point of abolishing it mydriatics other than anticholinergics and for
1 introduction 115
drugs that constrict the pupil (miotics), the properties that disturb patient recovery be-
appropriate chapter should be consulted. cause of their anticholinergic effects on the
Mydriatics and cycloplegics are special CNS (11).These effects are termed central an-
types of antispasmodics. In clinical practice ticholinergic syndrome (CAS) and are dis-
mydriasis is produced by local instillation of cussed in different chapters on centrally act-
the chosen drug into the conjunctival sac. This ing drugs (volume 4). These effects can be
enables one to produce the desired effects on reversed with physostigrnine, the centrally ac-
the eye with minimal systemic effects. How- tive cholinesterase inhibitor, which has a spar-
ever, such compounds should possess proper- ing effect on acetylcholine molecules at mus-
ties that allow them to penetrate the cornea in carinic receptor sites. An increased number of
effective concentrations. There are no signifi- acetylcholine molecules displace the mole-
cant differences between the muscarinic re- cules of anticholinergic drug from the musca-
ceptors of the guinea pig ileum or the rabbit rinic receptor sites.
iris, as judged by the binding characteristics of
potent anticholinergic agents. Muscarinic re- 1.6 Classification of Anticholinergic Agents
ceptors in both tissues are possibly of the M3 Based on Subtypes of Muscarinic Receptors
subtype. If anticholinergic drugs are available Acetylcholine produces its parasyrnpathomi-
that are selective for muscarinic receptors on metic effects by binding at cholinergic recep-
constrictor muscles and ciliary muscles, myd- tors of the muscarinic type. The classical anti-
riatic and cycloplegic effects can be produced cholinergic agent, atropine, binds to the same
by different drugs. muscarinic receptors and prevents acetylcho-
line from binding to these receptors and elic-
1.4 Anticholinergic Drugs in premedication iting muscarinic responses. Based on modern
during Anesthesia developments in the design of relatively sensi-
Prevention of some undesirable side effects tive antagonists for muscarinic receptors in
during anesthesia has been considered a func- different tissues (12-15), muscarinic receptors
tion of premedication with anticholinergic have been subdivided into three (possibly five)
drugs. For example, atropine is a popular an- subtypes M1 to M5 (Table 3.1). All muscarinic
ticholinergic agent that has been used for its receptors are glycoproteins of molecular
antisialogogic, antibradicardia, and anti- weight of 80,000 and have seven membrane-
emetic effects (10). The emphasis of using an- spanning regions. All of the receptors have a
ticholinergic drugs during premedication has slow response time (100-250 ms) and are cou-
been changing over the years because of the pled to G-proteins (13, 14). They act directly
availability of inhalational anesthetic agents on ion channels or are linked to second-mes-
that are better than ether. An anticholinergic senger systems, attenuation CAMPformation
agent (e.g., atropine), although no longer re- (16, 17), and formation of inositol triphos-
garded as an essential premedicant under all phate and diglyceride (16, 18). The final effect
circumstances, does have specific applications of activation of these receptors can be to open
for injured patients and children. Atropine or close K' channels, Ca2+ channels, or C1-
(0.6 mg, i.v.1 blocks the muscarinic actions of channels. These multiple channel activities
suxamethonium (succinylcholine), bradycar- lead to either depolarization or hyperpolariza-
dia, and salivation during crash induction of tion of the cell membrane. The final responses
anesthesia in an injured patient (10). Admin- are either excitatory or inhibitory. Atropine
istration of an anticholinergic drug to prevent blocks all of these activities and does not dis-
bradycardia in children in response to suxam- tinguish subtypes. Selective muscarinic ago-
ethonium or tracheal intubation is desirable. nists and antagonists that will distinguish dif-
ferent subtvpes are needed. Further, it will be
" a
containing water and attached to a kymo- ligation and the stomach contents are col-
graphic recording system in the fistula and re- lected for examination. The stomach is opened
cording the pressure waves and their alter- along the greater curvature and the ulcers are
ations by the action of drugs; or (3) placing a examined and scored by a suitable scheme
French catheter in the aboral end of the fis- such as 0 = normal, 1 = scattered hemorrhagic
tula, connecting it to a suitable recording sys- spots, 2 = deeper hemorrhagic spots and some
tem, and thus making a record of normal pres- ulcers, 3 = hemorrhagic spots and ulcers, and 4
sures and those occurring after treatment. = perforation. Variable results have been re-
Other qualitative and quantitative meth- ported by investigators using this technique.
ods to study the antispasmodics have been de- Production of chronic experimental peptic
scribed (19). These include (1)the fluroscopic ulcers in dogs (or rats) by the Mann-William-
study of the gastrointestinal motility and (2) son procedure (36) is one of the standard
the use of an ingestible pressure-sensitive ra- methods. The gastric juice is diverted into the
dio-telemetering capsule (Transensor) for intestine some distance from the pancreatic
measuring the pressure in the gastrointesti- and biliary secretions. The objective is
nal tract. achieved by isolating the duodenum from the
The subtype of muscarinic receptor in the pylorus and the jejunum. The oral end of the
smooth muscle has been characterized as M3 duodenum is closed and its distal end is anas-
by use of selective anticholinergics and differ- tomosed with a loop of ileum, so as to dis-
ent smooth muscle preparations from differ- charge the pancreatic and biliary secretions
ent species. These smooth muscle tissues in- into the lower portion of the bowel. The cut
clude (1)trachea (22), ileum (23, 241, uterine end of the jejunum is then anastomosed to the
artery (25), and submucosal arterioles of pylorus. About 95% of dogs so prepared de-
guinea pig (26); (2) aorta (27) and coronary velop typical chronic peptic ulcers just distal to
artery of rabbit (28); and (3) trachea (291, the gastric anastomosis with the jejunum.
aorta (30), and iris (31) of rat. Human uterine With similar operative procedures 85% of rats
arteries (32),airways (331, and ciliary muscles develop gastric, marginal, or jejunal ulcers.
(34) have also been shown to contain the M3 The complete reversal of the duodenum in
type of muscarinic receptors. dogs produces chronic peptic ulcers in about 6
months (19). These animals maintain their
2.2 Antiulcer Activity
weight until the development of ulcerations
The problems encountered in testing drugs for and might become a useful preparation for de-
antiulcer activity result in part from a lack of tecting and comparing antiulcer activity.
complete understanding of the physiological Stress produces ulcers in the rats, which
and biochemical mechanisms involved in the could be used to test the antiulcer activity of
formation of ulcers, and in part from the test- drugs (37). Rats fasted for 48 h and immobilized
ing of drugs for activity on normal or quasi- in a galvanized screen cage under light ether
normal animal preparations, although they anesthesia develop ulcers in the glandular re-
are ultimately applied to abnormal or patho- gion of the stomach after 4 h of restraint. The
logical human states. The various methods estimate of severity can be all or none, or may be
differ in producing ulcers in experimental an- coded in the same way as the Shay preparation.
imals (19). One of the side effects of adrenocortico-
A preparation developed by Shay et al. (35) tropic hormone (ACTH)and corticoid therapy
has been used to test for antiulcer activity on in humans is the development or reactivation
an all-or-none basis. The ligation of the pylo- of gastroduodenal ulcers. Daily subcutaneous
rus of rats, previously fasted for 48-72 h, leads administration of cortisol or A'-cortisol to rats
to the accumulation of acid gastric contents for 4 days results in the regular development
and ulceration of the stomach 17-19 h after of gastric ulcers (38). This procedure has been
the operation. The antiulcer agents are given adapted to testing antiulcer activity (39).
subcutaneously or intraduodenally at the time There are certain differences between steroid
of ligation of the pylorus, or orally 1 h before. ulcers and "natural" ulcers in localization,
The animals are killed 17-19 h after pyloric rate of development, and severity (40).
2 Biocomparative Assay of Anticholinergics
The antisecretory activities of anticholin- jury and finally results in gastric metaplasia.
ergics are as important as their antiulcer ac- In the presence of antral H. pylori, the gastric
tivities for their therapeutic usefulness. The metaplasia becomes colonized and inflamed.
Pavlov gastric pouch (41) with intact vagal The inflammation and infection disrupts mu-
and sympathetic nerve supply and a modified cosal defense and regenerating mechanisms,
Heidenhain pouch (421, which is essentially resulting in ulceration. The combination of in-
denervated, are prepared from dog stomach flammation, protective deficiencies, and mod-
and have been used for determining the action erate amounts of acid and pepsin may be
of drugs on gastric secretion. Histamine or a enough to induce ulceration. Several groups of
test meal is usually used as a stimulus. Similar drugs including anticholinergic agents have
methods for the preparation and use of been developed to antagonize risk factors
chronic total gastric fistulas and chronic de- causing ulcer disease. A good animal model,
nervated gastric pouches have been described which incorporates all variable causes of ulcer
for determining drug action on gastric secre- disease, is yet to be developed.
tion in rats (43-45). The muscarinic receptors of the parietal
There are a significant number of reports cells are of the M1 subtype. The specific anti-
in which antisecretory and antimobility ef- cholinergic agents for M1 receptors are con-
fects of anticholinergic drugs have been eval- sidered to be effective for the treatment of ul-
uated in ulcer patients (9,46). The antisecre- cer disease (51). The muscarinic receptors on
tory potency can be measured best in the the duodenal smooth muscle are possibly of
duodenal ulcer patient in whom the acid out- the M3 subtype. Anticholinergics at M3 may
put is already high. Ability of the drug to abol- partially decrease pain of duodenal ulcers by
ish or diminish acid output under histamine decreasing the motility of the duodenum (52).
stimulation is a stringent test of activity, al-
though the test has limited physiological rele-
2.3 Mydriatic and Cycloplegic Activities
vance. The effect of the drug on the amount of
acid secreted under ordinary clinical condi- A simple and relatively accurate test for myd-
tions is the most pertinent of all tests in rela- riatic activity has been described (52). The
tion to therapeutic application. method requires mice and a binocular micro-
Despite extensive research, certain aspects scope, magnifying about 10 times and pro-
of ulcer disease are not clearly elucidated. Be- vided with a scale in the eyepiece with which
cause of the multiple processes that control to examine and measure the diameter of the
acid and pepsin secretion and defense and re- pupil of the mouse. A strong light shining into
pair of gastroduodenal mucosa, it is more the eye of the mouse must be attached to the
likely that causes of ulceration differ among microscope. The diameter of the pupil is mea-
individuals (Section 3.5). Two other factors sured at the peak effect after administration of
have been acknowledged as risk factors in the the anticholinergic agent by intraperitoneal
pathophysiology of peptic ulcers: nonsteroidal injection. The duration of the effect is also im-
anti-inflammatory drugs (NSAIDs) and Heli- portant, given that one of the most character-
cobacter pylori infection (47-50). NSAIDs in- istic and valuable properties of atropine and
duce a significant number of gastric and duo- analogous compounds is the prolonged effect
denal ulcers, possibly because of inhibition of that they produce in the eye.
prostaglandin synthesis with consequent loss Entopic pupillometry is an accurate and
of protective effects. H. pylori has been recog- practical method for measuring the size of the
nized as a risk factor in the ulcerative process, pupil in humans (53). With a Cogan entopic
similar to acid and pepsin. Duodenal ulcer is pupillometer, the normal size of the pupil and
typified by H. pylori infection and duodenitis the near and far points before and after instil-
&d possibly impaired duodenal bicarbonate lation of the drug in the conjunctival sac can
secretion in the face of moderate increases in be measured at different time intervals. The
acid and peptic activity. Increased peptic ac- amount of light entering the eye is quite small
tivity with decreased duodenal buffering ca- and the movements of the eye during the mea-
pacity possibly leads to enhanced mucosal in- surement do not interfere with the test.
Anticholinergic Drugs
2.4 Miscellaneous Anticholinergic Activities Apollo in his temple at Delphi. Here the priest-
ess of the god Pythia sat on a tripod uttering
A number of other methods are available for
incoherent words in a divine ecstacy, in reply
comparing the activities of anticholinergic
to the questions that were asked. Pythia was
agents, of which the antitremor and antisali-
intoxicated by the fumes from burning datura
vary effects are widely used. Arecoline or pilo-
leaves; her replies were interpreted by a priest
carpine may be used to induce tremor or sali-
in the form of a verse. The more common uses
vation in a suitable species that can be blocked
of datura were for robbery or conspiracy. In-
by an anticholinergic agent. There seems to be
dian courtesans were known to place datura in
good correlation between anticholinergic and
their visitors' wine, so that they could be
antitremor effects (54). Recovery of the sali-
robbed without interference. As recently as
vary gland from cholinergic block may con-
1908, there was a plan to poison the European
ceivably precede that of the gastric glands and
garrison in Hanoi in Vietnam using datura.
the two effects may therefore not necessarily
Those in the conspiracy intended to stupefy
parallel each other in duration (9).
the soldiers, and then to kill them.
The pharmacological actions of atropine
3 SOLANACEOUS ALKALOIDS and related alkaloids are intimately connected
with our knowledge of the organization and
The older anticholinergic drugs are the vari- function of the autonomic nervous system.
ous galenical preparations of belladonna, hyo- Schmiedeberg and Koppe (56) were the first in
scyamus, and stramonium, all of which are de- 1869 to focus attention on the similarity be-
rived from plants of the potato family, the tween a drug effect and electrical stimulation,
Solanaceae. The species used as drugs include when they pointed out that muscarine and va-
Atropa belladonna, one of several plants gus stimulation affected the heart in the same
known colloquially as "deadly nightshade"; fashion and the actions of both were antago-
Hyoscymus niger (black henbane); and Datura nized by atropine. Further, they recom-
stramonium (jimsonweed, jamestown weed, or mended atropine as an antidote for mushroom
thorn apple). The active principles in all these poisoning. As early as 1887, Kobert and Sohrt
plants consist mostly of (-)-hyoscyamine, with (57) provided experimental proof for both sim-
smaller variable amounts of (-)-scopolamine ilarities and dissimilarities between atropine,
(hyoscine).Atropine is (?)-hyoscyamine. and scopolamine.
Atropine was isolated by Mein in 1831 (58),
3.1 History and since then the synthesis of both atropine
The poisonous nature of solanaceous alkaloids and scopolamine has been achieved (59,60). A
has been known for many centuries (55). The biogenetic scheme for the synthesis of atro-
toxic properties of deadly nightshade were ev- pine-like alkaloids in datura species starting
ident when children ate the blackberries, from ornithine has been described (61).
which looked attractive in a fall hedgerow in
England. The children became delirious and 3.2 Chemical Structure
their eyes had widely dilated pupils. The
deadly nightshade was used by the poisoners All the solanaceous alkaloids are esters of the
of the Middle Ages to induce obscure and often dicyclic amino alcohol 3-tropanol (tropine, 1).
delayed poisoning. Therefore, Linn6, in 1753, Atropine is an ester of (+)-tropic acid and tro-
named the shrub Atropa belladonna after At- pine. In scopolamine the organic base is sco-
ropos, the oldest of the Three Fates, who cuts
the thread of life. "Belladonna" does not refer
to Atropos, who is considered as a grim and
-
pine. Scopine differs from tropine in having an In tropine, the axially oriented hydroxyl
oxygen bridge between C-6 and C-7. group, trans to the nitrogen bridge, is desig-
There are some other alkaloids that are nated as a! or anti, and the alternate, equato-
members of the solanaceous alkaloids (e.g., rially oriented hydroxyl group as P or syn. It is
apoatropine, noratropine, belladonnine) but generally considered that cycloheptane is fixed
they are not of sufficient therapeutic value to through an -N(CH3)-bridge in the struc-
be discussed in this context. tures of tropine and pseudotropine. Therefore,
The carbon cr to the carboxyl group of tropic a chair conformation is ascribed to the piperi-
acid is asymmetric and is easily racemized dine ring system in tropine and pseudotro-
during the isolation of the solanaceous alka- pine. However, there is only a seeming differ-
loids. Atropine and atroscine are racemic ence between the two conformations of
forms. The corresponding lev0 isomers, (-1- tropane derivatives (66). The tropane system
hyoscyamine and (-)-scopolamine (hyoscine), can be considered with equal justification as a
occur naturally in the solanaceous plants. piperidine twisted through the -CH2CH2-
The absolute configuration of (-)-tropic bridge or as a cycloheptane fixed through an
acid has been established by its correlation -N(CH3)-bridge. When the tropane system
with (-)-alanine (62). According to the Cahn- is structured by the chair form of piperidine, it
Ingold-Prelog convention (631, natural (- )- also represents the boat form of cycloheptane.
tropic acid possesses the (5')configuration. Ac- Similarly, the boat form of piperidine is at the
cordingly, (-)- hyoscyamine and (-)-hyocine same time a chair form of the cycloheptane
have an (S) configuration (64). ring. Therefore, it may be assumed that both
The piperidine ring system can exist in two forms are present in a state of equilibrium
principal conformations. Its chair form has (65a)65. Based on the conformations of the
the lowest energy requirement. However, the tropane system, the structure of atropine (4)
alternate boat form can also exist, because the can be represented by (5)and (61, of which (5)
energy barrier is not great. The formula of is more generally accepted.
3-hydroxytropine (1) indicates that, even
though there is no optical activity because of H
the plane of symmetry, two stereoisomeric H2C-C- CH2 CH20H
I I I
forms, tropine (2) and pseudotropine (31, can NCH3 CHOCOCH
exist because of the rigidity imparted to the I I I
molecule through the ethane chain across the H2C-C- CH2 C6H5
H
1,5 positions (65a).
(chair)
(2)
Anticholinergic Drugs
oq
I
H H
CH2 CHzOH
0
P - 7 - I I
NCH3 CHOCOCH -
- H, F6H5
\ c-c- I I
CH2
I
C6H5
C
1\
H H O--CI I CH20H
The amino alcohol derived from scopol- through its quinine salt and the separated en-
amine (7), that is, scopine (8), has the axial antiomorph~can be converted into (+)- and
orientation of the 3-OH group but in addition (-)-hyoscyamines.
has a P-oriented epoxy group bridged across (-)-Scopolamine (hyoscine) is isolated
the 6, 7 positions. from the mother liquor remaining after the
isolation of hyoscyamine, and is marketed as
H H its hydrobromide. Scopolamine is readily race-
CH2 mized to atroscine, when subjected to treat-
I
NCH3 CHOH ment with dilute alkali.
I 0
C-C-CH2 The synthesis of scopolamine differs from
H H OH that of atropine in the synthesis of the amino
alcohol, scopine portion of the molecule. Fod6r
and coworkers (60, 73, 74) have synthesized
scopine starting from 6-P-hydroxy-3-tropanone.
3.3 Preparative Methods Esterification of scopine with 0-acetyltropyl
Conventional methods of alkaloid isolation are chloride and mild hydrolysis of the acetylsco-
used to obtain a crude mixture of atropine and polamine give scopolamine.
(-)-hyoscyamine from the plant products.
This crude mixture of alkaloids is racemized to 3.4 Molecular Factors in the Absorption,
atropine by refluxing in chloroform or by Fate, and Excretion of Atropine and
treatment with cold dilute alkali (67). Related Compounds
Atropine can be synthesized from tropi-
none and tropic acid as starting materials. The belladonna alkaloids are absorbed rapidly
Tropinone can be prepared by Robinson's syn- after oral administration (75). They enter the
thesis (68) and reduced under proper condi- circulation when applied locally to the muco-
tions to tropine. (?)-Tropic acid can be pre- sal surfaces of the body. Atropine absorbed
pared from ethyl phenylacetate (69, 70) or from inhaled smoke of medicated cigarettes
acetophenone (71). The 0-acetyl derivative of can abolish the effects of intravenous infusion
tropyl chloride reacts with tropine to yield O- of methacholine in humans. The transcon-
acetyl of atropine hydrochloride, from which junctival absorption of atropine is consider-
the acetyl group hydrolyzes spontaneously in able. About 95% of radioactive atropine is ab-
aqueous solution (72). sorbed and excreted following subconjunctival
One of the commercial sources for (-)-hy- injection in the rabbit. The total absorption of
oscyamine is Egyptian henbane (Hyoscyamus quaternary ammonium derivatives (Section
muticus) in which it occurs to the extent of 3.5) of the alkaloids after an oral dose is only
0.5%. Another method for extraction of the about 25%. The liver, kidney, lung, and pan-
alkaloid uses Duboisia species. It is prepared creas are the most important organs that take
from the crude plant material in a manner up the labeled atropine. The liver probably ex-
similar to that used for atropine and is purified cretes metabolic products of atropine by way
as the oxalate. (?)-Tropic acid can be resolved of bile into the intestine (in mice and rats).
,
3 Solanaceous Alkaloids
Carbon dioxide
(+ noratropine 2%)
Atropanol
(aldehyde)
Rabbit liver
in vitro
1I Rabbit,
guinea pig
Rabbit
guinea pig
Tropine
tropic acid
Noratropine,
apoatropine
Rat liver
in vitro
Atropine
I
(25%) Man,
mouse
Tropine-modified
atropines (10%)
- Tropicacid %)
Mouse
Because most of the synthetic antispas- At least four types of molecular modification6
modic and antiulcer agents are administered occur for the urinary excretion of atropine
orally, their absorption through the gastroin- (Fig. 3.2). Cleavage of the ester bond takes
testinal tract limits their therapeutic useful- place in the rabbit and the guinea pig (84),
ness. There are striking diferences in the ab- whereas para and meta hydroxylation of the
sorption of tertiary amines and quaternary benzene ring of tropic acid occurs in the mouse
ammonium compounds (76-78). The tertiary and the rat (80, 82). The tropine moiety of
amines (e.g., noroxyphenonium, mepiper- atropine is also chemically modified for excre-
phenidol; Section 4) are absorbed completely tion in man and mouse and, though unidenti-
from rat intestinal loops. The maximal ab- fied, "tropine-modified atropines" are ex-
sorption of the corresponding quaternary am- creted in humans and in mouse (83). Tropic
monium compounds is about one-fifth of the acid itself does not undergo metabolic alter-
total dose. The poor absorption of quaternary ation for urinary excretion in all species men-
ammonium compounds may be partly attrib- tioned above. The metabolic conversions of
utable to the positive charge that promotes tropine itself are not fully investigated. How-
the formation of a nonabsorbable complex ever, demethylation of atropine- N-14CH3(or
with mucin. The ready absorption of tertiary tropine- N-14CH3)has been reported in a num-
amines may be explained partly by their per- ber of species with exhalation of 14C0, (90).
meability through lipid membranes (79). The possible metabolic changes of atropine are
Considerable species variations have been schematically represented in Fig. 3.2.
reported for the metabolic detoxification of at- After intravenous injection of atropine, ap-
ropine in mammals (80-91). These differences proximately 25% of the dose is excreted in
seem to be more quantitative than qualitative. mouse urine as atropine, more than 50% as
Anticholinergic Drugs
conjugates with glucuronic acid, and the re- tropic acid. In this way a series of tropeines
maining 20-25% as intermediate oxidation have been synthesized, among which a num-
products (probably p-hydroxyatropine and ber of active compounds have been found (86-
3,4-dihydroxyatropine)and "tropine-modified 92). Of the tropeines, mandelyl tropeine (9,
atropines." Rats are known to metabolize at-
ropine in a manner similar to that in mice. In
humans, about 50% of the administered dose
of atropine is excreted unchanged in the urine
and about 33% as unknown metabolites that
are esters of tropic acid. Neither hydroxyla-
tion of the tropic acid moiety nor glucuronide
formation has been demonstrated in humans
(83). Only less than 2% appears as tropic acid
in urine. homatropine), has survived as a thrapeutic
It has been known for more than a centurv agent to the present.
"
that rabbits can tolerate large quantities of Methylatropine nitrate (10)(or bromide) is
atropine (84, 85). The cause of this observa- a synthetic quaternary derivative of atropine.
tion is the ability of the serum of some, but not Atropine oxide (atropine N-oxide) is known as
all, rabbits to hydrolyze atropine into tropic a genatropine (11)and may be prepared by oxi-
acid and tropine. The hydrolysis is attributed dation of the alkaloid with hydrogen peroxide.
to an enzyme, atropinesterase, which is found
in most other tissues as well as the serum of
these rabbits. The highest activities are found
in the liver and intestinal mucosa; only the
brain and aqueous humor of the eye contain
no enzyme. The enzyme is also found in the
liver of the guinea pig and accounts for the
appearance of tropic acid in the urine of the
rabbit or the guinea pig, but not other ani-
mals, following the administration of atro-
-pine.
The presence of atropinesterase in rabbits
is inherited through an incompletely domi-
nant gene (84). This gene is associated with
another gene that influences the color of the
fur, causing "extension of black pigment in the
The derivatives of scopolamine (7) pre-
fur."
pared by similar methods are available com-
Atropinesterase can also hydrolyze homat-
mercially. These include methscopolamine
ropine and scopolamine. This enzyme is ste-
bromide (121, methscopolamine nitrate, and
reospecific for (S)-(- )-hyoscamine, which is
genoscopolamine (scopolamine N-oxide, 13).
split; the more inert (R)-(+) isomer is not
readily hydrolyzed (84).
HC-CH CH2 CHzOH
3.5 Semisynthetic Derivatives / I I I
of Solanaceous Alkaloids 0 N+(CH3)2 CHOCOCHCsH5. Br-
\ I I
Early attempts to modfy the atropine mole- HC-CH CH2
cule (4) were aimed at converting the solana-
ceous alkaloids containing the tertiary nitro-
gen into quaternary ammonium compounds
and N-oxides. Later developments have been Homatropine (9) is prepared by evaporat-
to retain the tropine (or scopine) portion of the ing tropine with mandelic and hydrochloric
molecule and substitute various acids for acids. Homatropine methylbromide (14) may
3 Solanaceous Alkaloids
Br-
pounds, the standard textbooks or review simpler to synthesize a fairly complex mole-
in pharmacology should be consulted (75,97- cule from two halves by esterification than by
99). any other method. Therefore, many esters of
amino alcohols and carboxylic acids have been
synthesized as atropine substitutes, in which
4 SYNTHETIC ANTICHOLINERCICS
the structures of either one or both halves
have been changed. For example, in homatro-
Although atropine and its related alkaloids are
pine, the tropic acid moiety has been replaced
potent anticholinergics, they have a wide spec-
by mandelic acid. The amino alcohol moiety
trum of pharmacological activities. Therefore,
(tropine, 1)of atropine has afforded unusually
therapeutic administration of these alkaloids
rich opportunities for the synthesis of anticho-
to elicit a particular desired activity invariably
linergics (Fig. 3.3). Scission of its piperidine
results in some undesirable side effects. For
ring at point X gives the derivatives of hy-
this reason, the search for compounds possess-
droxyalkylpyrrolidines (16),and scission of its
ing one or another of the specific desirable ac-
pyrrolidine ring at point Y makes it possible to
tions has been an active field of investigation
proceed to derivatives of 4-hydroxypiperidine
in medicinal chemistry. The ideal specificity of
(17).The scission of both rings at Z leads to
action has not been attained in these at-
dialkylaminoalkanol derivatives (18). Fur-
tempts; perfect atropine substitutes with pre-
thermore, simplification and alteration of
dominant antispasmodic, antisecretory, or cy-
these three groups of amino alcohols has re-
cloplegic actions have yet to be synthesized.
sulted in the synthesis of esters containing
However, some progress has been made since
structural features more or less similar to
the discovery of multiple subtypes of func-
those of atropine.
tional muscarinic receptors (MI-M5) and the
Antagonists of acetylcholine often have
cloned muscarinic receptors (m,-m,) have
chemical structures resembling that of acetyl-
been identified (Table 3.1). Several antago-
choline, although they differ from it by greater
nists, which show selectivity to one subtype of
complexity of the molecule and higher molec-
muscarinic receptors over others, have been
ular weight. Acetylcholine is a quaternary am-
introduced and they have become useful in the
monium compound; atropine and tropine con-
delineation of subtypes of muscarinic recep-
tain a tertiary nitrogen. Therefore, a number
tors in various tissues. Some of these agents
of atropine-like compounds having quater-
may become useful as antiulcer agents, anti-
nary nitrogen atoms have been synthesized.
spasmodics, or mydriatics.
In some of them, the acetyl group of acetylcho-
line has been replaced by acid moieties con-
4.1 Analogs of Atropine
taining blocking groups (e.g., diphenylacetic
The synthetic anticholinergic drugs can be acid).
considered as analogs of atropine or antago- The principles used in the design of antime-
nists of acetylcholine. Most of these com- tabolites have been applied to synthesize atro-
pounds were designed using broad principles pine-like compounds. The ester group in atro-
of molecular modification such as (1)scission pine-like compounds has been replaced by a
of the atropine molecule into simpler mole- thioester, an amide, an ether group, or a chain
cules containing the essential pharmacophoric of methylene carbons (Table 3.3).
groups; (2) molecular modification by intro- All synthetic anticholinergic agents have
ducing "blocking" moieties into cholinergics; some structural features in common. In most,
and (3) changes in other anticholinergics us- the molecule has bulky "blocking moieties,"
ing principles of bioisosterism. often cyclic radicals, linked by a chain of atoms
The structure of atropine has been the ba- of limited length, to a positively charged
sis for a large number of synthetic anticholin- m i n e nitrogen (Fig. 3.4). The length and
ergic agents. However, no significant changes structure of the main chain have considerable
have been made to affect the "ester-complex" influence on the anticholinergic activity of the
grouping because of atropine-like properties. substance. At the same time the chemical na-
Another probable consideration is that it is far ture of the main chain determines the class of
4 Synthetic Anticholinergics
p H 3
HOCHR'CH2CHR"N
\
R"' Figure 3.3. "Scissions" of tropane
(18) ring.
organic substances to which a given substance However, several examples from drugs used as
belongs. Therefore, the classification of syn- therapeutic agents are discussed at appropri-
thetic anticholinergics in Table 3.3 is based on ate places in the following pages. These com-
the structure of the main chain of the mole- pounds may be classified differently, and the
e, taking into consideration wherever nec- same compound may be placed in more than
ary the presence or absence of any addi- one group. Each one of them may be consid-
It is beyond the scope of this text to consider ergic activities among a series of structurally
all compounds that belong to each group. related compounds whose structure-activity
II
C C - 0 - C -OH
0
I1
Thioester CC--S-C -OH
0
II H
Amide -C-N-€ -OH
0
II
Carbarnate )N-C--O--C
Alkane --C-C-C--
(a)Amino alcohols -OH
(b) Amides -4ONH,
Anticholinergic Drugs
-- -
opened and probably intracellular calcium is
mobilized and, like many other transmitters,
acetylcholine increases the turnover of phos-
H J
phoinositides. Therefore, there is much expec-
L V
tation that selective agents will be found
among muscarinic antagonists that will be
Acyl Ester Alkamine Quaternary
radical group chain amino function useful to block one particular physiological or
Acetylcholine (19) biochemical response to acetylcholine. Thus,
- -*
several agents have been synthesized that
Cyclic Esteratic Cationic have structural features similar to those of at-
radical group portion ropine-like agents, cyclic blocking moieties
linked by a chain of atoms of limited length to
,H a positively charged nitrogen atom.
Based on the cyclic-blocking moieties and
other substituent groups, subtype-selective
muscarinic antagonists can be classified into
eight groups: (1)tricyclic benzodiazepines, (2)
benzothiazepines, (3) quinuclidines, (4) poly-
methylene tetramines, (5) indenes, (6) sila-
difenidols, (7) diphenylacetyloxy derivatives,
~ a i chain
n and (8) himbacine akaloids.
Atropine (20)
Figure 3.4. Structural features of acetylcholine 4.2.1 Tricyclic Benzodiazepines. Sigdicant
and atropine. The asymmetric carbon in atropine is side effects of tricyclic antidepressants, like
marked with an asterisk (*). imipramine, are antimuscarinic effects. Ben-
zodiazepines also cause some antimuscarinic
effects like dry mouth at therapeutic concen-
relationships have been evaluated for differ-
trations. Some of the well-known anticholin-
ent types of pharmacological effects. Com-
ergic agents, Banthine, Probanthine, and
pounds with the same or similar structural
Trest, have tricyclic bulky moieties at the end
features may exhibit other pharmacological
of their molecules. Some molecular features of
effects as side effects. For example, a large
these three types of phamacological agents are
number of compounds have been synthesized
present in tricyclic pyrido-benzodiazepines.In
containing an ether link in the main chain.
these compounds, portions of benzene and
These compounds are useful as antiparkinso-
pyridine (or other rings) are fused to a seven-
nian drugs and antihistaminic agents.
membered diazepine ring in the middle. The
trycylic bulky moiety containing benzene, di-
4.2 Receptor-Subtype-Selective
azine, and pyridine rings (or other rings) at
Anticholinergics
the end of a molecule satisfies one of the re-
The muscarinic actions of acetylcholine can be quirements for an anticholinergic agent. Fur-
either stirnulatory or inhibitory. Acetylcholine ther substitutions on the imino-nitrogen atom
stimulates secretion and contraction of the have resulted in selective M1 receptor antag-
gut, but it inhibits the contraction of the heart onists that are useful as antiulcer agents.
and relaxes the smooth muscle of blood ves- Pirenzepine (21) was the first M1 receptor
sels. Acetylcholine can inhibit adenylate cy- antagonist shown to inhibit gastric secretion
clase and activate guanylate cyclase. In the (100, 101). This drug (100-150 mg/day) is
cortical neurones, muscarinic agents cause a used in several countries to decrease gastric
slow depolarization mediated by closing potas- secretion and achieve maximal rates of ulcer
4 Synthetic Anticholinergics 129
$'
AQ-RA 741 (25) is an analog of pirenzepine
0 that exhibits higher affinity to chimeric m2
and M4 receptors than for m5 receptors (105).
VH-AH-37 (26), a pirenzepine derivative, ex-
hibits higher affinity to chimeric m5 receptors
N than to m2 receptors.
4
-
0CH3
CH2-NH-(CH2)s-NH-
(32)
l2(CH&
Receptor
Subtype"
MI
M1
M2
M2
Test System
Rabbit vas deferensb
Rat duodenumb
Guinea pig atria
Rabbit vas deferens
pA, Values
of Isomers
(R)
5.81
5.49
6.25
6.22
(s)
6.83
6.36
7.86
7.74
tors are dependent on a tetramine backbone M3 Guinea pig ileum 5.61 6.92
and the nature of substituents on the terminal M3 Guinea pig trachea 5.59 6.96
nitrogens (33,34).This selectivity is improved
"Different selective agonists were used to stimulate the
by introduction of N-methyl groups into the receptors.
tetramine backbone and introduction of a tri- bSome test systems contain more than one subtype of
cyclic system on the terminal nitrogens of the muscarinic receptor.
Anticholinergic Drugs
q €{I: CH-C-0
activity and higher Kd (181 nM).It appears
that himbacine is a potent muscerinic antago-
nist at M2 or M4 receptors compared to M1 or
M3 receptors (116,117).
d II
(37)
1-
5 STRUCTURE-ACTIVITY RELATIONSHIPS
Pavatrine 2-Diethylaminoethyl-9-
fluorene-carboxylate
hydrochloride
Eucatropine Euphthalmine
di-methylpiperidinium
methylsulfate
4-(1,2,2,6-Tetramethyl-
(C6H5)2C
€ Nf(CH3)2
132,133
piperidyl) mandelate
hydrochloride
-.L C6H5CH(OH)C02
W
Q,
\
C3H5
Pipethanate Sycotrol 2-(1-Piperidino)ethyl benzilate
hydrochloride (C6H5)2C(OH)C02CH2CH2N
Quaternary Ammonium Compoundsdharacteristic Group in the Main Chain: Ester
Glycopyrrolate ND Robinul 3-Hydroxy-1,l-dimethyl-
pyrrolidinium bromide
a-cyclopentylmandelate
-0
Pipenzolate NND Piptal N-Ethyl-3-piperidyl benzilate
methobromide (CGH~)~C(OH)C~Z
Nt CH3
\
I
Valethamate ND Murel 2-Diethylaminoethyl-3-methyl- CH3CHCHC02(CHZ)
2N'(CzH5)zCH8
2-phenylvalerate 1
methobromide C6H5
4
W
w Mepiperphenidol Darstine 5-Methyl-4-phenyl-141-
methy1piperidinium)-3-
hexanol bromide
Tricyclamol NND Elorine, l-Cyclohexyl-l-pheny1-3-
Tricoloid pyrrolidino-1-propanol
methochloride
Tridihexethyl NF Pathilon
1-cyclohexyl-1-propanol
ethiodide
140 Anticholinergic Drugs
traction of the oppositely charged groups, the and partition between aqueous and organic
weaker dipole-dipole, hydrophobic, and van phases. Alkylation of the oxygen atom con-
der Valls forces go into action; if there are verts the N-oxides into typical quaternary am-
many of them, especially in the case of anti- monium compounds. By this procedure, both
cholinergics, they contribute to the stability of the basicity and anticholinergic activity (Ta-
the drug-receptor complex. In such an interac- ble 3.8) of the substance increase sharply.
tion not only the charge of the cation head but The influence of a steric factor is more evi-
also its size and shape are of vital importance. dent among compounds in which the size of
The basicity of different amino derivatives, the substituents at the nitrogen atom is varied
and consequently the degree of their ioniza- both in the series of anticholinergic and cho-
tion at physiological pH, varies over a broad linergic compounds. Progressive replacement
range. The more ions of the anticholinergic of the N-methyl groups of acetylcholine with
ammonium compound or amine in solution, ethyl groups leads to a stepwise reduction in
the greater the probability of their interaction muscarinic activity (167). Likewise, maximal
with the anionic center of the muscarinic re- anticholinergic or blocking activity (Table 3.9)
ceptor to form the drug-receptor complex. In is obtained by replacing the N-methyl groups
addition, the stability of the drug-receptor of P-dimethylaminoethyl benzilate methyl-
complex that has formed should depend on the chloride with ethyl groups (119). Further in-
basicity, given that the rate of hydrolysis of creases in size to butyl or larger alkyl groups
salts is inversely proportional to the base reduce or abolish the activity (119, 168-172).
strength. Therefore, it seems that for stimulant activity,
Thus, high basicity should favor the anti- the small cationic head must fit into a definite
cholinergic activity of a substance. Although space and must aid the neutralization of the
the logic of this conclusion is simple, its proof charge of the anionic site of the receptor. The
involves great difficulties. In a series of anti- inhibitory action is obtained when large
cholinergics, transition from one derivative to enough groups are substituted on the cationic
another is associated with stepwise changes in portion to prevent close contact with the re-
basicity, as well as steric factors. In this re- ceptor and hence the neutralization of the
spect the N-oxides, which are obtained charge (173,174). Thus, the cationic portion of
through the oxidation of the corresponding the blocking agents provides the electrostatic
tertiary amines, have lower basicities and also forces necessary to orient the molecules to-
lower anticholinergic activities (164-166). ward the receptor and hold them in place.
The N-oxides are closer to the corresponding The anticholinergic activity depends not
quaternary ammonium compounds than to only on the number and the molecular weight
the tertiary ammonium ions in steric respect of the alkyl radicals that are connected to the
5 Structure-Activity Relationships 141
Table 3.9 Influence of the Number, Size, and Structure of Alkyl Groups in the Cationic Head
on the Anticholinergic Activity
nitrogen atom but also on their structure. In Besides the charge on the cationic head of
contrast to di-n-propylamino derivatives, di- anticholinergics (and cholinergics), other fac-
isopropylamino derivatives have an anticho- tors seem to contribute to the interaction be-
linergic activity close to or higher than the tween the muscarinic receptor and the anticho-
activity of diethylamino derivatives (136,175- linergics. The substituents at the nitrogen atom
178). The close correlation of the activities of apparently participate actively in the process.
the diethyl and diisopropyl derivatives could This is evident from the anticholinergic action of
be related with the equal linear lengths (from the 3,3-dimethylbutyi ester of benzilic acid,
the nitrogen atom) of these radicals. (C,H,),C(OH)C02CH,CH2C(CH3)3, which con-
In the case of cyclic amino alcohols where tains no nitrogen and consequently is not ion-
nitrogen enters into the composition of the cy- ized, but which has in the corresponding posi-
cle, the optimal anticholinergic effect is pro- tion a t-butyl radical that sterically imitates the
duced not by the N-ethyl, N-isopropyl, or N- trimethylammonium group (191). A similar re-
allyl, but by N-methyl radical, as is apparent placement of a trimethylammoniumgroup with
from a comparison of the esters of tropine (Ta- a t-butyl radical in acetylcholineleads to its "car-
bles 4-6). It may be that the elements of the bon analog," CH,CO,CH,CH,C(CH,),, which
cyclic structure occupy a sufficiently large is similar to acetylcholine in its behavior toward
space besides the nitrogen atom. cholinesterase (192).
As a general rule, quaternization with a 5.2 Cyclic Moieties
small alkyl group increases activity (Table
3.10),although a few exceptions have been re- The introduction of two phenyl groups into a
ported (181, 182). molecule of acetylcholine or a cholinergic
142 Anticholinergic Drugs
Table 3.10 Differences Between the Anticholinergic Activities of Tertiary and Quaternary
Ammonium Compounds and Atropine-like Agents
Series A: Tertiary Activity
Compound Ammonium Series B: Quaternary Ratio
Pair Compounds Ammonium Compounds Test System BIA Ref.
Atropine Methylatropine Guinea pig: ileum
Mouse: mydriasis
Mouse: mydriasis
Guinea pig: ileum
Mouse: mydriasis
Tertiary analog Rabbit: intestine
of methantheline
XN(C,H&
Tertiary analog Rabbit: ileum
of penthienate Rabbit: salivation
XN(C,H&
(5)-Procyclidine Guinea pig: ileum
Mouse: mydriasis
Methyl analog Guinea pig: ileum
+ of (?I-benzhexol Mouse: mydriasis
RN(CH& Cat: salivation
Cat: blood pressure
Mouse: mydriasis
Cat: Salivation
Cat: blood pressure
Mouse: mydriasis
"For complete structures see Table 3.7.
*For complete structures see Table 3.7.
'1-Piperidino-3-pheny1-3-cyc1ohexy1-propan-1-o1.
dR = (C6H5)2C(OH)C02CH2CH2-.
antagonists (65d)65. Thus, anticholinergic ac- ciently. A great importance was attached to
tivity depends not only on the length of the the complex
- esters in the initial period
- of the
main chain of the molecule but also on its abil- search for atropine-like substances, when ac-
ity to adopt a certain conformation that is fa- tive compounds were known only among the
vorable for the interaction of the substance esters of amino alcohols and carbowlic - acids.
with the receptor. However, the presence of this grouping is not
There is some information about the influ- necessary - for the manifestation of anticholin-
ence of branching of the main chain on the ergic activity. Presently, a large number of
anticholinergic activity. Esters with a methyl substances are known that belong- to different
group CY to the ester oxygen in the amino chemical structures and that possess high an-
alcohol part are less active than compounds ticholinergic activity (Table 7).
without the methyl group (175, 198, 201). The influence of an ester link can be as-
Similarly, the derivatives of 1,3-aminopropanol, sessed by comparing similar compounds that
aminopropane, and y-arninobutyronitrile (201, do not contain pharmacophoric groups other
203),which contain a branch at the carbon atom than the anchoring groups (amino nitrogen,
p to the nitrogen, are less active anticholinergics cyclic radicals). Comparative data on the
than the compounds without the branching. anticholinergic activities of the 2-diethyl-
The negative influence of such a side chain has aminoethyl ester of diphenylacetic acid
been explained by steric hindrance at the recep- [(C6H5),CHC0,CH,CH,N(C2H5)2] and 1,l-
tor (174). diphenyl-5-diethylaminopentane [(C6H5),-
The inclusion in the main chain of opti- CHCH,CH,CH,CH,N(C,H5),] indicate that
mum length of other atoms such as oxygen, they are equally active (65d). Thus, the com-
sulfur, nitrogen, and other functional groups plex ester group is not essential for anticholin-
changes any anticholinergic activity (65). ergic activity; however, it may contribute to
However, such compounds are considerably optimal activity when it is present in atropine-
potent (see Section 5.4). like compounds (123). It may influence the
conformation of a molecule that in turn deter-
5.4 Esteric Linkage
mines the effectiveness of the interaction of
The question of the importance of complex es- the essential anchoring groups, the cationic
ter grouping in anticholinergics, and even head and the cyclic radicals (65d), with the
more of its role, has not been cleared up suffi- muscarinic receptor.
5 Structure-Activity Relationships
located in the acyl moiety and is connected 3-quinuclidinyl diphenylacetate, the carbon
with the cyclic and the hydroxyl groups (di- atom p to the nitrogen is asymmetric; the (-)
rectly or through a methylene group). The (-1 isomer has more atropine-like activity than
isomers are often more active than (+) iso- does the (+) isomer.
mers (Table 3.14), indicating some apparent
stereospecificity with respect to the carbon 5.7.2 Derivatives of Tropine and Pseudo-
atom a to the carbonyl group of atropine. tropine. The configuration of the 3-OH group
The atropine-like activities of some com- in the tropine part of the molecule has signif-
pounds in which the asymmetric carbon atom icant influence on the activity at the musca-
is considerably closer to the amino group have rink receptor (Table 3.15). The derivatives of
been described. In 1,l-diphenyl-3-piperidino- +-tropine (pseudotropine, 3) are less active;
1-butanol, the carbon a to the nitrogen is +
the activity ratio for the compound relative
asymmetric. In this case the (+) isomer seems to the isomeric tropine varies from 2 to 13, but
to be more active than the (-) isomer. In more information is needed on this point.
Table 3.15 Relative Anticholinergic Activities of the Esters of Tropine and Pseudotropine
Intrinsic
Activity
a P& Test System Ref.
Rat: intestine 225
Rat: intestine
Rat: intestine
Rat: intestine
Rat: intestine
Guinea pig: ileum
Guinea pig: ileum
Guinea pig: ileum
Guinea pig: ileum
Guinea pig: ileum
Guinea pig: ileum
Rat: intestine 225
Anticholinergic Drugs
and anticholinergic drugs at the muscarinic istence on the receptor of two active sites sep-
receptors see original papers and reviews on arated by a distance 3.2 + 0.2 A (219, 237-
the subject (230-236). 239). One of them is an anionic site with which
6.1 Kinetic Basis for the Mechanism of the quaternary ammonium group interacts to
Action of Anticholinergics induce stimulant or blocking actions. The
ether oxygen of muscarine and the ester oxy-
The major action of a number of anticholin- gen of acetylcholine interact with the second
ergics is a competitive antagonism to acetyl- site. There are some similarities between the
choline and other cholinergic agents. The an- active sites on acetylcholinesterase and the
tagonism can therefore be overcome by muscarinic receptor. The amine portion of an-
increasing the concentration of acetylcholine ticholinergics interacts at the same anionic
at receptor sites of the effector organs. Thus site as the quaternary group of acetylcholine
anticholinesterases partially reverse the an- and atropine. Several facets of acetylcholine-
tagonism of anticholinergics by sparing acetyl- atropine antagonism are well known:
choline at the receptor sites. The anticholin-
ergics can inhibit all muscarinic actions of 1. One molecule of atropine blocks one mole-
acetylcholine and other choline esters. Re- cule of acetylcholine. Atropine is a larger
sponses to postganglionic cholinergic nerve molecule than acetylcholine and either me-
stimulation may also be inhibited, but less chanically or electrostatically inactivates
readily than responses to administered cho- receptors engaged by it.
line esters. The differences in the ability of
2. Atropine has greater affinity than acetyl-
anticholinergics to block the effects of exoge-
choline for the receptor. Its intrinsic activ-
nous choline esters and the effects of endoge-
ity is not significant, whereas acetylcholine
nous acetylcholine liberated by the postgangli-
has high intrinsic activity. Substances with
onic parasympathetic nerves may result from
intermediate intrinsic activities behave ei-
the release of the chemical transmitter by the
ther as cholinergics or as anticholinergics,
nerve at the receptors in relatively inaccessi-
depending on the nature of their influence
ble sites where diffusion limits the concentra-
on the receptor. Among such substances
tion of the antagonist.
are partial agonists with "dual action";
6.2 Specificity of Antagonism cholinergic activity precedes the anticho-
linergic activity. The partial agonists can
Atropine is a highly selective antagonist of
be detected in a homologous series by grad-
acetylcholine, muscarine, and other cholin-
ually proceeding from agonists to antago-
ergic agents on the smooth and cardiac mus-
nists with increasing molecular weight.
cles and glands. This antagonism is so selec-
tive for cholinergic agents that atropine 3. Besides the cationic head, bulky cyclic
blockade of the actions of other types of drugs groups are essential constituents of com-
has been taken as evidence for their actions pounds with anticholinergic activity. It
through cholinergic mechanisms. For exam- seems clear that the van der Wads or hy-
ple, the smooth muscle of guinea pig ileum is drophobid binding of the planar cyclic
stimulated by muscarine, 5-hydroxytrypta- groups together with the binding of the
mine, histamine, and barium chloride. Atro- amine group produce a strong drug-recep-
pine is more specific in blocking the stimulant tor complex, which effectively blocks the
effects of muscarine and acetylcholine at lower close approach of acetylcholine to the
dose levels than those of the other three stim- receptor.
ulant agents. 4. Acetylcholine increases potassium efflux
and causes depolarization of the mem-
6.3 Molecular Basis for the Interaction of brane, both of which effects are blocked by
Acetylcholine and Anticholinergics at the atropine.
Muscarinic Receptors
5. The receptor proteins on the membrane
Structure-activity relationships among mus- may undergo molecular disorientation dur-
carinic agents (or cholinergics) indicate the ex- ing the interaction of acetylcholine with
7 Therapeutic Uses of Anticholinergics
the cholinergic receptor, and this change in The mydriatic and cycloplegic activities of
the receptor proteins may be prevented by anticholinergics in humans are listed in Table
a suitable blocking agent (240). Given that 3.18. Atropine is recommended in situations
five subtypes of muscarinic receptors have requiring complete and prolonged relaxation
been isolated and their amino acid se- of the sphincter of iris and the ciliary muscle.
quences have been determined, future in- Mydriatics, like cyclopentolate, eucatropine,
vestigations may reveal the molecular na- and homatropine bromide, with a shorter du-
ture of interactions of anticholinergics ration of action, are usually preferred for mea-
with muscarinic receptors. suring refractive errors because of the relative
rapidity with which their cycloplegic effects
7 THERAPEUTIC USES OF are terminated.
ANTlCHOLlNERGlCS Atropine and scopolamine are used for pre-
medication before the administration of some
The chief use of most of the antispasmodic inhalation anesthetics, to reduce excessive sal-
agents is as an adjunct in the management of ivary and bronchial secretions. Atropine and
the peptic ulcer; this group of drugs includes related agents have been used in the treat-
adiphenine, aminopentamide, amprotropine, ment of renal colic and hyperhidrosis, and to
dibutoline, diphemanil, glycopyrrolate, hexo- control sweating that may aggravate certain
cyclium, homoatropine methylbromide, meth- dermatologic disorders. Atropine also may be
scopolamine bromide, methscopolamine nitrate, used to counteract the toxicity of certain cho-
oxphencyclimine, oxphenonium, penthienate, linergic drugs and anticholinesterase agents.
pipenzolate, piperidolate, pipethonate, propan- Certain drugs with anticholinergic effects
thelin, tricyclamol, and trihexethyl (241). To are used for the symptomatic treatment of
this group of several M1 receptor antagonists Parkinson's disease (paralysis agitans) and re-
that decrease acid secretion should be added lated syndromes of the extrapyramidal tracts.
pyrenzipine, which is a leading compound in this (Of the presently available drugs, none is use-
group of agents. The anticholinergic agents that ful in all cases of Parkinsonism.) Despite
are useful as adjuvants in the management of claims of superiority for newly introduced syn-
the functional disorders of the bowel (e.g., irri- thetic agents, none possesses outstanding effi-
table colon, spastic colitis, ulcerative colitis, and cacy and freedom from adverse side effects
diverticulitis) include dicyclomine, hexocyclium, when compared clinically with atropine and
mepenzolate, and valethamate. scopolamine (241).
Anticholinergic Drugs
8 MOLECULAR BASIS FOR THE SIDE of euphoria, dizziness, and delirium may be
EFFECTS OF ANTlCHOLlNERClCS observed because the drugs can cross the
blood-brain barrier.
The most widely used mode of approach in the Many synthetic quaternary ammonium
design of anticholinergics is based on the use compounds may block acetylcholine at ganglia
of tropine alkaloids as models of prototypes, at high doses. Ganglionic-blocking agents
from which congeners or homologs or analogs cause impotence as a side effect. High doses of
have been designed. Tropine alkaloids have methantheline may also cause impotence, an
many pharmacological activities and interact effect rarely produced by pure antimuscarinic
at many cholinergic sites. In drug design the drugs and indicating ganglionic blockade.
main purpose is to increase one pharmacolog- Toxic doses of quaternary ammonium com-
pounds (e.g., menthantheline, propantheline,
ical action at one particular site of action while
and oxyphenonium) block acetylcholine at the
concomitantly suppressing other pharmaco-
somatic neuromuscular junction and paralyze
logical activities at other sites. It is not always respiration.
possible to abolish all pharmacological effects Adiphenine and amprotropine have local
other than the desired activity by molecular anesthetic activities, and anesthesia of the
modification. Though the desired activity is oral mucosa results when tablets of these
useful in its therapeutic applications, other drugs are chewed. It should be remembered
pharmacological activities manifest them- that local anesthetic esters and amides exert
selves as side effects. For example, atropine, their action by anticholinergic mechanisms,
scopolamine, and cocaine are structurally re- probably essentially at the nodes of Ranvier.
lated, each having atropine nucleus. They dif- The central side effects have appeared
fer in some of their pharmacological activities. among children even when cyclopentolate,
Atropine stimulates the CNS, scopolamine de- tropicamide, and other anticholinergics are
presses the CNS, and cocaine is a local anes- used as mydriatics. All anticholinergics in-
thetic and CNS stimulant. crease intraocular pressure in most patients
By molecular modification, it has been pos- with simple glaucoma.
sible to produce a series of anticholinergics Some of the cyclic groups in anticholin-
having qualitative effects resembling those ergics are pharmacophoric moieties for other
produced by parasympathectomy to a particu- types of activities. For example, the com-
lar organ. Although these drugs exert specific pounds containing a phenothiazine nucleus
therapeutic effects at one organ, they exert exhibit central depressing and antihistaminic
side effects at other organs. Recent develop- side effects. These side effects are of advan-
ments on the design of anticholinergics selec- tage in the treatment of Parkinson's syn-
tive for subtypes of muscarinic receptors and drome. The side effects of certain drugs that
identification of subtypes of muscarinic recep- result from their anticholinergic activities are
tors in a number of organs has partially pro- prominent among some analgesics (e.g., me-
vided a solution for this problem. peridine), antihistamines (e.g., prometha-
The untoward effects associated with the zine), psychosedatives (e.g., benactizine), and
use of anticholinergics are manifestations of psychotomimetics (e.g., dexoxodrol).
their pharmacological actions, and usually oc-
cur on excessive dosage. The effects include 9 PROFILE O F ANTlCHOLlNERClC
dryness of mouth, blurred vision, difficulty in ACTIVITIES OF VARIOUS AGENTS
urination, increased intraocular tension,
tachycardia, and constipation. Most of these The relative anticholinergic activities of the
side effects are lessened when the quaternary well-known therapeutic compounds are listed
anticholinergics are administered orally in the in Table 3.19. Although it is very difficult to
treatment of peptic ulcer because of low ab- justify collecting the results of a wide variety
sorption into the systemic circulation. In the of experiments, it seems likely that the table
case of tertiary amines the central side effects gives some idea of their relative antisecretory,
10 Nonanticholinergics as Antiulcer Agents 155
Penthionate
Pipenzolate
Poldine methyl sulfate
Propantheline
Valethamate
Esters, Tertiary
Adiphenine
Amprotropine phosphate
Benactyzine
Carbofluorene
Cyclopentolate
Dicyclomine
Eucatropine
Oxyphencyclimine
Piperidolate
Propivane
Thioesters, Tertiary
Triphenamil 6.0rb
Carbamates, Quaternary
Dibutoline sulfate
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CHAPTER FOUR
CNS Stimulants
DAVID E. NICHOLS
Department of Medicinal Chemistry and
Molecular Pharmacology
School of Pharmacy and Pharmacal Sciences
Purdue University
West Lafayette, Indiana
Contents
1 Introduction, 168
1.1 Ephedra and Khat, 168
1.2 Caffeine, 169
2 History, 172
3 Clinical Use of Agents, 172
3.1 Therapeutic Applications, 172
3.1.1 Attention Deficit Hyperactivity
Disorder (ADHD), 172
3.1.2 Narcolepsy, 173
3.1.3 Use for Depression in Terminal Illness,
173
3.1.4 Use in Obesity, 173
3.1.5 Apnea in Premature Infants, 173
3.2 Side Effects, Adverse Effects,
and Drug Interactions, 174
3.2.1 Methylphenidate, 174
3.2.2 Pemoline, 174
3.2.3 Cocaine, 175
3.2.4 Caffeine, 176
3.3 Absorption, Distribution, Metabolism, and
Elimination, 176
3.3.1 Amphetamine Metabolism, 176
3.3.2 Methylphenidate Metabolism, 177
3.3.3 Cocaine Metabolism, 177
3.3.4 Diethylpropion Metabolism, 177
4 Physiology and Pharmacology, 179
4.1 Where and How These Drugs Work, 179
4.2 Biochemical Pharmacology: Receptor Types
and Actions, 180
4.2.1 A Role for Serotonin, 182
4.2.2 A Role for Norepinephrine, 183
5 Structure-Activity Relationships, 184
5.1 Amphetamine, 184
Burger's Medicinal Chemistry and Drug Discovery 5.1.1 Length of the Side-Chain, 185
Sixth Edition, Volume 6: Nervous System Agents 5.1.2 Nitrogen Substituents, 185
Edited by Donald J . Abraham 5.1.3 Stereochemistry a t the a! Carbon, 186
ISBN 0-471-27401-1 O 2003 John Wiley & Sons, Inc. 5.1.4 The a!-alkyl Substituent, 186
167
CNS Stimulants
5.1.5 Other Side-Chain Substitutions, 186 5.3.5 Substitutions on the Aromatic Ring at
5.1.6 Aromatic Ring Substitution, 187 Position 3, 191
5.2 Methylphenidate, 187 5.3.6 Requirement for the Intact Tropane
5.3 Cocaine, 188 Ring System, 192
5.3.1 N-substituents, 188 6 Recent and Future Developments, 192
5.3.2 Basic Nitrogen Atom, 189 6.1 Web Site Addresses and Recommended
5.3.3 Substituent at C(2),189 Reading, 194
5.3.4 The Ester Linkage at C(3),191
active component in Khat is a substituted where it occurs to the extent of about 3.5%, by
phenethylamine derivative known as (-)- weight), of coffee beans (Coffea arabica, where
cathinone (2) (3). it constitutes about 1-2% by weight), and tea
(Camellia sinesis, where it makes up 1-4% of
the mass of dried leaves). The annual con-
sumption of caffeine has been estimated at
120 million kilograms, the approximate equiv-
alent of one caffeine-containing beverage per
day for each of the world's six billion plus in-
habitants. As a beverage, the worldwide con-
sumption of tea is surpassed only by water.
The structurally related dimethylxanthines,
theophylline (5) and theobromine (61, have
less of a CNS stimulant effect, and are princi-
pally important for their ability to relax
smooth muscle. Cocoa and chocolate have lit-
tle caffeine, but do contain theobromine.
1.2 Caffeine
From an economic standpoint, the most im-
portant CNS stimulant is caffeine (1,3,7-tri- A regular cup of coffee contains between 40
methylxanthine, 4). It occurs naturally and is and 176 mgof caffeine, with a mean content of
a product of kola (cola) nuts (Cola nitida, about 85 mg. Tea contains less caffeine, with
Table 4.1 Psychostimulant and Anorexigenic Preparations
Generic Name Dosea
(structure) Trade Name Originator Chemical Class (mgtday)
Psychostimulants
Cocaine HCl(3) Cocaine HCl powder Mallinckrodt Ecgonine methyl ester NA
benzoate
Amphetamine (7) Adderall Shire Richwood Phenethylamine 5-30 mg
Amphetamine Amphetamine sulfate Lannett 5 mg
sulfate (7)
Dextroamphetamine Dextroamphetamine sulfate Various 5-10 mg
sulfate (10)
d
4'
Dexedrine SmithKline-Beecham
0 Richwood
Dextrostat
Methamphetamine Desoxyn Abbott 5 mg
HCl(11)
Desoxyn Gradumet Abbott 5-10 mg
Methylphenidate Methylphenidate HC1 Various a-Phenyl-2-piperidineacetic 5-20 mg
HCl(8) acid methyl ester
Ritalin Ciba-Geigy
Methylin Mallinckrodt
Metadate ER Medeva 10 mg, ER
Concerta Alza 18 mg, ER
Modafinil(44) Provigil Cephalon Diphenylmethyl-sulfinyl-2- 100-200 mg
acetamide
Pemoline (9) Pemoline 2-Amino-5-phenyl- 18.75-75 mg
4(5H)oxazolone
Cylert Abbott
PemADD Mallinckrodt
Caffeine (4) Quick Pep; Caffedrine; NoDoz; Thompson; Thompson; Bristol-Myers; Trimethylxanthine 75-200 mg
Stay Awake; Vivarin; Stay Major; SK-Beecham; Apothecary;
Alert; Enerjets; Starbucks Chilton
Anorexiants
Benzphetamine (22) Didrex Upjohn Phenethylamine 25-50 mg
Diethylpropion (16) Diethylpropion various Phenethylamine 25-75 mg
Tenuate Aventis Phenethylamine
Phendimetrazine Phendimetrazine various Phenethylamine 35 mg
(21)
Bontril PDM Carnrick
Plegine Wyeth-Ayerst
Phendimetrazine various 105 mg, SR
Adipost Jones
Bontril Slow-Release Carnrick
Dital UAD
Dyrexan-OD Trimen
Melfiat-105 Unicelles Numark
Prelu-2 Boehringer-Ingelheim
Rexigen Forte ION Labs
Phentermine (19) Phentermine Various Phenethylamine
Ionamin Medeva
-.L Fastin SmithKline-Beecham
V Ion
d zantqd
Adipex-P Lemmon
Obe-Nix 30-P Holloway
Decongestants and
Bronchodilators
Ephedrine sulfate Pretz-D 0.25% spray Parnell Phenethylamine NA
(1)
Ephedrine sulfate West-Ward Phenethylamine 25 mg
Ephedrine sulfate Various Phenethylamine 50 mg/mL
aAdministered orally unless otherwise noted.
CNS Stimulants
an average of about 27 mg per cup, and an narcolepsy (7) and, by 1936, orally active
ounce of sweet chocolate typically contains be- Benzedrine tablets were available without
tween 75 and 150 mg of combined methylxan- prescription (8). By 1937 it was being used
thines (4). recreationally by the general population,
The reader should be aware that the use of with particular popularity among American
the term CNS stimulation encompasses sev- college students (9).
eral physiological mechanisms of action and It is not clear when or by whom metham-
many different types of biologically active phetamine was first synthesized. Various ac-
substances. A number of different agents, in- counts indicate its first preparation some-
cluding caffeine (see below), affect these phar- where between 1888 and 1934 (5). In any case,
macological mechanisms and cause CNS stim- Hauschild (10) published the first studies of
ulation. Other diverse examples include the pharmacology of methamphetamine in
strychnine (causing CNS stimulation by 1938, characterized its stimulant effects in an-
blockade of inhibitory glycine receptors) and imals, and also carried out a self-experiment.
benzodiazepine inverse agonists (causing CNS
stimulation by decreasing the inhibitory ef-
3 CLINICAL USE OF AGENTS
fects of GABA on inhibitory chloride chan-
nels). It is not the intent of this chapter to
3.1 Therapeutic Applications
provide an encyclopedic treatment of all the
possible substances that can cause "CNS stim- Psychostimulants generally increase the level
ulation," but rather to focus primarily on the of activity, alleviate fatigue, increase alert-
psychostimulants (i.e., drugs that affect brain ness, and elevate mood (or cause euphoria in
monoaminergic systems). high doses). Unfortunately, the ability to pro-
duce euphoria leads these compounds to have
a high potential for abuse and dependency.
2 HISTORY The principal clinical indications for psycho-
stimulants are in the treatment of attention
The historical development of amphetamine deficit hyperactivity disorder (ADHD) and the
and methamphetamine is described in inter- sleep disorder known as narcolepsy. A less
esting detail by Angrist and Sudilovsky (5). commonly recognized use, but one that' is
The discovery of psychostimulants differs gaining importance, is in the treatment of de-
somewhat from the usual drug discovery pro- pression in terminal patients or the chroni-
cess because there was a long folkloric history cally ill (11-13). There is also need for psycho-
of the use of khat, coca leaves, and ma huang stimulants in certain occupations (e.g., in the
(ephedra). Although there may not have been military), as a countermeasure to fatigue from
a formal pharmacologicalclassification of CNS irregular or prolonged work hours, where a
stimulants at that time, the ability " of these high level of vigilance and alertness must be
agents to alleviate fatigue was well recog- maintained (14, 15). Some specific clinical ap-
nized. plications include the following.
Amphetamine itself was synthesized in
1887 and first studied as early as 1910, but its 3.1 .I Attention Deficit Hyperactivity Disor-
stimulant effects were not discovered until der (ADHD). ADHD is a diagnosis applied
about 1930. Amphetamine was independently mostly to children, but one that persists into
resynthesized in 1927 by the noted psycho- adulthood for many people. It is reflected in a
pharmacologist Gordon Alles in a program to persistent pattern of inattention and/or hy-
develop synthetic substitutes for ephedrine, a peractivity-impulsivity that is more frequent
drug then being used as a bronchodilator for and severe than typically observed in individ-
the treatment of asthma (6).
. . The central stim- uals at a comparable level of development (16).
ulant effects of amphetamine were probably Inattention prevents ADHD patients from
noted about 1930, when it appeared in nasal keeping their mind on one thing and focusing
inhalers in Germany. The first medical use their attention; they are easily bored with a
for amphetamine was in the treatment of task after only a short while. They have no
3 Clinical Use of Agents
difficulty devoting attention to activities that that may occur without warning and are often
they enjoy, but find it hard to focus conscious irresistible. Another hallmark symptom of
attention to organizing or completing a task, narcolepsy is cataplexy, which is a sudden loss
or learning something new. They may forget of voluntary muscle control, often triggered by
to plan ahead and tasks are rarely completed, emotions such as laughter or surprise. Cata-
or are filled with errors. plexy occurs more frequently during stress or
Children with ADHD (particularly of fatigue. The attack may involve only a feeling
school age) have great difficulty being still, of weakness and limp muscles or it may result
they may be in and out of their seats, and talk in total muscular collapse, during which the
incessantly. The inability to focus makes person can appear unconscious, but actually
learning tasks boring, and exacerbates the de- remains awake and alert. Attacks may be very
sire to move around and become involved in brief or may last for tens of minutes. Another
distractions. ADHD children may squirm, characteristic symptom of narcolepsy is hyp-
shake their legs, touch everything, or make nagogic hallucinations. These are vivid, realis-
distracting noises. Hyperactive teens and tic, and often frightening, reminiscent of
adults may feel intensely restless, and may try nightmares, and are usually accompanied by
to do several things at once, going from one sleep paralysis, a temporary inability to move.
activity to the next. Impulsivity is another Whereas the psychostimulants can have a
characteristic of ADHD, with patients often beneficial effect, they are likely to be sup-
acting without thinking about the conse- planted by newer drugs that are more specific
quences. They may have difficulty curbing and have fewer side effects.
their immediate reactions to situations, mak-
ing inappropriate remarks without thinking 3.1.3 Use for Depression in Terminal 111-
what they are saying. They find it hard to wait ness. Although this indication for psycho-
for things they want or to wait to take their stimulants is not as widely recognized, agents
turn. such as amphetamine and methylphenidate
In normal subjects, psychostimulants can are preferred because they do not suffer from
increase activity and talkativeness, especially the weeks-long delay in onset of action that is
at higher doses. Paradoxically, in ADHD suf- characteristic of traditional antidepressant
ferers, stimulants appear to have a calming medications. Thus, a rapid antidepressant re-
effect, and allow an increased focus and atten- sponse can be achieved in severely ill patients,
tion to tasks. Although appearing paradoxical, who in some cases may not survive long
it is now believed that the decreases in activity enough for a traditional antidepressant medi-
in ADHD are secondary to improvements in cation to begin to have an effect (11, 17-19).
attention. This beneficial effect of low doses of
the stimulants has led to a large number of 3.1.4 Use in Obesity. As noted earlier,
children being prescribed methylphenidate many of the psychostimulants have also been
(Ritalin) or various amphetamine prepara- used as anorectics (anorexics; anorexigenics),
tions for the treatment of ADHD. This, in that is, as appetite suppressants. A few of
turn, led to great concern about the fact that them are still useful in this regard, but the
these drugs were overprescribed for ADHD, high abuse potential of psychostimulants, cou-
and that children who are merely highly ener- pled with the development of tolerance to
getic were routinely being given them for be- their anorectic effects, has meant that pre-
havior management. The reader should be scribing psychostimulants for weight control
aware of this social issue, but it requires no has generally fallen into disfavor.
further comment in the context of this chap-
ter. 3.1.5 Apnea in Premature Infants. Apnea of
prematurity (AOP) occurs in about 90% of
3.1.2 Narcolepsy. Narcolepsy is a condi- premature neonates weighing less than 1 kg
tion that includes as its predominant symp- at birth, and in 25% of infants with a weight of
tom, excessive daytime sleepiness (EDS), per- less than 2.5 kg (20). The first-line pharmaco-
sistent drowsiness, and daytime sleep attacks logical therapies for the management of AOP,
CNS Stimulants
to stimulate respiration, are the methylxan- been recently discussed extensively by Angrist
thines, with theophylline (5) presently being (27). Interestingly, psychostimulants can in-
most extensively used. Recent studies suggest, duce a psychotogenic response in schizophren-
however, that caffeine (4) should be consid- ics, in doses that are subpsychotogenic in nor-
ered the drug of choice because of similar effi- mal subjects, and methylphenidate was found
cacy, longer half-life, fewer adverse effects, to have greater potency in this regard (28).
and better brain penetration than that of the- Activation of psychotic symptoms by methyl-
ophylline (21). phenidate was found to be a predictor of risk of
relapse (29). These, and other studies, are all
3.2 Side Effects, Adverse Effects, consistent with the dopamine hypothesis of
and Drug Interactions schizophrenia.
Generally, psychostimulants like amphet-
3.2.1 Methylphenidate. Methylphenidate
amine (7) and methylphenidate (8) can be
(Ritalin, 8) is widely prescribed for the treat-
used safely with most classes of medications
ment of ADHD. Approximately 90% of chil-
and with few contraindications (22). The
dren treated for ADHD are given methyl-
acute adverse reactions to stimulants can gen-
phenidate (30), representing about 2.8% of all
erally be understood from the perspective of
U.S. children aged 5-18 years (31). It is both
their pharmacology. Psychostimulants act as
well tolerated and efficacious in the treatment
indirect sympathomimetic agents; they either
of attention deficit hyperactivity disorder, and
directly release stored catecholamines, includ-
is associated with few serious adverse effects
ing those in peripheral adrenergic neurons re-
(32). Although there are rare reports of drug
sponsible for vascular tone, or else block their
interactions between methylphenidate and
reuptake. These actions affect the cardiovas-
certain other drugs, they are so infrequent
cular system in fairly predictable ways. In ad-
that there is no consistent pattern that can be
dition, cocaine produces a local anesthetic ef-
identified. Toxic concerns with methylpheni-
fect by the blockade of sodium channels (23).
date would principally revolve around the
Although this would normally be the pharma-
abuse of this drug to obtain a stimulant high,
cological basis for a class I antiarrhythmic
and the consequent possibility of developing
drug, it paradoxically induces proarrhythmia
dependency. A further concern with the long-
(24). term use of methylphenidate is the possibility
that patients may be at increased risk for psy-
chostimulant abuse. Although when taken
orally methylphenidate has a low euphori-
genic potential (33), when used intravenously
it has an abuse pattern and symptoms of tox-
icity similar to those of cocaine and amphet-
amine (34).
. . Recent studies in rats have also
In addition to acute effects, however, pro- shown that animals treated with methyl-
longed usage of amphetamines (and other psy- phenidate develop behavioral sensitization,
chostimulants) can produce an "amphetamine suggesting that human users may have in-
psychosis." This syndrome was first clearly creased susceptibility to psychostimulant
documented by Connell(25)and is regarded as abuse (35).
very similar to paranoid schizophrenia, char-
acterized by "paranoid psychosis with ideas of 3.2.2 Pemoline. Pemoline (91, an agent
reference, delusions of persecution, auditory used in treatment of ADHD, has been associ-
and visual hallucinations in a setting of clear ated with hepatotoxicity, with the majority of
consciousness" (25). The psychosis clears cases occurring in pediatric patients. From its
quickly after the drug is withdrawn. Psychosis marketing in 1975 up to 1989, 12 cases of
has been induced experimentally in normal acute hepatic failure and six deaths associated
subjects by continuous amphetamine admin- with pemoline hepatotoxicity had been re-
istration (26). Amphetamine psychosis has ported to the FDA (36). Death generally oc-
3 Clinical Use of Agents
parent children by using conduct norms that correspond to functional antagonism of this
are structured to produce individuals who receptor, similar to the effects of caffeine (57).
have reduced chances to become conventional 3.3 Absorption, Distribution, Metabolism,
adults (48). and Elimination
3.2.4 Caffeine. The psychostimulant ac- All substituted amphetamines are strong or-
tion of caffeine generally is accepted as well ganic bases, with pKa values ranging from 9.5
established. Caffeine quickens reaction time to 10 (58). The pKa of both cocaine and phen-
and enhances vigilance, increases self-rated metrazine is somewhat lower, at 8.5, and
alertness, and improves mood. There is, how- methylphenidate has a pK, of 8.8 (58). Thus,
ever, little unequivocal evidence to show that these bases are all significantly protonated at
physiological pH. Binding to their biological
regular caffeine use is likely to benefit sub-
targets also probably occurs with the proton-
stantially either mood or performance. In-
ated species [e.g., (5911. These drugs are all
deed, one of the significant factors motivating
administered as their water-soluble salts, usu-
caffeine consumption appears to be "with- ally as hydrochlorides or sulfates. At physio-
drawal relief" (49). logical pH, of course, these bases exist in an
Caffeine can produce adverse and unpleas- equilibrium between the protonated ionized
ant effects if doses are increased. Caffeine has form and the unprotonated un-ionized spe-
weak reinforcing properties, but with little or cies. The latter free bases are relatively lipid
no evidence for upward dose adjustment, pos- soluble and readily penetrate the brain, where
sibly because of the adverse effects of higher they exert their CNS stimulant effects. Many
doses. Withdrawal symptoms, although rela- of these drugs are eliminated in the urine un-
tively limited with respect to severity, do oc- changed because acidic urine leads to a higher
cur, and may contribute to continued caffeine fraction of protonated species, thus decreasing
consumption (50). Health hazards are small if reabsorption of the unchanged drug in the re-
any, and caffeine use is not associated with nal tubules. Decreasing urinary pH by, for ex-
incapacitation (51). Acute intake of caffeine ample, administering ammonium chloride
increases blood pressure, with the strongest leads to the anticipated increased urinary ex-
pressor response in hypertensive subjects. cretion and reduced duration of action (60). A.
Some studies with repeated administration of comparison has been reported of the urinary
caffeine have shown a persistent pressor ef- excretion pattern of methamphetamine in hu-
fect, whereas in others chronic caffeine inges- mans, guinea pig, and rat (61). In humans,
tion did not increase blood pressure (52). 23% of the dose was excreted unchanged.
Epidemiologic studies have produced contra- Ring-hydroxylated and N-demethylated me-
dictory findings regarding the association be- tabolites were excreted as 18 and 14%of the
tween blood pressure and coffee consumption. dose, respectively.
Duringregular use, tolerance to the cardiovas-
cular responses develops in some people, and 3.3.1 Amphetamine Metabolism. The me-
therefore no systematic elevation of blood tabolism of (+)-amphetamine (10)is variable,
pressure can be shown either in long-term or depending on the species studied. Possible
in population studies. Thus, regular caffeine metabolic transformations involve hydroxyl-
consumption may be harmful to some hyper- ation at the a or p side-chain carbon atoms,
tension-prone subjects (52). The hemody- the nitrogen atom, and thepara position ofthe
namic effects of chronic coffee and caffeine aromatic ring. These metabolites would then
consumption have not been sufficiently be further oxidized, or conjugated and ex-
studied. creted. One or more of these pathways pre-
Finally, caffeine may provoke a panic at- dominates, depending on which animal spe-
tack in individuals who suffer from panic or cies is being studied. In humans, the half-life
anxiety disorders (5356). Recently, an aden- of (+)-amphetamine has been reported as 7 h
osine &, receptor knockout mouse has been (62). About 30% of the dose of racemic am-
developed that has behavioral symptoms that phetamine is excreted unchanged, and acidifi-
3 Clinical Use of Agents
cation of the urine can decrease the half-life methylphenidate to between 10% and 50%
significantly (63,641.In humans, the principal (68). Ritalinic acid is not pharmacologically ac-
metabolite is benzoic acid (65). The details of tive. A ring-hydroxylated ritalinic acid metab-
the sequences of metabolic reactions of am- olite (2%) has also been identified. Other mi-
phetamine that lead to benzoic acid have not nor pathways involving oxidation of the
been elucidated (62), but the P-hydroxylated piperidine ring (0x0-ritalin) and conjugation
metabolite, norephedrine, has also been iden- reactions represent less than about 1%of the
tified as a metabolite in humans (66). administered dose (30).
The metabolism of methamphetamine (11)
involves both N-demethylation and ring hy- 3.3.3 Cocaine Metabolism. Because co-
droxylation. Caldwell et al. (61) reported that caine (3)has two ester functions, both can be
23% of the administered dose was excreted as hydrolyzed in vivo to generate metabolites.
unchanged drug, 18% as the 4-hydroxylated Hydrolysis of the methyl ester leads to benzo-
compound, and 14% as the N-demethylated ylecgonine (12), and hydrolysis of the benzoyl
amphetamine. ester leads to ecgonine methyl ester (13). Tro-
pan-3P-01-2P-carboxylicacid is known as ecgo-
3.3.2 Methylphenidate Metabolism. The me- nine (14). In cocaine users who also consume
tabolism and pharmacokinetics of methyl- significant amounts of ethanol, a transesteri-
phenidate have been studied extensively. fication product (cocaethylene, 15) is also de-
Methylphenidate (8)is administered as the ra- tected. Cocaethylene is also a potent psycho-
cemic threo isomer, but the (-)-threo enantio- stimulant, with about four times higher
mer is more rapidly metabolized. potency as a local anesthetic than that of co-
Methylphenidate is an ester, and the caine itself (69),and can enhance the cardio-
methyl ester is rapidly cleaved. The ester hy- toxicity associated with cocaine use.
drolysis product, called ritalinic acid, com-
prises about 80% of the urinary metabolites 3.3.4 Diethylpropion Metabolism. Diethyl-
after an orally administered dose (67). The la- propion (16) is used most extensively as an
bility of the ester function is probably the ma- appetite suppressant. It possesses the core
jor factor limiting the oral bioavailability of phenethylamine structure characteristic of
CNS Stimulants
many psychostimulants, but is a tertiary arni- apparently N-dealkylated readily and, thus,
noketone. It is extensively metabolized in hu- the principal metabolite is the N-deethylated
mans, with only about 3-4% of the drug ex- compound (17),constituting about 35%of the
creted unchanged. a-Alkylaminoketones are administered dose. Reduction of the carbonyl
4 Physiology and Pharmacology
is less important, with about 20% of the dose begun to focus attention on glutamate sys-
going that route, to afford N,N-diethyl- tems as potential key components of the ac-
norephedrine. About 30% of the dose cannot tions of psychostimulants. For example,
be accounted for as an amine product in the Swanson et al. (79) have shown that re-
urine and is probably a deaminated metabolite peated cocaine administration leads to long-
(70).Studies by Yu et al. (71) found that the term attenuation of group I metabotropic
N-deethylated metabolite (17) was probably glutamate receptor function in the nucleus
responsible for the pharmacological effects of accumbens. In particular, this functional re-
diethylpropion (16). These workers reported duction was related to significantly reduced
that the N-monoethyl metabolite (17) was a mGluR5 immunoreactivity in the medial nu-
substrate at the norepinephrine and serotonin
cleus accumbens. Even more exciting is the
transporters and an inhibitor at the dopamine
recent report that mGluR5 knockout mice
uptake transporter, whereas (1R,2S) and
(1S,2R)-N,N-diethylnorephedrine as well as do not display the reinforcing and locomotor
diethylpropion itself were inactive in those as- effects of cocaine, in spite of the fact that
says. cocaine administration increases extracellu-
lar dopamine in the nucleus accumbens of
these mice to levels that do not differ from
4 PHYSIOLOGY A N D PHARMACOLOGY those of wild-type animals (80).In the near
future, the role of glutamate systems in the
4.1 Where and How These Drugs Work
actions of psychostimulants will no doubt be
Neurons in the central nervous system com- more fully elucidated, resulting in new ap-
municate by chemical transmission. Of rele- proaches to the treatment of conditions that
vance to the present discussion are mono- now respond to classical stimulants.
amine neurons that release dopamine, There are two principal mechanisms for in-
norepinephrine, or serotonin as one of their creasing synaptic monoamine levels. One is to
transmitters in response to an action poten- block the reuptake of neurotransmitter after
tial. Reuptake transporter proteins embedded its excitation-coupled release from the neuro-
in the neuronal plasma membrane then clear nal terminal. Thus, blocking the action of the
the synapse of monoarnines, typically taking uptake carrier protein prevents clearance of
up 70-80% of the released transmitter. This the neurotransmitter from the synapse, leav-
reuptake is thought to be the major termina- ing high concentrations in the synaptic cleft
tion mechanism for the monoamine chemical that can continue to exert a signaling effect.
signaling process. This mechanism is the one invoked to explain
All psychostimulants appear to elevate the action of cocaine, a potent inhibitor of
synaptic levels of dopamine and norepineph- monoamine reuptake at the dopamine, seroto-
rine. In addition, cocaine and, to a lesser ex- nin, and norepinephrine transporters, and of
tent, some of the other agents also raise syn- methylphenidate, which is a reuptake inhibi-
aptic levels of serotonin. It is the current tor at the dopamine and norepinephrine
consensus that elevated dopamine levels transporters (81).It should be noted, however,
lead to CNS stimulation and are responsible that methylphenidate also has the ability to
for the reinforcing properties of stimulants induce the release of catecholamines stored in
(72-78). Nevertheless, recent studies have neuronal vesicles (82,83).
180 CNS Stimulants
Figure 4.1. Amphetamine interacts with the dopamine transporter protein (1)and is transported
inside. Na+ and C1- are cotransported, and Kf is countertransported in the process. After being
transported inside the terminal, high concentrations of amphetamine can displace dopamine from
vesicular storage sites (2),leading to elevated cytoplasmic levels of dopamine (3).After amphetamine
dissociates on the intraneuronal surface, dopamine binds to the carrier (4). The carrier then trans-
ports dopamine to the extracellular face (5), driven by the favorable concentration gradient, where
the dopamine dissociates and leaves the carrier available for another cycle.
The second mechanism is the one more rel- uptake. Not surprisingly, cocaine and amphet-
evant to the action of amphetamine and re- amines have effects on the cardiovascular
lated agents. This mechanism is illustrated in system, by virtue of their ability to enhance
Fig. 4.1. Amphetamine, and other small mo- indirect adrenergic transmission at peripheral
lecular weight compounds with similar struc- sites. Knowledge of the physiology of the sym-
tures, are substrates at the monoamine up- pathetic nervous system and the functions of
take carriers and are transported into the peripheral adrenergic nerve terminals allows
neuron. The uptake carrier has an extracellu- a relatively straightforward prediction of the
lar and intracellular face, and after transport- types of drug effects possessed by monoamine
ing a substrate (amphetamine, etc.) into the uptake inhibitors or releasing agents.
neuron, the intracellular carrier face can bind
to dopamine and transport it back to the ex- 4.2 Biochemical Pharmacology: Receptor
tracellular face. This exchange diffusion Types and Actions
mechanism is calcium independent, and is ca-
pable of robustly increasing synaptic trans- The monoamine reuptake carrier proteins
mitter levels. This process is often described (targets of the psychostimulants) are mem-
as a "reversal" of the normal uptake carrier bers of a larger Nat/C1- transporter family
process. that includes a number of other proteins, in-
Whereas the CNS stimulant effects of these cluding the GABA transporters, amino acid
molecules depend on an action in the brain, transporters, and orphan transporters (84).
uptake inhibitors and substrates at peripheral The primary amino acid sequence of the
monoamine carrier sites can obviously exert monoamine transporters is highly conserved,
other physiological effects. Cocaine is an excel- with several regions of these proteins having
lent local anesthetic agent. Furthermore, its high homology. It is presently believed that all
potent inhibition of norepinephrine reuptake of the members of this family possess a mem-
leads to stimulation of a-adrenergic receptors, brane-spanning 12 a-helix motif, with a single
causing local vasoconstriction that delays the large loop containing glycosylation sites on the
diffusion of the anesthetic agent out of the tis- external face of the membrane (Fig. 4.2).
sue. Similarly, users who chronically insuf- Members of this family of proteins have been
flate cocaine into their nasal passages often identified not only in mammalian species, but
develop necrotic lesions as a result of the local also in eubacteria and archaebacteria, indicat-
vasoconstricting effect of cocaine, again aris- ing their very early emergence in the evolution
ing from the blockade of norepinephrine re- of life.
gy and Pharmacology 181
Inside
-e 4.2. Representation of the 12-helix transmembrane transporter protein family. Both the
I- and the carboxyl-terminus are intracellular, with the second extracellular loop being larger,
uman norepinephrine uptake trans- stimulant drug of interest. The imaging tech-
1s first sequenced and then expressed nique then is used to determine how much of
cells in 1991 (85) and found to have the labeled ligand has been displaced from its
IS identical to those of the native receptors by competition from increased ex-
ber. The cloning and sequencing of tracellular endogenous dopamine. Based on
nine transporter (86-88) and the se- the known affinity of the labeled ligand for its
ransporter (89, 90) were reported in dopamine receptor, calculations can be used to
year. There are a number of excel- determine the increased concentration of do-
!w articles written about monoamine pamine that must have been available at the
;ers (84, 91-93). receptors. These definitive studies have
lacological studies of the mechanism clearly established a role for dopamine in the
For psychostimulants in animals have effects of stimulants in humans (72, 74).
liformly pointed to the importance of This type of approach has recently been ap-
pathways for the increases of loco- plied to the study of methylphenidate. For ex-
tivity and reinforcing properties (94, ample, Booij et al. (96) used SPECT imaging
:onclusions of those studies have gen- and an [1231]benzamidedopamine D, receptor
!n extrapolated to humans, with little ligand antagonist ([123111BZM)to measure sig-
vidence until recently to clearly sup- nificant displacement of the ligand by endoge-
e ideas. In the past several years how- nous dopamine that had been released in re-
ical studies of several stimulants, us- sponse to administration of methylphenidate.
yo brain imaging either with single In related work, Volkow et al. (74) used [llC]-
lission computed tomography (SPECT), (+I-threo-methylphenidateto show that greater
)n emission tomography (PET) tech- than 80% occupancy of the dopamine trans-
lave provided evidence for elevated porter was required to produce the stimulant
~ l adopamine
r in response to psycho- "high." With the dopamine D, receptor antag-
t administration. In essence, these onist [llClraclopride, Volkow et al. (97, 98)
!mploy either a single photon- or showed that the intensity of the methylpheni-
emitting dopamine receptor antago- date "high" was quantitatively correlated
labeled antagonist is administered with the levels of released dopamine and dopa-
ie absence and in the presence of the mine D, receptor occupancy. Subjects who
CNS Stimulants
perceived the most intense high had the high- postsynaptic receptor isoforms that are impor-
est increases in extracellular dopamine. Con- tant in the various actions of psychostimu-
versely, no high was experienced by subjects lants.
when methylphenidate did not increase dopa-
mine levels. In a second study, using the same 4.2.1 A Role for Serotonin. Although the
methodology, this same group (99) found that conventional wisdom is that stimulants ele-
subjects who "liked" the effects of methyl- vate synaptic dopamine, it is not at all clear
phenidate had significantly lower dopamine that this sole mechanism is responsible for the
D, receptor levels than those of subjects who spectrum of effects seen with the psycho-
disliked its effects. The authors speculated stimulants. In addition, it is becoming evident
that lower D, receptor density might be a fac- that animal models used to understand the
stimulants must consider mechanisms under-
tor contributing to psychostimulant abuse, by
lying effects on locomotor activity somewhat
providing a more pleasurable response. These
differently from those that govern either re-
imaging studies illustrate how data from ani-
ward or drug discrimination phenomena (101,
mal research can now be validated in humans. 102). The use of mice genetically deficient for
Because the stimulants cause increased the serotonin or dopamine transporter
synaptic levels of dopamine, and other mono- ("knockout mice") has produced some partic-
amine neurotransmitters, they indirectly lead ularly interesting findings. For example,
to stimulation of various postsynaptic recep- knockout mice lacking the DA transporter
tors, through the increased concentrations of have high levels of extracellular dopamine, a
neurotransmitter. A large number of animal condition that would presumably mimic the
studies have been reported that used various pharmacological action of cocaine and display
agonists and antagonists to elucidate the role spontaneous hyperlocomotion (103). Surpris-
of different dopamine receptor isoforms. Until ingly, these mice still self-administered co-
recently, however, only nonspecific ligands caine (104). Further experiments in these
(i.e., with effects on both the Dl-like and D,- mice indicated the probable involvement of
like families) were available. In drug discrim- the serotonin transporter. In addition, condi-
ination studies, rats have been trained to rec- tioned place preference, another animal model
ognize and discriminate the interoceptive cue of the reinforcing quality of a drug, could be
produced by injection of amphetamine or co- established for cocaine in mice lacking either
caine (100). Administration of a partial but the dopamine transporter or the serotonin
selective Dl-receptor agonist SKF 38393 was transporter (105). Place preference could also
partially recognized by these cocaine-trained be established for methylphenidate, another
stimulant that is thought to work through do-
rats, but not by amphetamine-trained ani-
pamine mechanisms, in mice lacking the dopa-
mals. Yet, both amphetamine- and cocaine-
mine transporter.
trained rats discriminated the cue produced
Experiments with knockout mice often
by a dopamine D, agonist bromocriptine as produce unexpected results. It must be kept in
being similar to their training drugs. A dopa- mind, however, that when a key protein is
mine D,-selective agonist produced cocaine re- missing during neural development, the off-
sponses, but was only partially recognized by spring often have some type of adaptation that
amphetamine-trained rats. Following addi- is not seen in the wild-type organism. Some
tional experiments with dopamine receptor caution, therefore, must be exercised in inter-
subtype selective antagonists, the authors preting the results. For example, Belzung et
concluded that the dopamine D, receptor al. (106) found that mice lacking the serotonin
played an essential role, but that both the Dl 5-HT,, receptor failed to display conditioned
and D, receptors might have some less impor- place preference. However, when these knock-
tant function. There is an extensive present out mice were compared in studies using clas-
research effort under way in many labora- sical pharmacological antagonists of the
tories that is attempting to elucidate both 5-HT,, receptor, divergent results were ob-
the anatomical substrates and the specific tained. The 5-HT,, receptor knockout mice
4 Physiology and Pharmacology
had an increased locomotor response and in- their ability to release NE, and not DA. Fur-
creased propensity to self-administer cocaine ther, their ranking in subjective effects did not
(107). By contrast, a 5-HT,, receptor antago- correlate with decreased plasma prolactin, a
nist attenuated cocaine-induced locomotor ef- response that is mediated by dopamine recep-
fects but had no effect on cocaine self-admin- tor stimulation. These authors suggested that
istration (108). The authors point out that NE might contribute to the amphetamine-like
compensatory changes during development of psychopharmacology of stimulants, at least in
the knockout mice may have rendered them humans.
more vulnerable to the effects of cocaine. In a related vein, the subjective psycho-
Nonetheless, there is a vast body of litera- stimulant effects of amphetamine were atten-
ture documenting interactions between dopa- uated following a 2-h pretreatment with a ty-
mine and serotonin pathways in the brain rosine- and phenylalanine-free amino acid
(109-111). Clearly, however, if a drug (e.g., mixture (118).These amino acids are biosyn-
cocaine) releases multiple transmitters, then a thetic precursors of the catecholamines, and
behavioral interaction is not surprising. Inhi- deprivation would be expected to produce
bition of presynaptic reuptake of serotonin, transient reductions in endogenous dopamine
for example, could lead to postsynaptic activa- and norepinephrine. The authors concluded
tion of a variety of other receptors, some of that tyrosine depletion attenuates the release
which could modulate dopamine function. In of dopamine required for the psychostimulant
addition to potential effects on 5-HT,, recep- effect. Interestingly, the pretreatment did not
tors, other studies have implicated serotonin reduce the subjective appetite-suppressant
5-HT, receptors (112), 5-HT,, receptors (anorectic) effect of amphetamine. The study
(113),and 5-HT,, receptors (114). 5-HT,, re- authors attributed this latter finding to a con-
ceptors can also modulate the locomotor ef- tinued release of norepinephrine by amphet-
fects of cocaine (115). amine. Tyrosine depletion, however, would
also attenuate norepinephrine biosynthesis
4.2.2 A Role for Norepinephrine. Although and it may be more reasonable to conclude
the vast majority of studies of psychostimu- that the anorectic effect might be related to
lants have focused on the role of dopamine the often-overlooked ability of amphetamine
andlor serotonin, the importance of norepi- to release neuronal serotonin.
nephrine (thought to be paramount 30 years This chapter makes no attempt to review
ago) is generally now overlooked. Details of all the literature that focuses on the role of
the mechanism of action of psychostimulants norepinephrine and serotonin in the actions of
have been developed primarily through the psychostimulants. At the time of this writing,
use of animal models, in which dopamine the general consensus seems to be that effects
seems to be the key player, and these results on dopamine systems are necessary, but per-
then have been extrapolated to humans. Yet, haps not sufficient, conditions to explain all
cocaine also is a potent NE uptake inhibitor, the different actions of stimulants. There ap-
and the potency of amphetamine for norepi- pears to be increasing awareness, spurred ini-
nephrine release is similar to that for dopa- tially by studies of cocaine, that serotonin may
mine release. Indeed, in the rat prefrontal cor- be a much more important player than was
tex, amphetamine and cocaine increased heretofore recognized. In the next few years
extracellular norepinephrine to an extent that this role likely will be studied and elucidated
was quantitatively similar to that of dopamine in much greater detail.
(116).Further, it appeared that the increase in The role of norepinephrine in the actions of
prefrontal cortical norepinephrine was actu- stimulants has largely been overlooked, al-
ally attributable to the blockade of the norepi- though a few studies suggest that this trans-
nephrine transporter by both drugs. Recently, mitter may be of major significance. On the
Rothman et al. (117) reported that the oral other hand, until clinical studies are carried
doses of several stimulants required to pro- out using receptor blockers and specific nor-
duce amphetamine-like subjective effects in epinephrine transporter inhibitors, this area
humans were most closely correlated with will remain muddy, at best. In virtually every
CNS Stimulants
example, from amphetamine to cocaine, the of locomotor activity in animal models, studies
compounds have significant effects at the nor- have implicated the dopamine D, receptor
epinephrine transporter, in some cases equal (129, 130). It has not been clear, however,
to, or even greater than, those at the dopamine whether effects mediated by striatal adeno-
transporter. When behavioral or mood changes sine &, receptors absolutely depend on the
are correlated with levels of extracellular do- presence of dopamine D, receptors. To study
pamine, and dopamine is highly correlated this problem, Chen et al. (131) employed ge-
with changes in extracellular norepinephrine, netic knockout mice deficient either in dopa-
one cannot be certain which underlying phar- mine D, receptors or adenosine &, receptors,
macology is ultimately more important with- or a double knockout mouse deficient in both
out experiments using specific blockers of both types of receptors. These studies found that
dopamine and norepinephrine transporters A, receptors may affect neuronal activity in a
and receptors. It may be that effects on dopa- manner that is partially independent of the
mine are necessary, but not sufficient, and presence of dopamine D, receptors, such that
that both norepinephrine and serotonin play endogenous adenosine may be most accu-
modulatory roles. Because the stimulants rately viewed as a facilitative modulator of
have such diverse effects, including increasing striatal neuronal activity rather than simply
activity, mood, appetite suppression, and so as an inhibitory modulator of D, receptor
forth, it seems likely that serotonin and nor- neurotransmission.
epinephrine play more or less important mod- These studies, and many others, conclude
ulatory roles, depending on which aspects of that the acute locomotor stimulant effects of
the specific drug's effects are being studied. caffeine in animal models are mediated in part
Caffeine and the other methylxanthines in- by dopaminergic systems and dopamine recep-
hibit phosphodiesterases, the enzymes that tors. Recent studies suggest that tolerance to
degrade CAMP.For many years it was believed the locomotor stimulant effects of chronic caf-
that the stimulant effect of caffeine was attrib- feine may also be related to specific changes in
uted to this enzyme inhibition. At the plasma dopaminergic function (128). Thus, in spite of
concentrations obtained after two to three the fact that methylxanthines are structurally
cups of coffee ( ~ 1 @),
0 however, antagonism different from other psychostimulants, and do
of adenosine A, (and A,) receptors in brain is not directly affect dopamine transporters or
believed to be the most relevant action to ex- receptors, in fact their stimulant action is de-
plain the stimulant effects of caffeine (119, rived from effects on central dopamine path-
120). Perhaps not surprisingly, in view of ear- ways.
lier discussion in this chapter, caffeine admin-
istration has been shown to lead to elevated
5 STRUCTURE-ACTIVITY RELATIONSHIPS
levels of brain dopamine (121, 122). It is
thought that adenosine receptor stimulation
Examination of the structure-activity rela-
facilitates GABA- or acetylcholine-mediated
tionships (SARs)of several of the classic stim-
inhibition of dopamine receptors in striatopal-
ulants provides not only an understanding of
lidal and striatonigral neurons (123), with the
the development of other drugs, but provides
end result of decreased dopaminergic func-
important clues as to the underlying mecha-
tion; adenosine antagonists would thus have a
nisms involved in interaction with the target
reverse action. Many studies have examined
protein(s). The following sections will hope-
the interaction between adenosine A,, recep-
fully illustrate both of these points.
tors and dopamine receptors, both of which
are highly concentrated and colocalized in the
5.1 Amphetamine
striatum and have reciprocal antagonistic in-
teractions (124-127). There is abundant evi- There are a number of related structures that
dence for pre- and postsynaptic interactions are often referred to as "amphetamines," al-
between adenosine and dopamine receptors, though the name amphetamine refers to one
by which adenosine inhibits doparninergic ac- specific molecular entity. Grouped in this class
tivity [e.g., (128)l. With respect to stimulation would be (+)-amphetamine (101,N-methyl-
5 Structure-Activity Relationships
alkyl groups of p-aminoketones are readily leling the potency difference found with the
cleaved metabolically. Thus, the N,N-di- enantiomers in vitro, using rat brain striatal
methyl cathinone analog is likely converted in synaptosomes (149). The two isomers were of
vivo to the N-monomethyl compound meth- nearly equal potency in their effects on norepi-
cathinone. This argument is based on evi- nephrine accumulation by rat hippocampal
dence that for diethylpropion, the N,N-diethyl synaptosomes (149). This stereochemical re-
congener of cathinone, it is the N-monoethyl quirement applies to p-keto derivatives as
metabolite that is the active species (70, 71). well; the corresponding active isomer has the
Although longer N-alkyl groups lead to less S-(-) configuration (136).
active compounds, one exception to this gen-
eralization is benzphetamine (Didrex),N-ben- 5.1.4 The a-alkyl Substituent. The a-alkyl
zyl-N-methylamphetamine (22). Despite the group cannot be much larger than a methyl.
N,N-dialkyl groups in benzphetamine, in hu- Phenethylamine itself, lacking the side chain
mans it produces subjective effects character- a-methyl group, is inactive in vivo because of
istic of amphetamine-like drugs such as phen- its rapid inactivation by monoamine oxidase.
metrazine (20) (143). Althoughpara-hydroxy- Addition of the a-methyl group retards metab-
N-benzylamphetamine is a major metabolite olism by this route, leading to the orally bio-
of benzphetamine, methamphetamine and available drug amphetamine. The uptake
amphetamine are also detectable in urine and transporter, however, cannot tolerate large
hair following administration of benzphet- groups in this region and the a-ethyl analogs
m i n e (144-146). It is not clear from the liter- of both amphetamine and methamphetamine
ature whether the reinforcing effects of benz- had markedly attenuated activity in a drug
phetamine are attributable to metabolic discrimination assay with rats trained to dis-
formation of amphetamine or methamphet- criminate (+)-amphetamine (150). a,a-Di-
amine. Based on the studies with N,N-dimeth- methyl groups, as in phentermine (19),
ylamphetamine by Witkin (138),however, one though giving an active compound, still reduce
would predict that the parent molecule has activity.
some pharmacological activity. Attempts to incorporate the side chain into
ring structures also led to compounds with at-
5.1.3 Stereochemistrv at the a Carbon. tenuated activity. For example, in drug dis-
The stereochemistry at the a carbon atom, crimination assays using rats trained to recog-
when enantiomers exist, is homochiral to that nize the effect of (+)-amphetamine (lo),
of S-(+)-amphetamine (lo), shown earlier. compounds (23) and (24) either failed to pro-
Both the releasing actions at dopamine and duce amphetamine like effects, or had much
norepinephrine transporters in isolated rat lower potency (150, 151). When n = 3, the
brain slices (147) and the locomotor and ste- compound lacked any amphetamine-like ac-
reotypic effects in rodents (148) are more po- tion.
tently affected by the S-(+) isomer of amphet-
amine than by the R-(-1 isomer. In this latter 5.1.5 Other Side-Chain Substitutions. Lim-
study, the (+) enantiomer was about five ited substitution of the side chain is tolerated.
times more potent than the (-1 isomer, paral- A p-hydroxy group on methamphetamine
5 Structure-Activity Relationships
monoamine transporters (161). In phenyl- would suggest that this interaction should be
tropane analogs where the ester linkage has very weak.
been removed (281,extensions of the N-alkyl Replacement of the nitrogen atom with ox-
out to n-butyl have no effect on dopamine ygen as in (30) gives compounds that retain
transporter affinity (162). Effects a t the se- high affinity for the dopamine transporter
rotonin transporter are variable, but affinity (165).This finding was accommodated by pro-
only decreases modestly. At the norepineph- posing that the oxygen atom could act as a
rine transporter, affinity drops about three hydrogen bond acceptor at the transporter
times with the longer N-alkyl group. (165),a conclusion that would at least be con-
sistent with the activity of the N-sulfonated
derivatives (29).
It was even more surprising, therefore,
when the report appeared that even a polar
oxygen was not required for good uptake in-
hibitors. Carbocyclic compounds such as (31)
proved to have transporter affinities nearly
equal to those of their amine-containing coun-
terparts (166)!
(33) R = alkyl
ing the monoamine transporters at the molec- may block the actions of cocaine without
ular level. Whereas it has been known in markedly affecting the normal transport func-
pharmacology for many years that cocaine tar- tion of the protein. Hence, there is presently
geted an energy-dependent reuptake pump, intense interest in such compounds because
the cloning and expression of these transport- they may provide new avenues for the treat-
ers has given access to high "purity" proteins ment of cocaine and psychostimulant addic-
that are amenable to more detailed study. tion.
These proteins have all been sequenced, and There also is a need for improved drugs to
shown to be membrane bound with 12 mem- replace existing CNS stimulants, as treat-
brane-spanning helical segments. A large ments for medical conditions like narcolepsy,
number of site-specific mutations have been ADHD, obesity, and for general attentional
used to correlate specific residues in the pro- purposes. Yet, virtually all of the existing
tein with specific functions. Nevertheless, it stimulants have the capacity to produce en-
will likely take a technical breakthrough to hanced mood, or euphoria. This side effect
obtain a crystal structure of one of these trans- means that they all possess abuse potential to
porters or one of their homologs, an accom- a greater or lesser degree. Advances in medic-
plishment that will no doubt lead to much inal chemistry and molecular neurobiology
greater understanding of how transporters have provided hope, however, for new genera-
function. Inability to crystallize these proteins tions of drugs. For example, recently the non-
is not a problem that is unique to monoamine amphetamine, nonstimulant drug modafinil
transporters, but continues to plague the (Provigil, 45) was approved for use in narco-
study of all membrane-bound transporters lepsy (see, e.g., Ref. 187). This drug has been
and receptors, and other membrane-bound shown to be more effective than amphet- -
proteins. amine, with fewer side effects, although its
Site-directed mutagenesis, cysteine-scan- mechanism of action has not yet been eluci-
ning accessibility methods, high field NMR, dated. The discovery that mutations of either
homology modeling, and continued develop- the gene for the novel neuromodulator orexin,
ment of structure-activity relationships will or the orexin receptor, can cause narcolepsy,
no doubt lead to better and better models of leads to the hope for even better and more
the monoamine transporters. These ap- specific drugs to treat that disorder, as well as
proaches are having the greatest impact on to the possibility of better treatments for obe-
studies of uptake inhibitors, rather than stud- sity (188-191).
ies of substrates.
Particularly interesting recent advances
indicate that ligands from different chemical
classes may bind in novel ways to the dopa-
mine transporter. Using site-directed mu-
tagenesis and photoaffinity labeling probes,
investigators have produced results suggest-
ing that the substrate uptake and cocaine
binding sites are probably not identical (184).
Indeed, it also now seems likely that different
chemical classes of uptake inhibitors may even
bind to distinct regions of the transporter
(185, 186), leading to different overall confor- Even those actions of the stimulants that
mations in the transporter protein and per- are absolutely dependent on activation of
haps subtly altered mechanisms of inhibition. monoamine systems may also be amenable to
These different transporter conformations breakthroughs in medicinal chemistry. For
would explain the observed differences in the example, the beneficial effects of stimulants
pharmacology of different chemical classes of on ADHD may be attributable primarily to ac-
DAT inhibitors. New derivatives that have se- tivation of only certain receptors. Recently,
lective affinity at these alternate binding sites the anatomical and functional substrates of
CNS Stimulants
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that certain drugs [e.g., selective D, dopamine 12. J. Olin and P. Masand, Psychosomatics, 37,
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the beneficial effects of the stimulants without 13. H. B. Rothenhausler, S. Ehrentraut, G. von
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CHAPTER FIVE
Sedative-Hypnotics
JULIUSVIDA
Vida International Pharmaceutical Consultants
Greenwich, Connecticut
JOSEPH YEVICH
Pharmaceutical Research Institute
Bristol-Myers Squibb Company
Wallingford, Connecticut
Contents
1 Introduction, 202
1.1 Classification of Sedative-Hypnotics, 202
1.2 The Ideal Hypnotic Drug, 203
2 Clinical Use of Agents, 203
2.1 Current Drugs on the Market, 203
2.2 Side Effects, Adverse Effects, and Drug
InteradionslContraindications, 203
2.2.1 Barbiturates, 203
2.2.2 Benzodiazepines, 206
2.2.3 Halogenated Sedative-Hypnotics, 208
2.2.4 Heterocyclic Sedative-Hypnotics, 210 .
2.2.5 Antihistamines, 211
2.3 Absorption, Distribution, Metabolism, and
Elimination, 211
2.3.1 Barbiturates, 211
2.3.1.1 Absorption, 211
2.3.1.2 Distribution, 211
2.3.1.3 Metabolism, 214
2.3.1.4 Elimination, 216
2.3.2 Benzodiazepines, 217
2.3.2.1 Absorption, 217
2.3.2.2 Distribution, 217
2.3.2.3 Metabolism, 218
2.3.2.4 Elimination, 218
2.3.3 Halogenated Sedative-Hypnotics, 220
2.3.3.1 Chloral Hydrate, 220
2.3.3.2 Ethchlorvynol, 221
2.3.4 Heterocyclic Sedative-Hypnotics, 221
2.3.4.1 Glutethimide, 221
2.3.4.2 Clomethiazole, 221
2.3.4.3 Zopiclone, 221
Burger's Medicinal Chemistry and Drug Discovery 2.3.4.4 Zolpidem, 221
Sixth Edition, Volume 6: Nervous System Agents 2.3.4.5 Zaleplon, 223
Edited by Donald J. Abraham 2.3.5 Antihistamines, 223
ISBN 0-471-27401-1 O 2003 John Wiley & Sons, Inc. 3 Physiology and Pharmacology, 233
201
Sedative-Hypnotics
Average
Duration Adult
Trade Other of Action Onset of Hypnotic
Barbituric Acid Generic Name Name RS R Modifications (h) Action (h) Dose (g)
5-Ethyl-isopentyl Amobarbital Amytal 2-8 0.25-0.5 0.1-0.3
5-Allyl-5-isopropyl Aprobarbital Resedorm 2-8 0.25-0.5 0.065-0.13
5,5-Diethyl Barbital Barbitalum 4-12 0.5-1 0.3-0.5
5-(1-Cyclohexen-1- Hexobarbital Sombulex 1-4 0.25 0.25-0.4
y1)-1,5-dimethyl
5-Ethyl-l-methyl-5- Mephobarbital
phenyl
5-Allyl-5-(1-methyl- Methohexital Brevital
2-pentynyl)
5-Ethyl-5-(1- Pentobarbital Nembutal
methylbutyl)
5-Ethyl-5-phenyl Phenobarbital Luminal
5-Isopropyl-5-ethyl Probarbital Ipral
5-Allyl-5-(1-methyl Secobarbital Second
butyl)
5-Allyl-5-see-butyl Talbutal Lotusate
5-Allyl-5-(1-methyl Thiamylal surital
buty1)-2-thio
5-Ethyl-5-(1-methyl Thiopental Pentothal 4-12 30 Seconds 0.1-0.15
butv1)-2-thio
*The principal route of administration for barbiturates is oral. However many barbiturates (hexobarbital, methohexital, pentobarbital, thiopental, etc.) are also available in
injectable formulation but indicated for anesthesia (not as sedative hypnotics).
Table 5.2 Sedative-Hypnotic Barbiturates in Clinical Use Outside the United States
Average
Duration Adult
Trade Other of Action Onset of Hypnotic
Barbituric Acid Generic Name Name R5 R Modification (h) Action (h) Dose (g)
Allobarbital Dial,
Curral
Brallo barbital Vespoerone
The rate of metabolism of several drugs (war- sion. Tolerance to benzodiazepines often de-
farin, phenytoin, tricyclic antidepressants, velops but occurs less frequently than that
oral contraceptives, etc.) is increased. with barbiturates.
Barbiturates are rarely used today, and Withdrawal of benzodiazepines can cause
when barbiturate treatment is initiated, it rebound of the original symptoms. A transient
should be restricted to 2 weeks because of the worsening of these symptoms may occur espe-
risk of tolerance developing. cially if benzodiazepines are used in high dose
and for a long duration.
2.2.2 Benzodiazepines. Benzodiazepines In 2001, Flunitrazepam has been subjected
(2) (see Table 5.3) in low doses have a sedative to the same prescribing restrictions as narcot-
effect; however, in higher doses, they induce ics in several countries because of its continu-
sleep and may even lead to coma. The sedative
ing abuse by drug addicts.
effect of benzodiazepinesimpairs motor skills,
The adverse effects of benzodiazepines in-
attention, memory, judgment, and if severe,
clude dizziness, vertigo, light-headedness,
may lead to confusion and incoordination. The
risk of accidents is increased, including an in- headache, changes in libido, ataxia, tremor,
crease of road traffic accidents. Consumption and urinary retention or incontinence.
of alcohol potentiates the effects of benzodiaz- The sedative and hypnotic effects of benzo
epines. diazepines are accentuated by other drugs act-
Benzodiazepines also cause anti-anxiety, ing on the gamma-aminobutyric acid,
muscle relaxant, and anticonvulsant effects, (GABA,) receptor, especially barbiturates
amnesia, depression, and respiratory depres- and alcohol. However, there are fewer drug
(2)
Benzodiazepine RI & % RS
Cinolazepam OH
Delorazepam H
Doxefazepam OH
Flunitrazepam H
Flurazepam H
Lorazepam OH
Lormetazepam OH
Nimetazepam H
Nitrazepam H
Nordazeparn H
Potassium clorazepate COOK
Quazepam H
Temazepam OH
Table 5.4 Benzodiazepines
No. Generic Structure Trade Name Originator Chemical Class First W/WLaunch Dose (mglday)
- - - - -
Chloralodol or
chlorhexadol
Carbochloral
Ethyl N-(2,2,2-trichloro-1-
hydroxyethyl carbarnate
Dichloralphen-
4-Dimethylamino-2,3-dimethyl- azone
1-phenyl-3-pyrazolin-5-one
compound with chloral
hydrate
Triclofos
2,2,2-Trichloroethanol, di-H-
phosphate, sodium salt
Ethchlorvynol
interactions because benzodiazepines do not and dependence and their danger in overdose.
induce hepatic microsomal enzyme produc- Chloral hydrate (3) (see Table 5.5) seems the
tion. least problematic with relatively low abuse po-
The advantages of benzodiazepines over tential; it is still used as an alternative to
older hypnotic drugs and especially over bar- benzodiazepines. The adverse effects include
biturates are as follows: safer in overdose, less gastric irritation, light headedness, ataxia,
tendency to tolerance and dependency, less nightmares, excitement, confusion, allergic
adverse potential, greater sedation-to-anxioly- reactions, and skin rash. It also causes drows-
sis ratio, and fewer drug interactions. iness and motor incoordination; therefore,
chloral hydrate should not be used concomi-
2.2.3 Halogenated Sedative-Hypnotics. tantly with other CNS depressant drugs such
Most of these agents are only rarely used be- as alcohol. The adverse effects of ethchlorvy-
cause of their high risk of causing tolerance no1 (4) (see Table 5.5) are similar to those of
Table 5.6 Halogenated Sedative Hypnotics
Generic Name
No. (Structure) Trade Name Originator Chemical Class Dose (mgld)
1. Chloral hydrate Noctec Squibb 2,2,2-Trichloro-1,l-Ethandiol p.0. 500-1000
2. Chloralodol or Lora Wallace (USA) 2-Methyl-2(2,2,2-trichloro-1-hydroxy p.0.800-1600
chlorhexadol ethoxy)S-pentanol
3. Carbochloral Prodorm Parke-Davis Ethyl N-(2,2,2-trichloro-1- p.0. 400-800
hydroxyethyl carbamate
4. Dichloralphenazone Welldorm Smith & Nephew (UK) 4-Dimethylamino-2,3-dimethyl-1- p.0. 225-700
Chloral01 Homer (Canada) phenyl-3-pyrazolin-5-one
Bonadorm Ferroson (Denmark) compound with chloral hydrate
5. Triclofos Triclos Marion Merrell Dow 2,2,2-Trichloro ethanol, di-H- p.0.500-1000
phosphate, sodium salt
6. Petrichloral Petrin Parke-Davis Pentaerythritol hemiacetal with p.0. 1200-2000
chloral
7. Carbromal Carbrital Parke-Davis 2-Bromo-2-ethyl-butyryl urea p.0. 250-1000
Somben Chinoin (Hungary)
Talambrol Kwizda (Austria)
8. Ethchlorvynol Placidyl Abbott 1-Chloro-3-ethyl-pent-1-en-4-yn-3-01 p.0. 100500
210 Sedative-Hypnotics
5-(2-Chloroethy1)-4-methylthiazole Clomethiazole
(6)
N f; A
OC-N
wN-CH3
(1)Imidazo[l,2-alpyridine-3- Zolpidem
acetamide,N,N,6-trimethyl-2-(4- (8)
methylpheny1)-,[R,-(R*,R*)]-2,3-
dihydroxybutanedioate (21);
N-(3-(3-Cyanopyrazolo[1,5-a] Zaleplon
pyrimidin- (9)
7-ylpheny1)l-N-ethylacetamide
chloral hydrate. Ethchlorvynol is contraindi- ment, hangover, blurred vision, and skin
cated in patients with porphyria, hepatic, and rashes.
renal failures. Overdose of glutethimide may cause even a
greater danger to life than barbiturate over-
2.2.4 Heterocyclic Sedative-Hypnotics. dosage (4); glutethimide has the highest
The adverse effects of glutethimide (5) (see mortality of drug-induced comas (5).Like bar-
Table 5.7) include nausea, headache, excite- biturates, glutethimide induces microsomal
2 Clinical Use of Agents
hepatic enzymes and can cause increased me- nausea, vomiting, diarrhea or constipation,
tabolism of anticoagulants and other drugs anorexia, or increased appetite. They may also
with reduced effects. produce antimuscarinic effects such as
Clomethiazole (6) (see Table 5.7) may pro- blurred vision, dysuria, micturition, dryness
duce nasal irritation and sneezing - on admin- of mouth, tightness of the chest, hypotension,
istration, conjunctival irritation, headache, muscular weakness, tinnitus, euphoria, and
gastrointestinal disturbances, nausea, vomit- occasionally headaches. Antihistamines should
ing, fever, cough, and tachycardia. It is contra- not be given to neonates and are contraindi-
indicated in patients with chronic pulmonary cated during acute attacks of asthma. All an-
insufficiency, renal failure, or liver disease. tihistamines used as sedative-hypnotics be-
Zopiclone (7) (see Table 5.7) has a short half- long to the class of HI-receptor antagonists.
life but can cause daytime sedation. However, it
causes less morning-after drowsiness and fa- 2.3 Absorption, Distribution, Metabolism,
tigue than longer-acting hypnotics. Rebound and Elimination
phenomena following withdrawal have not
proved a serious problem. Zopiclone causes min- 2.3.1 Barbiturates
imal impairment of psychomotor performance 2.3.1.1 Absorption. Barbiturates are well '
and mental alertness the morning after night absorbed after oral administration usually
time administration (6). given as sodium salts. They have a long dura-
Zolpidem (8) (see Table 5.7) rarely causes tion of action, usually 6-10 h. Peak plasma
daytime
- sedation because of its short duration levels occur in 2-12 h and are dose related.
of action. However, it may cause nausea, vom- 2.3.1.2 Distribution. The lipid solubility
iting, diarrhea, headaches, and dizziness. (lipophilicity)of barbiturates varies and deter-
Tolerance to its use can develop, but this is mines how readily they cross the blood-brain
less than with benzodiazepines. It may also barrier.
cause mild respiratory depression. With- It is possible to calculate the amount of bar-
drawal symptoms associated with zolpidem bituric acid in the undissociated form at vari-
are unusual. ous pH values from the equation:
Zaleplon (9) (see table 5.7) is a very short-
acting hypnotic that occasionally causes head-
aches and dizziness and may lead to respiratory
depression. It rarely causes daytime sedation be-
cause of its short duration of action. Psychomo- where [A-I = Concentration of the anion
tor impairment is unusual. The risk of tolerance
is unknown; the withdrawal symptoms are rare, [HA] = Concentration of the undis-
and dependency is unlikely. sociated acid
2.2.5 Antihistamines. Antihistamines may The values of several examples are given in
produce gastrointestinal disturbances such as Table 5.13.
Table 5.9 Antihistamines
Structure Chemical Name Generic Name
9 CH-0-CH2-CH2-N
/CH3
2-Benzhydryloxy-N,N, -dimethylamine Diphenhydramine
d \
CH3
q C-0CH2CH2N
/CH3
\
Doxylamine
CH3
3
l . t
1-(p-Chloro-a-phenylbenzy1)-4-[2-[2-(2- Etodroxizine
hydroxy-ethoxy)ethoxy]-
ethylldiethylenediamine
Hydroxyzine
f.7 2-[2-[4-[(4-Chloropheny1)phenyl-methyl]-
N- C H ~ - CH~-- 0- C H ~ - C H ~ - OH 1-piperazinyll- ethoxylethanol
N-[3-[4-(p-Fluropheny1)-1-piperazinyl]-1- Niaprazine
methyl-propyllnicotinamide
CH3
N,Na-Trimethyl-1OH-phenothiazine-10- Promethazine
ethanamine
Propiomazine
Pyrilamine
214 Sedative-Hypnotics
Among the given examples, only 5-ethyl-5- from 0.36 to 0.67 Llkg. Phenobarbital enters
phenylbarbituric acid and l-methyld-ethyl-5- the brain in a manner reflecting blood flow
phenylbarbituric acid have a proper ratio of patterns, and in steady state, it is evenly dis-
dissociated forms present at physiological pH tributed in gray and white matter. The bind-
to enable them to cross the blood-brain barrier ing to plasma proteins of barbiturates plays a
and exert an effect in the CNS. minor role in distribution.
Lowering serum pH increases the non-ion- 2.3.1.3 Metabolism. The principal site
ized (undissociated) portion in the serum, en- of metabolic inactivation is in the liver. In
hancing diffusion into tissue, whereas higher the metabolism, the lipophilic character of
serum pH has the opposite effect (7). the barbiturates decreases, which in turn
Estimates of the apparent volume distribu- decreases the ability of the barbiturates to
tion for phenobarbital vary nearly twofold penetrate into the CNS. There are four pri-
N-[2-(5-Methoxy-lH-indol-3-yl)ethyl] Melatonin
acetamide
2 Clinical Use of Agents 215
) Bmrnazepam I
Brotizolam
Loprazolarn Midazolam
Estazolam: X=H.Y=H
Figure 6.1. Benzodiazepine Triazolam: X = CH,. Y = CI
structures.
0 0
Pentobarbital 5-Ethyl-5-(3-hydroxyl-1-
methylbutyl) barbituric
acid
CH3
I
\
Acetylurea
cleavage derivative 2.3.2.1 Absorption. The rate of absorption
along b (5.3) from the gastrointestinal tract after oral dos-
age determines the speed of the onset of action
of a benzodiazepine. For a quick onset of ac-
tion, the benzodiazepine must dissolve com-
pletely in the stomach and cross the stomach
mucosa into the systemic circulation. The dif-
Acetamide ferent dissolution and absorption kinetics of
derivative benzodiazepines will affect their onset of ac-
tion. Once the benzodiazepine is in the sys-
dent of concentration. From 11% to 50% of temic circulation, it must also cross the blood-
phenobarbital is eliminated from the body per brain barrier to enter the CNS. Therefore, the
day, corresponding to a half-life range of 24- lipophilicity of the benzodiazepine is important
140 h. Average half-lives after single doses in determining the entry into the CNS and the
range from 75 to 126 h and are not influenced onset of clinical action. Most benzodiazepines
by route of administration. Alkalization of are highly lipophilic with the 3-hydroxy-substi-
urine increases phenobarbital excretion. tuted benzodiazepines (lormetazepam, loraz-
epam, temazepam, and doxefazepam); triazo-
2.3.2 Benzodiazepines. The pharmacoki- lam is the least lipophilic.
netic properties of benzodiazepines are most 2.3.2.2 Distribution. Distribution is one of
important in selecting a specific drug for the the important factors contributing to the du-
treatment of sleep disorders. In fact, pharma- ration of action of benzodiazepines. Benzodi-
cokinetic parameters provide a rational azepines normally cross the blood-brain bar-
method for selecting a benzodiazepine specifi- rier quite easily and exert a pharmacodynamic
Sedative-Hypnotics
CI %'5
0
0
CH3
Minor pathway
- cl
0
CH3
OH - Glucuronide
Diazepam Temazepam
t
0
CI
0
Clorazepate
Figure 5.4. Biotransformation pathways of potassium clorazepate, diazepam, nordazepam, oxaze-
Pam, and temazepam.
Half-life also determines the time necessary tor impairment produced next day after drug
to attain steady state. Long half-life hypnotics administration. In general, these residual ef-
accumulate slowly but extensively, whereas fects occur more often after long-acting drugs
short half-life benzodiazepines reach steady (such as nitrazepam or flurazepam) than after
state rapidly and accumulate to a small extent. intermediate (temazepam) or short-acting
Half-life also determines the time required benzodiazepines (triazolam).
for drug washout after multidose treatment is Drug dose is a critical issue in determining
discontinued. Short half-life benzodiazepines unwanted daytime sedation. Thus, 5 mg of ni-
disappear quickly, whereas long half-life com- trazepam produces few effects compared with
pounds are eliminated slowly following the 10 mg. Similarly, large doses (over 0.5 mg) of
last dose. triazolam produce definite hangover effects.
These factors are important when consid- After stopping benzodiazepines, patients
ering the daytime drowsiness and psychomo- may experience rebound insomnia or anxiety.
Sedative-Hypnotics
Bromazepam 3-Hydroxy-bromazepam
Br a""'
- A"".
- CI = O Br C1 = O Conjugates
2-Aminod- 2-Arnino-3-hydroxy-5-
bromobenzoylpyridine bromobenzoylpyridine
Rebound insomnia occurs more often with EEG changes in many subjects on the morning
short-acting and intermediate-acting benzodi- after drug administration (13). Subjects given
azepines, when given in high doses and with-
-
benzodiazepines should be warned of this fad.
drawn abruptly. Conversely, very long-acting
benzodiazepines (such as flurazepam and 2.3.3 Halogenated Sedative-Hypnotics
quazepam) show milder rebound effects. 2.3.3.1 Chloral Hydrate. Chloral hydrate
Ideally, a hypnotic drug should not produce is rapidly absorbed from the stomach and
unwanted drowsiness or heavy-headedness on starts to act within 30 min. However, no chlo-
awakening. Several studies in humans have in- ral hydrate blood levels are found in humans,
dicated that benzodiazepines produce morning- because the metabolic conversion of chloral
after effects visible in EEG recordings. Nitraz- hydrate is very fast (Eq. 5.4).
epam (5-10 mg) and flurazepam (15-30 mg), for
example, produce a shift to low voltage, high fre-
quency activity in the EEG (15,161, which is still
present 12-18 h after a single dose of the drug.
There is disagreement whether benzodiazepine
administration also produces an impairment of Chloral hydrate Trichloroethanol
performance of intellectualand motor function.
Several investigators observed impaired perfor- The long-lasting hypnotic effect is caused
mance (15-18), whereas others have not con- by its metabolite, trichloroethanol(20).
firmed these findings (19). Trichloroethanol passes into the cerebro-
It is generally agreed, however, that even spinal fluid and has a half-life of 7-11 h. In
though the symptoms of hangover may be rec- addition to trichloroethanol, chloral hydrate is
ognized, benzodiazepines, like all other seda- also metabolized to trichloroacetic acid, which
tive-hypnotics, do produce psychomotor and is inactive. Both trichloroethanol and trichlo-
2 Clinical Use of Agents
Delorazepam Lorazeparn
Lormetazeparn
Figure 5.6. Bic>transformationpathways of delorazepam, lormetazepam, antd lorazepam.
roacetic acid are excreted in the urine and in 2.3.4.2 Clomethiazole. Clomethiazole is rap
the bile, in part, as glucuronides. idly absorbed from the gastrointestinal tract
2.3.3.2 Ethchlorvynol. Ethchlorvynol is rap- and produces peak plasma concentrations in
idly absorbed from the gastrointestinal tract about 15-45 min after oral administration. It
and is extensively metabolized mainly in the is broadly distributed in the body and is exten-
liver and to a lesser extent in the kidney. The sively metabolized in the liver and excreted in
half-life of ethchlorvynol is biphasic with a the urine with only very small amounts of un-
rapid initial phase and a 10- to 25-h-long ter- changed drug appearing in the urine.
minal phase. Only traces of unchanged drug 2.3.4.3 Zopiclone. Zopiclone is well ab-
appear in the urine. The metabolic half-life is sorbed from the gastrointestinal tract with
5.6 h, and it disappears from the plasma with a peak plasma concentration reached within 2 h
rapid a phase and a much slower P phase, be- after administration. It has a half-life of 4-6 h
cause of extensive tissue redistribution. and a duration of action of 6-8 h. It is metab-
olized in the liver to N-desmethylzopiclone
2.3.4 Heterocyclic Sedative-Hypnotics and zopiclone N-oxide, and these two metabo-
2.3.4.1 Glutethimide. Glutethimide is ir- lites constitute about 36% of the dose excreted
regularly absorbed from the gastrointestinal in the urine along with small amounts of zopi-
tract and is extensively metabolized in the clone. Excretion of drug and metabolites was
liver. It is about 50% bound to plasma proteins essentially complete 48 h after the final dose.
and has a biphasic plasma half-life. It is ex- 2.3.4.4 Zolpidem. Zolpidem is rapidly ab-
creted in the urine with only up to 2% of un- sorbed from the gastrointestinal tract and has
changed drug and 98% as metabolites. Glu- a half-life of 1.5-2.5 h. It has a rapid onset of
tethimide is highly lipid soluble and may be action that is detectable within 15-30 min.
stored in adipose tissue. The peak plasma level is reached within 1-2 h
Sedative-Hypnotics
Quazepam \ Oxoquazepam
mean area under the curve and C,, were de- larger molecular size or their electrostatic
creased by up to 25%. charge. The relative contribution of these fac-
2.3.4.5 Zaleplon. Zaleplon is rapidly ab- tors is unknown.
sorbed from the gastrointestinal tract and Serum elimination half-life values differ
reaches peak plasma level within 1 h after in- greatly from one antihistamine to another.
gestion. It has avery fast onset of action. It has Most of the antihistamines are metabolized by
a half-life of 30 min and a short duration of the hepatic cytochrome P450 system and give
action, less than 5 h. Zaleplon has a terminal rise to metabolites that are usually excreted in
phase elimination half-life of approximately the urine.
1 h. Zaleplon does not accumulate with once-
daily administration, and its pharmacokinet- 3 PHYSIOLOGY AND PHARMACOLOGY
ics are dose proportional in the therapeutic
range. The absolute bioavailability of zaleplon The clinical effects of sedative-hypnotics in-
clude sedation and sleep. Sedative-hypnotic
is approximately 30% because it undergoes
drugs depress the function of the CNS and in a
significant presystemic metabolism. Zaleplon
dose-dependent fashion produce drowsiness
is a lipophilic compound. The blood-to-plasma
(sedation). Several sedative-hypnotic drugs,
ratio for zaleplon is approximately 1, indicat- especially the older ones, produce sedation,
ing that zaleplon is uniformly distributed sleep, unconsciousness, surgical anesthesia,
throughout the blood with no extensive distri- coma, and ultimately may cause fatal depres-
bution into red blood cells. sion of respiration and cardiovascular regula-
Zaleplon is primarily metabolized by alde- tion.
hyde oxidase to form 5-0x0-zaleplon. Other Because these drugs facilitate the onset
metabolites include desethylzaleplon and and maintenance of a state of sleep that re-
5-0x0-desethylzaleplon.Approximately 70% of sembles natural sleep, the physiology of sleep
the administered dose is recovered in urine is of significant importance and is described
within 48 h, almost all as zaleplon metabolites below (Section 3.1). In the second part, the
and their glucuronides. An additional 17% is pharmacology is addressed (Section 3.2).
recovered in feces within 6 days, mostly as
3.1 Physiology of Sleep
5-0x0-zaleplon. The effects of zaleplon on
sleep onset are reduced if it is taken with or
3.1.1 Wakefulness and Sleep. Sleep and
immediately after a high-fat or heavy meal.
wakefulness are two easily recognizable dis-
tinct functional states. The person in sleep
2.3.5 Antihistamines. Antihistamines are shows a reduced awareness and responsive-
rapidly absorbed from the gastrointestinal ness both to internal and external stimulation
tract. After oral administration the onset of and hisher motor functions are inhibited. The
action of HI-receptor antagonists is prompt, sleeping person seems quiescent. However,
occurring within 0.5 h, although the peak his- the sleeping person exhibits some movements,
tamine blockade may not occur until 5-7 h indicating that sleep is an active process.
after oral administration. The cerebral activity of a human in wake-
Bioavailabiity has not been well studied be- fulness and in sleep display significant differ-
cause of the lack of intravenous formulations. ences. In the human electroencephalogram
Most fmt-generation histamine HI-receptor an- (EEG), wakefulness is characterized by low
tagonists readily cross the blood-brain barrier, amplitude waves and a rhythm. On the other
which consists of the endothelial lining of the hand, the EEG in sleep consists of high ampli-
capillaries of the CNS. The first-generation an- tude slow waves and spindles, indicating slow
tihistamines listed in Section 2.1.5 are used as synchronized, idling neural activity. Eledro-
sedative-hypnotics. oculographic (EOG),electromyographic (EMG),
The second-generation HI-receptor antag- and electrocardiographic (ECG) data have
onists, such as fexofenadine, loratadine, and lent additional support to the finding that
cetirizine, are relatively lipophobic and pene- both wakefulness and sleep are active pro-
trate poorly into the CNS because of their cesses.
Sedative-Hypnotics
3.1.2 Sleep Studies. As early as 1907, Leg- sleep consisted of two distinct organismic
endre and Pieron discovered the existence of states. As a result the following three physio-
endogenous sleep factors (21). Injection of logical states exist:
blood serum from sleep-deprived dogs could
induce sleep in dogs who were not deprived of 1. Alert wakefulness
sleep. The search for endogenous sleep factors 2. Nonrapid eye movement (NREM) sleep
is still continuing. 3. REM sleep
In the 1920s and 1930s, Kleitman studied
the effects of sleep deprivation (22) and con- 3.1.4 Alert Wakefulness. In wakefulness,
cluded that the build-up of endogenous sleep the EEG shows a high level of low voltage fast
factors did not exceed certain limits, and hu- (LVF) frequency activity (9-10 Hz a activity
mans became as impaired as they would get mixed with low amplitude P activity). The
after approximately 2.5 days of wakefulness. EMG shows a high level of tonic activity and
The first EEG recordings were reported by the EOG shows frequent eye movements.
Berger in 1930 (23). He discovered differences
in the electrical rhythms of subjects when 3.1.5 NREM Sleep. This consists of four
awake or asleep. stages:
In the late 1940s,sleep research in animals
was conducted by implantable electrodes. This 1. Stage 1 NREM Sleep. The first state of
led to the designation of the ascending reticu- sleep (also called drowsy state). This is
characterized by a 50% diminution of the a
lar activating system by Moruzzi and Magoun
waves of wakefulness in the EEG and low
(24). They discovered that an important struc-
amplitude, mixed frequency activities (in
ture of the brain stem was interposed between the range of 3-7 Hz) consisting of 8 and
sensory input and the higher centers of the some p waves. The muscular tone in the
brain. High frequency electrical stimulation of EMG becomes slightly relaxed, and slow
the brain stem reticular formation through rolling eye movements (SEM) are observed
implanted electrodes produced activation of in the EOG. The state of drowsiness
the EEG, wakefulness, consciousness, and be- quickly changes to definite sleep.
havioral arousal. On the other hand, reticular 2. Stage 2 NREM Sleep. The onset of stage 2
deactivation produced by EEG synchroniza- NREM sleep is observed by bursts of 12- to
tion leads to sleep and lack of consciousness. 16-Hz sleep spindles in the EEG. The EMG
In the early 1950s,Aserinsky and Kleitman activity is less than in wakefulness and
(25) deployed the method of EOG for the mea- stage 1, and the EOG does not show eye
surement of eye mobility. Soon thereafter, movements. Stage 2 sleep lasts about
Aserinsky and Kleitman (26) discovered that 30-60 min.
sleep onset is characterized by slow eye move- 3. Stage 3 NREM Sleep. Stage 3 sleep follows
ment, which in time, changes to REM sleep. stage 2 sleep and is characterized by slow
Subsequent research by Aserinsky and Kleit- wave sleep. The EEG shows 20-50% of 6
man led to the conclusion that REM sleep is waves of 2 Hz or less with amplitudes
associated with dreaming. greater than 75 pV from peak to peak. The
Dement and Kleitman (27) expanded their muscle tone in the EMG is more relaxed
research in all-night sleep recordings using than before, and there are no eye move-
EEG and EOG techniques. They observed a ments in the EOG.
sequence of patterns over the course of the 4. Stage 4 NREM Sleep. This stage is charac-
night that revealed a basic sleep cycle. terized by 6 waves of 2 Hz with amplitudes
greater than 75 pV, which constitute more
3.1.3 States of Sleep. EEG studies have re- than 50% of the total. Toward the end of
vealed that the activity of the brain is not con- the deep sleep (6 sleep), sleep lightens, of-
stant during sleep. EOG and EMG data have ten abruptly to stage 2 NREM sleep, ac-
lent additional support to the finding that companied by body movements. After a
3 Physiology and Pharmacology
brief period of stage 2 NREM sleep, the first than a normal amount of time in REM sleep
REM sleep occurs about 60-90 min after when allowed to follow a normal pattern. This
sleep onset. rebound affect is also observed after REM-
sleep suppressing drugs are withdrawn (29).
3.1.6 REM Sleep. This is characterized by Sleep laboratory studies are usually per-
rapid eye movements. The slow wave pattern in formed by applying the standardized methods
the EEG is desynchronized and changes to a low developed by Rechtschaffen and Kales (30).
amplitude mixed frequency 0 including some a
pattern and often displaying saw tooth waves. 3.1 .a Neurotransmitters and Sleep. Based
No sleep spindles are seen. The muscle tone be- on the very elegant studies performed in cats,
low the chin is totally relaxed or abolished. Jouvet postulated that there is a direct relation-
ship between 5-hydroxytryptamine(5-HT,sero-
3.1.7 Sleep Cycle. The first REM period tonin) and NREM sleep (31,321.
lasts a few minutes and changes to stage 2 and Correlations were also obtained for other
subsequently to stage 3 and stage 4 of NREM neurotransmitters as well.
sleep before the second REM sleep period oc-
3.1.9 Neurotransmitters and REM Sleep in
curs. Accordingly, a full sleep cycle consists of
Humans. Neurotransmitters have both a di-
a sequence of NREM sleep and REM sleep, rect and indirect action on REM sleep. The
and a cycle lasts about 90-110 min. Alto-
indirect action is manifested by a self-inhibi-
gether, three to five REM periods occur during
tory feed back mechanism (Equation 5.5).
the night in 90- to 110-min cycles alternating
Cholinergic transmission (acetylcholine) acti-
with NREM sleep. However the various
vate neurons in their pontine reticular forma-
NREM sleep stages change in duration. The
tion (PRF) and produce REM sleep. REM
first two cycles are dominated by stages 3 and
sleep gradually activates REM-off monoamin-
4 NREM sleep. Subsequent cycles are domi-
ergic neurons, which produce a self-inhibitory
nated by stage 2 NREM sleep and stage 3 and
feedback and eventually terminates REM
4 NREM periods are brief or do not appear at
sleep. As the REM sleep period changes to
all. NREM sleep, the REM-off neuronal activity
Conversely, the REM sleep duration in-
gradually decreases during NREM sleep, and.
creases from the first to the last cycle. The last
is at minimum at the onset of REM sleep.
REM cycle is usually the longest REM cycle
The role of REM sleep on and off switches
and may last as long as an hour. As a result,
are further accentuated by the effects of vari-
the first third of sleep is dominated by stages 3
ous drugs in humans in the NREMIREM sleep
and 4 NREM sleep and the last third of the
cycle (Equation 5.5 and Fig. 5.8).
sleep is dominated by REM sleep.
Sleep deprivation studies (28) have demon- 3.1 .I 0 Nonhypnotic Drugs Affecting REM
strated the need for both NREM and REM On Switch
sleep. Subjects deprived of sleep tend to spend
more than a normal amount of time in NREM 1. Muscarinic Agonists. The muscariniclnico-
sleep when allowed to sleep. Similarly, if the tinic agonist carbachol produces a long
subjects are awakened every time that REM lasting REM sleep that is blocked by the
sleep begins, they become selectively deprived administration of atropine. The same effect
of REM sleep, because every time they fall was obtained by using the selective musca-
asleep again, the sleep cycle begins with rinic agonist bethanecol. Further studies
NREM sleep. As a result, REM sleep occurs revealed that M, muscarinic agonists are
after shorter and shorter times. Furthermore, active, whereas the M, muscarinic agonists
subjects deprived of REM sleep spend more are without effects (33).
Cholinergic Monoaminergic
REM sleep
off switch (5.5)
Transmission Transmission
Sedative-Hypnotics
Serotonin
Norepinephrine
MA01
-
AMPT Amphetamine
REM sleep Apomorphine
Disulfiram & fusaric acid GABA + GABA.
on switch compounds
6-OHDA Opioids
IL-1
TN Fa
levels, had very few measurable effects on cat, and monkey by measuring drug-induced
NREM sleep. In general, GABA (inhibitory changes in the sleep-wakefulness cycle. These
amino acid) promotes NREM sleep and influ- studies have been reviewed (55).
ences sensory transmission and seems to have The ultimate answer for the usefulness of a
an opposite action to glutamate excitatory drug candidate is found in humans (56). Sleep
amino acid. laboratory studies in humans have been in-
creasingly used recently in the evaluation of
NREM On Switch Promoters the effectiveness of hypnotic drugs. These
studies have been reviewed (57, 58).
Serotonin DSIP
Melatonin CCK Briefly, evaluation of the effects of hypnot-
GABA Insulin ics on sleep physiology and sleep architecture,
Adenosine IL-1 as measured by electroencephalography, has
PGD, IFN received increased attention. Continuous all-
GHRH TNF-a! night electrophysiological measurement of
CCK, cholecystokinin; DSE', 6 sleep inducing peptide; sleep termed polysomnography (PSG) has
GHRH, growth hormone releasing hormone; IFN, interfer- made it possible to evaluate the action of hyp-
ons; IL-1, interleukin-1; TNF-or,tumor necrosis factor or. notics on sleep. PSG assessment as a means of
GAB& gamma-aminobutyric acid; PGD,, prostaglandin D,. evaluating the hypnotic effects of a drug can-
didate is now required by the U.S. FDA.
There is a mutual interaction of the factors This method is used to determine sleep pa-
controlling NREM and REM sleep. It is well doc- rameters including total sleep time, number of
umented that during selective REM sleep depri- awakenings during sleep, sleep latency, sleep
vation, there is also a significant reduction in the efficiency, effects on sleep stages, and sleep
low frequency activity of the NREM sleep EEG architecture (reduction of slow wave sleep or
(42). To explain the interaction between NREM REM sleep, changes in the latency of REM
and REM sleep, several models were proposed sleep, etc.). Assessment of hypnotics on sleep
(43). The reciprocal interaction models on the include measures of nighttime sleep and day-
NREM and REM sleep cycle (44) evolved from time function, and quality of sleep, which is
neurophysiological data obtained in animals. It measured by changes in stage 1 sleep, which
is postulated that NREM-REM sleep cycle is typically is increased in many insomnia
caused by the reciprocal interaction of two neu- conditions.
ronal systems in the brain stem with self-ex- In addition to showing statistically signifi-
citatory and self-inhibitory connections. For cant improvement on various parameters (for
further details, recent monographs on sleep example, latency of sleep, onset of sleep, or
medicine should be consulted (45-47). total sleep time in transient insomnia or main-
taining sleep and reducing wakefulness in
3.2 Pharmacology chronic insomnia) the adverse effects caused
Additional details are described in Section 5.3. by hypnotics are also measured. These include
effects on residual sedation, rebound insom-
3.2.1 Evaluation of Sedative-Hypnotics. The
nia (referring to increase in wakefulness or
methods usually employed in the evaluation of anxiety), amnesia, and adverse cardiopulmo-
nary effects.
sedative-hypnotic drug candidates in small ro-
dents (mouse or rat) involve measurements of The goal of sleep research is to help find
hypnotic drugs that will approximate the ideal
the levels of CNS depression. These involve
measurements of sleeping time (48), loss of hypnotic drug.
righting reflex (491, performance on the ro-
tarod (50), behavior in the activity cage (51- 3.2.2 Pharmacologic Effects of Sedative-
531, and finally, potentiation of other CNS de- Hypnotic Drugs
pressants (54). 3.2.2.1 Before Barbiturates. In ancient times,
Recently more emphasis has been placed on alcoholic beverages and herbal potions have
the evaluation of drug candidates in the rat, been used to induce sleep.
3 Physiology and Pharmacology
The first chemical used specifically as a sed- Barbiturates induce hepatic microsomal
ative and later as a hypnotic was bromide in drug-metabolizing enzymes resulting in an
the middle of the 19th century. increased degradation of barbiturates, ulti-
Bromides were followed by paraldehyde, mately leading to barbiturate tolerance.
urethane, and sulfonal. These medications Because of their enzyme-inducing effects,
were no great improvement because they barbiturates can cause increased inactiva-
could be addictive, and an overdose could kill. tion of other compounds (anticoagulants,
The launching of chloral hydrate was an im- phenytoin, theophylline, digoxin, glucocor-
provement because chloral hydrate is a rela- ticoids, etc.). This may lead to serious prob-
tively safe hypnotic drug. lems with drug interactions.
Pharmacologic effects of chloral hydrate:
3.2.2.3 Benzodiazepines, The launch of
It induces sleep in 0.5 h. Librium in 1960 was quickly followed by the
0 The sleep lasts about 6 h.
launch of diazepam in 1961, oxazepam in
1964, nitrazepam in 1965, clorazepate potas-
r It causes a relatively small reduction in sium in 1967, temazepam in 1969, lorazepam
REM sleep. in 1971, and so on.
r It is used mainly in children and the elderly. The benzodiazepines overtook the barbitu-
It is most effective when used for only 1-3 rates because of the following disadvantages
nights to treat transient insomnia. of barbiturates:
6. Benzodiazepines get attached to receptor All these have a short half-life with no active
sites that are highly specific. By combin- metabolite.
ing with receptor sites, they elicit a phar- There is no objective evidence of rebound
macologic effect. insomnia or tolerance in studies of up to
7. All the effects of benzodiazepines that are 30-40 nights at recommended doses.
mediated by receptors can be prevented or All these have a favorable safety and tolera-
reversed by drugs that act as selective bility profile.
benzodiazepine antagonists. They generally preserve the stages of nor-
8. Benzodiazepines show less tendency to mal sleep.
tolerance and dependency than other
older sedative-hypnotic drugs, especially 3.2.2.5 Antihistamines. The pharmacologic
barbiturates. Also, benzodiazepines pro- effects of antihistamines are summarized as
duce less abuse potential. follows:
9. Benzodiazepines are safer in overdose es- Effects of first generation HI-receptor an-
pecially compared with barbiturates. tagonists in the CNS are mediated by block-
10. Benzodiazepines produce fewer drug in- ade of endogenous histamine neurotrans-
teractions because they do not induce he- mitter.
patic microsomal enzymes. Conversely, antihistamines at high concen-
trations may inhibit the metabolism of his-
3.2.2.4 Heterocyclic Sedative-Hypnotics. tamine methyltransferase thus increasing
Despite the advantages of benzodiazepines the availability of histamine to act at CNS
compared with other sedative-hypnotic drugs, histamine receptor.
tolerance and dependency do occur. Rebound These agents also block a-adrenergic recep-
insomnia may also occur either during each tors, serotonin receptors, and/or cholinergic
night of treatment with benzodiazepines or muscarinic receptors, causing dysuria, mic-
during withdrawal of treatment extended for turition, dryness of the mouth, and tight-
several days. Cessation of benzodiazepines ness of the chest.
may also lead to recurrence of the original 0 High dose therapy may produce allergic re-
symptoms or even to transient worsening. actions and cross-sensitivity to related
Heterocyclic compounds were synthesized drugs.
and tested as sedative-hypnotics with the di- Continuous use of antihistamine may pro-
rect purpose of substituting them for both bar- duce tolerance, rendering the sleep-induc-
biturates and benzodiazepines (Table 5.7). ing effects essentially non-existent.
In quick succession, two heterocyclic
3.2.3 Clinical Pharmacology of Important
non-benzodiazepine sedativeihypnotics were
Sedative-Hypnotics
launched in 1985 by Rhone-Poulenc as zopi-
3.2.3.1 Benzodiazepines. Bromazepam is
and in lgg8 S~nthelaboas zol~idem' usually administered orally between 3 and 18
In 1999, W ~ e t hA ~ e r slaunched
t another het- mg daily in divided doses. a clinical trial in
erocyclic non-benzodiazepine sedativekyp- children, bromazepam suppos~toryand chlo-
notic, zaleplon. ral hydrate suppository were compared as an-
The ~harmacologiceffects esthetic premedication. Increasing doses of
sedative-hypnoticsare summarized as ~ O ~ ~ O W S : bromazepm were used in &Idren. The group
younger than 5 years were inadequately se-
e All these drugs, zo~iclone,zol~idem,and dated compared with those receiving chloral
zaleplon, display affinities to the benzodiaz- hydrate. However, in the group 5 years and
epine receptor. However, problems with de- older, bromazepam and chloral hydrate were
pendency and/or tolerance have not been re- equally effective (59).
ported. Brotizolam in 0.25- or 0.50-mg doses was
All these drugs elicit rapid onset of action. compared in 38 clinical and epidemiological
3 Physiology and Pharmacology
studies with triazolam, zopiclone, zolpidem, ioral impairment in humans, but no such ef-
midazolam, temazepam, lormetazepam, and fects were observed after a 1-mg dose or a
loprazolam. A total of 5506 patients partici- 100-mg dose of amobarbital (66). The plasma
pated in the parallel-design and crossover levels of delorazepam correlated well with
studies. To provide clinically relevant compar- sleep-inducing effects. In a single-blind study,
isons, only studies using comparator agents in delorazepam in a 2-mg dose proved to be a
doses equipotent to the triazolam doses were highly effective hypnotic agent in 22 patients
included. who suffered from long-lasting severe insom-
Two general findings emerged. First, sig- nia (67).
nificant CNS side effects such as excitement In 20 psychiatric patients, delorazepam (2
and violence were not observed with any of the mgld) was as effective as lorazepam (5 mgld) in
hypnotic agents including brotizolam. With the improvement of anxiety and insomnia. No
regard to other CNS side effects, depression, serious side effects were reported (68).
irritability, rebound insomnia, and early Doxefazepam in a 5-mg dose in a controlled
morning insomnia were observed for all hyp- clinical study in humans was equivalent to a
notics in these studies. Second, the authors 15-mg dose of flurazepam in terms of sleep
claim that remarkable similarities were found induction time, sleeping time, and quality of
among all these agents in terms of efficacy, sleep. Moreover, doxefazepam caused signifi-
side effects, and performance-related effects cantly less hangover effect compared with that
(60). caused by flurazepam (69).
However, epidemiologic studies do not In 40 patients, the efficacy of doxefazepam
carry the same significance as direct compar- in a 20-mg dose did not diminish over 1 year,
ative randomized double-blind studies con- and no major side effects were reported (70).
ducted against placebo and a comparator In a double-blind study involving 1139 pa-
drug. tients, estazolam in a 2-mg dose seemed to be
Brotizolam (0.25 mg) in randomized dou- equivalent to a 10-mg dose of nitrazepam in
ble-blind studies was as effective as flunitraz- efficacy. A higher dose of estazolam (4 mg) was
epam (2 mg) (61) and triazolam (0.25 mg) (62). more effective but also caused more incidence
However, brotizolam produced markedly of side effects than a 10-mg dose of nitrazepam
fewer adverse side effects than flunitrazepam. (71). The incidence of unsteadiness caused by
In addition, it was also reported (63) that the 2 mg of estazolam was the same as that caused
incidence of hangover and gastrointestinal by 5 mg of nitrazepam.
problems was greater in patients treated with In a double-blind crossover study, estazo-
2 mg flunitrazepam than in those treated with lam (2 and 4 mg) was compared with nitraz-
0.5 mg brotizolam. epam (5 and 10 mg). Administration of 2 mg of
Brotizolam (0.5 mg) was as effective as 2 estazolam prolonged total sleep time and de-
mg flunitrazepam in inducing sleep and was creased stage 1 and stage 2 sleep, whereas 4
superior in maintaining sleep in a double- mg of estazolam reduced stage 2 sleep and
blind trial involving 40 patients due to un- REM sleep. Nitrazepam (5 mg) produced pro-
dergo surgery (64). longation of total sleep and a decrease in fre-
Cinolazepam was evaluated in a double- quency of awakenings, whereas 10 mg of ni-
blind placebo controlled trial in 20 normal trazepam prolonged stage 2 sleep and reduced
subjects. The subjects were given either pla- the total wakefulness during the night (72).
cebo or 40 mg cinolazepam orally 30 min be- Flunitrazepam in a 2-mg dose was com-
fore bedtime. A significant improvement in pared with zolpidem in a 20-mg dose. Forty-
sleep maintenance was obtained with 40 mg two insomniac female in-patients between 30
cinolazepam, measured by sleep disruption and 65 years of age were included in a double-
using nocturnal traMic noise. Sleep architec- blind, parallel group trial and were randomly
ture was only minimally affected, and sleep allocated to the two treatments. Study dura-
quality improved significantly (65). tion was 9 days with 2 days of placebo run-in, 5
Delorazepam in 2-mg doses in a controlled days of active medication, and 2 days of pla-
trial produced a hangover effect and behav- cebo withdrawal. Sleep latency, sleep dura-
Sedative-Hypnotics
tion, number of awakenings, and time spent secutive nights in insomniac patients. Both
asleep during the night were given an ordinal flurazepam and estazolam significantly im-
score; condition in the morning was evaluated proved sleep. There was no significant differ-
by visual analogue scale (VAS), and the psy- ence in hypnotic effect between flurazepam
chomotor performance was evaluated by the and estazolam.
following tests: night-day for anterograde am- The percentage of patients reporting any
nesia, digit span for verbal recall, and Grun- adverse effect was greatest for flurazepam fol-
berger's fine motor function test. lowed by estazolam and placebo.
There was no difference between zolpidem Estazolam and flurazepam effectively, and
and flunitrazepam for any of the variables; the comparably, relieved insomnia when adminis-
drugs were significantly better than placebo tered for seven nights in adult patients com-
baseline for all the sleep efficacy variables.
plaining of insomnia. Estazolam demon-
The results of this study indicate that zolpi-
strated a more favorable side effect profile
dem is as effective as flunitrazepam in induc-
ing and maintaining sleep, but it does not in- than flurazepam (76).
duce a sense of weakness in the morning and Loprazolam (1mg) was compared with tri-
does not impair memory (73). azolam (0.25 mg) in a cross-over double-blind
In a double-blind study of 102 patients with trial. The drugs were administered by a design
a mean age of 70 years, flunitrazepam was of cross-over on the first two nights and con-
compared with zopiclone (74). The patients tinuation of the preferred treatment. Sixty-
rated their sleep in a diary. There was no sta- seven outpatients suffering from chronic in-
tistically significant difference between the somnia took part in the study.
relatively low dose of 5 mg zopiclone and 1 mg Both drugs provided improvement in sleep
flunitrazepam for 11of the 12 variables mea- quality (decreased sleep latency, increased to-
suring subjective sleep quality and quantity. tal duration of sleep, decreased number of
There was no difference between the drugs in night awakenings) and were equally well tol-
regard to patients' feelings of being rested or erated (77).
alertness. Lorazepam (1 mg) was compared with tri-
A multi-center, double-blind, randomized, azolam (0.25 mg), zolpidem (10 mg), zopiclone
placebo-controlled, parallel-group study com- (7.5 mg), and placebo in a randomized doub1.e-
pared the next-day residual effects, hypnotic blind study using 10 nocturnal polysomno-
efficacy, and sleep staging effects of fluraz- grams with at least 72-h washout intervals.
epam (30 mg) and zolpidem (10 and 20 mg) Six healthy middle-aged subjects who were
with those of placebo in patients with chronic normal sleepers (three men and three women)
insomnia. received single dose of the drugs both under
As measured by objective and subjective basal and under perturbed conditions. For
criteria, both zolpidem and flurazepam were each individual, five recordings were carried
effective hypnotics. Sleep stages were affected out under basal conditions (sound pressure
more by flurazepam than by zolpidem. The in- level not higher than 30 dB) and five record-
cidence of treatment-emergent adverse events ings under acoustically perturbed conditions
was approximately the same for zolpidem (10 (continuous white noise at 55 dB). Sleep qual-
mg), flurazepam, and placebo. The 20-mg dose ity was assessed by visual analogue scale
of zolpidem (twice the therapeutic dose) was (VAS). Zolpidem produced the highest protec-
associated with a higher incidence of adverse tive action of the four drugs during perturba-
effects. It was concluded that no next day re- tion (78).
sidual effects are associated with nightly in- Lormetazepam (1mg) was compared with
take (three nights) of the recommended dose midazolam (15 mg) and zopiclone (7.5 mg) as
of zolpidem. At this dose, zolpidem was an ef- night medication in patients scheduled for
fective and safe hypnotic (75). elective surgery the next morning. Sixty pa-
Flurazepam (30 mg) was compared withes- tients divided at random into three groups
tazolam (2 mg) and placebo in a multi-center, (double-blind) received the medication at one
randomized, double-blind study for seven con- time.
3 Physiology and Pharmacology
The three hypnotics were equally effective epate increased total sleep time and reduced
as sleep medication for sleep onset latency, du- the number of awakenings, and the time re-
ration of sleep. and condition on awakening, quired to fall asleep was decreased. A dose of 5
whereas zopiclone provided significantly mg of nordazepam had no effect on the dura-
fewer spontaneous awakenings. tion of sleep stages. Nordazepam (10 mg) and
On the other hand. the lormetazepam potassium clorazepate (15 mg) decreased the
group scored significantly better in an ocular duration of stage 2 sleep. During the recovery
imbalance test than the zopiclone group (79). night, stage 1 sleep was reduced and stage 2
Midazolam (7.5 mg), flunitrazepam (1mg), sleep was increased. No effects of stage 3 sleep
and placebo were compared in a double-blind were observed, but stage 4 sleep seemed to be
crossover trial to study the effects of drugs on suppressed.
sleep, nighttime respiration, and body move- Potassium clorazepate is decarboxylated
ments in five elderly insomniac patients. No rapidly at the pH of the stomach to form nor-
signs of increased respiratory resistance was dazepam (desmethyldiazepam), which is
seen with either of the drugs or placebo. There quickly absorbed. The peak plasma nordaz-
were no differences in the quality and quan- epam concentration is obtained 45 min after
tity of sleep induced by either drug. Only the clorazepate administration of 15 mg (93).
sleep onset latency was shorter with flunitraz- Potassium clorazepate is used in the
epam compared with midazolam and placebo United States in rather high doses of 15-60
(80). mg daily in two to four divided doses or as a
Nimetazepam is metabolized to nitraz- single dose at night. In the United Kingdom, a
epam. It is used as a hypnotic in 3- to 5-mg single dose of 15 mg potassium clorazepate is
doses. The features and characteristics of usually given at night or a dose of 7.5 mg is
nimetazepam are very similar to those of ni- given three times a day.
trazepam. Clorazepate in 22.5-mg daily doses admin-
Nitrazepam in doses as low as 5-10 mg pro- istered for 8 days decreased REM sleep, stage
duces a hypnotic effect in humans comparable 4 sleep, sleep latency, and total waking (94),
with a 100- to 200-mg dose of amobarbital(81- whereas total sleep time was increased. Dur-
83), 50- to 200-mg dose of butabarbital (18, ing recovery, REM sleep, and sleep latency
83-85), 100- to 200-mg dose of secobarbital were increased (95).
(86),and 250- to 500-mg dose of glutethimide Quazepam (15 mg) was compared with tri-
(87). Nitrazepam suppressed REM sleep (88) azolam (0.5 mg) and placebo in 65 insomniac
in humans but the extent of suppression de- patients. The patients were treated with pla-
creased with time (88).Sleep is longer lasting cebo for 4 days into the study, and if no ame-
and less broken while using nitrazepam, and lioration of insomnia was observed, they were
no tolerance was obvious after 2 months of allocated randomly to the drugs with 32 pa-
nitrazepam use (89). After stopping nitraz- tients receiving quazepam and 33 triazolam
epam, there is a rebound of REM sleep, which for 8 weeks and finally placebo for another
reaches a maximum in 1-2 weeks (90). Com- week. The sleep quality, sleep efficacy, un-
plete recovery after nitrazepam use takes 3-6 wanted side effects, and the rebound effects
weeks (91). Nitrazepam also produces hang- had been assessed by specific evaluation.
over effects with impairment of psychomotor Quazepam had significantly less night awak-
performance and difficulty in falling asleep enings. At the treatment's interruption, only
that may be longer lasting than those pro- the patients treated with triazolam had longer
duced by sodium amobarbital(17). awakenings and rebound symptoms. In con-
Nordazepam is the principal metabolite of clusion, quazepam has good hypnotic effect
diazepam. It has been administered as a hyp- without inducing rebound effects (96).
notic in 7.5- to 15-mg daily doses (92). Temazepam (15 mg) was compared with
The effects of nordazepam and a precursor, triazolam (0.125 mg), zolpidem (5 mg), and
potassium clorazepate, on sleep were evalu- placebo. After a single-blind placebo screening
ated in humans (92). A dose of 5 or 10 mg weak 335 elderly insomniacs were randomized
nordazeparn or 15 mg of potassium cloraz- to 28 days of double-blind treatment with the
Sedative-Hypnotics
drugs or placebo followed by a 14-day single- and that the two drugs are relatively safe and
blind placebo withdrawal period. The primary effective in the treatment of insomnia (99).
efficacy parameters were self-reported sleep Zolpidem (10 mg) or triazolam (0.25 mg)
latency (SSL) and self-reported total sleep du- were given to patients for 14 days in a random-
ration (SSD); they were measured by re- ized double-blind study. Data form 139 pa-
sponses on daily morning questionnaires. tients were used in the analysis. No statisti-
Compared with placebo, zolpidem and cally significant differences were found
temazepam produced significantly shorter between the two groups regarding sleeping
SSL over the 4 treatment weeks, but triazolam time, number of awakenings, or sleep quality.
did not. In the zolpidem group, SSL was sig- Morning feeling and day feeling were numeri-
nificantly shorter than in the placebo group in cally better for zolpidem, although not statis-
all 4 treatment weeks; in the temazepam tically significant. There was no statistically
group, SSL was significantly shorter than in significant difference in the number of pa-
the placebo group at weeks 1, 3, and 4. SSD tients experiencing side effects in the two
was increased above baseline levels in all treatment groups. On a short-term basis, ad-
groups. No tolerance to the subjective effects ministration of zolpidem (10 mg) seemed as
or rebound above baseline levels occurred in effective and well tolerated as triazolam (0.25
any of the treatment groups. Overall, the mg) (100).
drugs were well tolerated. No difference was Zaleplon was evaluated for efficacy and
found among the placebo and treatment safety compared with zolpidem. After a
groups in overall adverse event incidence 7-night placebo (baseline) period, 615 adult
rates. However, compared with zolpidem and patients were randomly assigned to receive, in
placebo, temazepam produced significantly double-blind fashion, one of five treatments
higher incidences of drowsiness and fatigue, (zaleplon: 5,10, or 20 mg; zolpidem: 10 mg; or
and triazolam produced a significantly higher placebo) for 28 nights followed by placebo
incidence of nervousness than zolpidem (97). treatment for 3 nights. Sleep latency, sleep
Triazolam was compared with zolpidem maintenance, and sleep quality were deter-
and placebo in a double-blind randomized mined from sleep questionnaires that patients
study in elderly insomniacs. The patients re- completed each morning. The occurrence of
ceived zolpidem (5 mg; 70 patients), zolpidem rebound insomnia and withdrawal effects. on
(10 mg; 74 patients), or triazolam (0.25 mg; 77 discontinuation of treatment were also as-
patients). The 3-week active treatment period sessed. All levels of significance were P I0.5.
was preceded by 3 days and followed by 7 days Median sleep latency was significantly lower
of placebo administration. Both patients and with zaleplon (10 and 20 mg) than with pla-
clinicians evaluated sleep quality. The im- cebo during all 4 weeks of treatment and with
provements between the end of placebo phase zaleplon (5 mg) for the first 3 weeks. Zaleplon
and the end of the active treatment phase (20 mg) also significantly increased sleep du-
were significant for all treatments. Overall ration compared with placebo in all but week 3
evaluation indicated that zolpidem and triazo- of the study. There was no evidence of re-
lam are both effective in geriatric insomniac bound insomnia or withdrawal symptoms af-
patients (98). ter discontinuation of 4 weeks of zaleplon
3.2.3.2 Heterocyclic Sedative-Hypnotics treatment. Zolpidem (10 mg) significantly de-
Zopiclone (7.5 mg) was compared to temaz- creased sleep latency, increased sleep dura-
epam (30 mg) and placebo in a double-blind tion, and improved sleep quality at most time
3-week study in insomniac patients. The pa- points compared with placebo; however, after
tients were assessed before and at the end of discontinuation of zolpidem treatment, the in-
each of the 3 weeks active treatment phase cidence of withdrawal symptoms was signifi-
and 1 week and 3 weeks posttreatment. The cantly greater than that with placebo and
results indicated that zopiclone and temaz- there was an indication of significant rebound
epam possess a clinically significant hypnotic insomnia for some patients in the zolpidem
activity with no rebound insomnia or anxiety group compared with those in the placebo
occurring during the week of drug withdrawal group. The frequency of adverse side events in
4 History
the active treatment groups did not differ sig- and Roche followed with the introduction of
nificantly from that in the placebo group. Zale- Noludar (methyprylone) in 1955. However,
plon is effective in the treatment of insomnia. these two sedative hypnotic drugs displayed
In addition, zaleplon seems to provide a favor- many of the unwanted side effects of the bar-
able safety profile, as indicated by the absence biturates. Noludar is no longer marketed and
of rebound insomnia and withdrawal syrnp- the sales of Doriden are insignificant today.
toms once treatment was discontinued (101). 4.1.4.2 The Second Phase. The second
phase of the development of heterocyclic sed-
ative-hypnotics was initiated after the devel-
4 HISTORY opment and launch of the benzodiazepine
sedative-hypnotics. Although the 1,Cbenzodi-
4.1 Discovery of Important Hypnotic Drugs azepines are safe and effective drugs, they too
have produced certain undesirable side ef-
4.1.1 Before Barbiturates. The introduc- fects. This compelled some companies to de-
tion of ether and chloroform in the 1820s led velop non-benzodiazepine sedative-hypnotic
to the synthesis of similar chemical com- drugs.
pounds. The famous German chemist Justus These include Rhone Poulenc (Aventis),
Liebig synthesized chloral in 1831 by passing the developer of zopiclone (launched in 19851,
chlorine through alcohol (hence its name). Synthelabo (Sanofi Synthelabo) the developer
This led to the development of chloral hydrate, of zolpidem (launched in 19881,and Wyeth Ay-
the first synthetic potent hypnotic drug that erst, the developer of zaleplon (launched in
was safe enough for routine use. Chloral hy- 1999).
drate was introduced in 1869. All three compounds elicit rapid onset of
In 1887, a number of new sulfur com- action and a full night's sleep. They have a
pounds were synthesized by Eugen Baumann short half-life with no active metabolites.
at the University of Freiburg, Germany and There is no objective evidence of rebound in-
evaluated by Alfred Kast. One of these, sul- somnia or tolerance in studies of up to 30-40
fond, was acquired and launched by Bayer in nights at recommended doses. They have a fa-
Germany. vorable safety and tolerability profile and gen-
,
erally preserve the stages of normal sleep.
4.1.2 Barbiturates. Based on the finding Zopiclone, zolpidem, and zaleplon have
that some compounds containing a quater- made major inroads in capturing significant
nary carbon (sulfonal and amylene) displayed market shares of the sedative-hypnotic mar-
hypnotic properties, in 1903, Emil Fischer and ket segment.
J. von Mehring synthesized 5,5-diethylbarbi-
turic acid. This was marketed by Bayer as 4.1.5 Benzodiazepines. ARer the first phase
Verona1 in the early 1900s. Since 1903, hun- of the launch of heterocyclic sedative-hypnotics,
dreds of barbiturates have been synthesized, as exemplified by glutethimide and methypry-
but only a few turned out to be useful. In ad- lone, most significant milestones were reached
dition, because of their side effects, the syn- by Roche. In 1960, Roche launched Librium
thesis of non-barbiturate hypnotics has been (chlordiazepoxide) and in 1963, Valium (diaz-
undertaken. epam). These two compounds were the first
two 14-benzodiazepine class compounds
4.1.3 Halogenated Sedative-Hypnotics. At- launched in the world. Subsequently a num-
tempts to develop effective halogenated seda- ber of companies launched several other 1,4-
tive hypnotic drugs without bothersome side benzodiazepines for a number of indications
effects have not met with success and resulted (sedative-hypnotic, anxiolytic, anticonvulsant,
in only marginally useful products. and muscle relaxant).
Nitrazepam (Mogadon) was the first benzo-
4.1.4 Heterocyclic Sedative-Hypnotics diazepine marketed in 1965 by Roche as a spe-
4.1.4.1 The First Phase. CIBA (Novartis) cific sedative-hypnotic drug. At that time it
introduced Doriden (glutethimide) in 1954 was already well known that diazepam mar-
Sedative-Hypnotics
keted in 1963 had also displayed sedative-hyp- azepines represented a major step forward.
notic activity, but diazepam was marketed pri- They replaced the barbiturates, which were
marily as an anti-anxiety agent. very effective but produced significant side ef-
The benzodiazepines became widely used fects. The biggest problem was the potential
and dominated the market for approximately fatal respiratory depression caused by over-
25 years. dose of barbiturates, which led to the barbitu-
However, the contention that benzodiaz- rates being used in suicide attempts.
epines have solved the problems usually asso- The launch of benzodiazepines eliminated
ciated with the use of barbiturates has been the risk associated with using barbiturates.
contraindicated. It has been pointed out that The benzodiazepines are well tolerated and
in humans, benzodiazepines produce a consid- produce only low toxicities. However, after ex-
erable reduction in REM sleep, and in addi- tensive use of benzodiazepines in millions of
tion, an appreciable reduction of stage 3 and patients, it became apparent that benzodiaz-
stage 4 sleep. Furthermore, it has also been epines also produce side effects, although to a
reported that a distinction between barbitu- considerably lower degree than barbiturates.
rates and benzodiazepines on the basis of The most important side effects of benzodi-
withdrawal effects on the sleep pattern as doc- azepines include the following:
umented by EEG measurements seems un-
warranted. On the other hand, it should also 1. Benzodiazepines disturb the natural archi-
be noted that, although in humans larger dose tecture of sleep, shortening REM sleep,
of the benzodiazepines suppress REM sleep, stage 3 and 4 (the deep sleep) periods, and
the extent of REM sleep suppression is usually lengthening stage 2 sleep.
smaller than with most other types of hyp- 2. Extensive use of benzodiazepines may lead
notic drugs. to psychological and physical dependence,
Benzodiazepines drugs are often taken in withdrawal syndromes, and rebound in-
suicidal attempts but have rarely been fatal somnia on discontinuation of benzodiaz-
following even a large overdose. In this re- epine treatment.
spect, benzodiazepines possess tremendous 3. Benzodiazepines may also cause memory
advantage over barbiturates and several other loss (anterograde amnesia), apneas, dis-
classes of hypnotic drugs. abling residual effects on alertness, and
may potentiate the CNS depressing effects
4.1.6 Antihistamines. A number of antihis- of alcohol.
tamines display sedative activity. Some of
these drugs have been employed as sedative- The recognition that benzodiazepines pos-
hypnotics. Several antihistamines are avail- sess these adverse effects caused Synthblabo
able on the OTC market, which explains their scientists to initiate research to develop a new
relative popularity. sleep-inducing drug that would be devoid of
4.2 Discovery of Zolpidem side effects.
In the early 1990s, the French company Syn- 4.2.2 The Development of Zolpidem. The
thblabo assembled a group of well-trained sci- fact that there are several benzodiazepine re-
entists to focus on research in the CNS area. ceptor (BZR) site subtypes preferentially lo-
The focus of the CNS department was to dis- cated in certain areas of the brain (see Section
cover innovative drugs that act through novel 5.2.2) encouraged Synthblabo scientists to
mechanisms and meet unfulfilled therapeutic search for new hypnotics that would lack the
needs. Another goal was to create new drugs unwanted pharmacologic activities generally
with improved efficacy or safety in the areas of associated with the hypnotic activity of benzo-
anxiety, depression, schizophrenia, sleep dis- diazepines and that would not cause tolerance,
orders, and neurodegenerative diseases. dependence, or rebound phenomena when
treatment was discontinued.
4.2.1 The Use of Benzodiazepines. In the The next step involved the search for com-
area of sleep disorders, the launch of benzodi- pounds that would display selective activity
5 Structure-Activity Relationships
for BZR subtypes. This research culminated in In Equation 5.6, C represents the moles of
the discovery of zolpidem, a selective agonist drug per kilogram of test animal producing
of the BZ-1 subtype that turned out to have hypnosis, P is the calculated partition coeffi-
selective hypnotic activity free of the disad- cient (based on the measured value for 5,5-
vantages of the benzodiazepines. diethylbarbiturate), and k , k t , and k" are con-
Synth6labo's strategy consisted of three stants derived by the method of least squares.
stages: The barbiturates were clustered into eight
groups depending on the species of test animal
1. Analysis of three-dimensional molecular and the means of measurement of hypnosis,
models helped determine the affinity of the such as ED,, or MED, as reported in the liter-
ligands for their receptors and elaborate ature from which the biological test data were
the chemical concept. taken. A constant termed log Powas derived
2. Synthesis of large number of heterocyclic by setting the derivative (d) log(1lC)ld log P
compounds corresponding to the chemical equal to zero and solving for log P. log Powas
concept led to the identification of the im- defined as representing "ideal liphophilic
idazopyridine class of compounds. character" for a set of congeners under specific
3. Lead optimization of imidazopyridines dis- test conditions. For the five groups of barbitu-
playing optimal hypnotic properties led to rates for which confidence intervals could be
the selection of SL 80, 0750 (zolpidem). calculated, there was good general agreement
among the Povalues with a mean value of 1.9.
Zolpidem showed powerful sleep-inducing Po values of about 2 were also obtained for
activity in EEG tests in rats with a short du- various non-barbiturate hypnotics (e.g., acety-
ration of action. In addition, zolpidem did not lenic alcohols, N, N'-diacylureas) and it was
alter the architecture of sleep and thus in- suggested that any organic compound with
duced physiological sleep. Clinical trials in hu- -
log P 2, which is not rapidly metabolized or
man with zolpidem began in 1983 and con- eliminated, would possess some hypnotic
firmed the results obtained in animals. properties.
Furthermore, in humans, zolpidem had a In a subsequent study, Hansch reported
short half-life combined with fast elimination that the induction of cytochrome P450 by a
from the body. Zolpidem was first marketed in series of 5,5-substituted barbiturates was di-
France in 1988 and subsequently it was mar- rectly related to their hydrophobicity (105).
keted worldwide. The features of zolpidem are Analysis of the in vitro data for barbiturate-
described elsewhere in the chapter. induced P450 in cultured chick hepatocytes
yielded Equation 5.7, where C is the drug con-
centration that caused a 50% increase in
5 STRUCTURE-ACTIVITY RELATIONSHIPS cytrochrome P450 and P is the octanol-water
partition coefficient.
5.1 Quantitative Structure-Activity
Relationships (QSAR)
(5.8)
chlordiazepoxide benzodiazepinones
useful pharmacological effects, must certainly ported by Sternbach and that bore a variety of
rank among the landmark achievements in substituents at positions 7 and 2' (108). Using
the annals of medicinal chemistry. Discovery CND0/2 methodology and calculated values
of the prototype compound, chlordiazepoxide, for dipole moment (p) and net charge on the
was serendipitous in that it, rather than the carbonyl oxygen (qo), analyses of the data for
simple substitution product, was formed by an several different in viuo tests were conducted.
unexpected ring enlargement from the Equations 5.9 and 5.10 were derived from data
treatment of a quinazoline N-oxide deriva- for the pentylenemetrazole test (a measure of
tive with methylamine (Equation 5.8) (106, anticonvulsant activity) and the "Cat" test ( a
107). After its structural elucidation, it was measure of sedative/muscle relaxing activity
found that this compound possessed desir- in cats), respectively.
able pharmacological properties including
anxiolytic, sedative, muscle relaxant, and
anticonvulsant activities. Ensuing preclini-
cal and clinical studies corroborated the ini-
tial findings and led to its introduction in
1960 as Librium. Synthetic investigations
aimed a t finding compounds with even bet-
ter pharmacological profiles led to the con-
version of N-oxides such as chordiazepoxide
to the classical 1,4-benodiazepin-2-one che-
motype (Equation 5.8); compounds of this
type were found to have activity at least
equivalent to that of chlordiazepoxide and Improvements in regression by qo were gen-
some, such as diazepam, showed severalfold erally not significant, and the introduction
greater potency in various tests. Thus, alter- of the Hansch lipophilic substituent con-
nate synthetic procedures were developed t o stant failed to produce improvement. It was
obtain a variety of analogues and study SAR. suggested that the negative term containing
A summary of the earlier qualitative SAR p might be caused by a binding process in-
findings based exclusively on in viuo data is volving dipole interactions that remove drug
graphically shown in Fig. 5.9. molecules from the active site.
A QSAR study was carried out on over 50 In the decades after the first synthesis of
1,4-benodiazepinonesthat were previously re- the prototypical agents chlorodiazepoxide and
5 Structure-Activity Relationships
Equation 5.15, which shows a positive contri- The summary conclusion drawn was that
bution to binding affinity by both hydropho- overall ligand hydrophobicity is important for
bicity (logP) and sterimol parameters, Bl-, BZR binding and that for the classical benzo-
and B1-,, for substituents at the 7 and 2' posi- diazepinone chemotype the hydrophobic1
tions, respectively. steric effects of moieties appended to positions
C7 and C2' are positively associated with re-
ceptor affinity.
The computational construction of artifi-
cial neural networks has also been applied to
relate physicochemical parameters of benzodi-
azepines with their receptor affinity and to
predict BZR properties and BZR ligand affini-
ties. In a study by Maddalena and Johnston,
back-propagation artificial neural networks
were used to examine the QSAR between sub-
stituent constants at six positions on 57 ben-
zodiazepinones with their empirically deter-
mined binding affinities (118). Among the
The significance of the Bl-,term was inter- findings of the study were the following:
preted as pointing to a steric effect of the f i s t
atom of groups in the 7 position; i.e., the larger 0 Position 7 is the most important location for
the atom attached to C7, the more effective the enhancing BZR affinity; increases in sub-
binding. It was suggested that atoms at this po- stituent lipophilicty and electronic charge
sition produce a conformational change in the were found to be directly related to in-
receptor that is conducive to ligand binding. The creases in receptor binding. The optimal C7
positive steric effect of C2' substituents implies substituents from best to worst were deter-
that receptor binding is enhanced by the twist- mined to be CH,CF, > I > Br > CF, > C1>
ing of the 5 phenyl ring out of the plane of the C(CH,), > NO, > F > N, > CH=CH,.
seven-membered ring. No role was found in 0 Substituents at position 2' are of second-
Equation 5.15 for electronic parameters. most importance in positively influencing
Hansch's reevaluation of the earlier QSAR BZR affinity, and increases in the polar na-
study of in vivo data (108) resulted in Equation ture of these substituents were shown to be
5.16 for the pentylenetetrazole test. In contrast beneficial, although this effect was dimin-
to Equation 5.9), which shows no role for hydro- ished if the groups were bulky.
phobic effects, Equation 5.16 is positively corre- 0 Substitution at positions 3 and 8 is disfa-
lated with both hydrophobicityand steric effects vored, and electrostatic influences at these
of the C7 and C2' substituents as well as with qo positions are important.
(charge on the carbonyl oxygen).
Figure 5.10 summarizes the findings of the
various QSAR studies. It is somewhat discon-
certing that there is considerable disagree-
ment among the key conclusions drawn from
several of the studies, e.g., some propose a hy-
drophobic interaction of C7 substituents,
whereas others suggest that hydrophilic
groups are favored or that electrostatic inter-
actions of C7 groups with a cationic subsite of
the receptor are important. However compar-
ison of Figs. 5.9 and 5.10 shows that the vari-
ous QSAR largely corroborate the earlier qual-
itative SAR reported by Sternbach. Because
Sedative-Hypnotics
[ ~ r o u plarger
s than methyl disfavored (112, 115)
Electron withdrawing
groups favored (111, 112, 114, 116);
hydrophilic groups favored (115);
hydrophobic and/or sterically bulky
favored (110, 116, 117, 118)
pfL R2'
Electron withdrawing
groups favored (111, 112);
hydrophobic groups favored (110);
polar groups beneficial if not too
bulky (118)
Substituents disfavored (112, 115)
the latter were based exclusively on in vivo nition sites of differing affinities for GABA
data, whereas most of the QSAR studies have (10)itself as well as for GABA agonists such as
used in vitro binding data, the general agree- the natural product muscimol (11)and the
ment between the two suggests that the phar-
macological effects of the benzodiazepines are
due mainly, if not solely, to their BZR interac-
tions.
GABAA
igonists Agonists Antagonists
rntagonists Bicuculline
lembrane
Barbiturates
Figure 5.11. Schematic represen-
Phenobarbital tation of the GABA, receptor com-
Pentobarbital plex. Reprinted with modification
'icrotoxin
butylbicyclophosphorothionate(TBPS) from P.A. Saunders and I.K. Ho,
Prog. Drug Res., 34, 261 (1990)
;opropylbicyclophosphate (IPPO)
'entylenetetrazole I with permission from Birkhauser'
Verlag AG.
sts, THIP has been found to be an effective been grouped into six classes based on se-
:dative-hypnotic in humans (120-122). quence homology. The 16 human GABA, re-
As shown in Fig. 5.11, the GABAAreceptor ceptor subunits (al- 6, pl-4, yl-4, 6, &) that
~mplexalso has binding sites for drugs such have been cloned to date show approximately
r barbiturates and benzodiazepines as well as 30% sequence identity among subunits and
r certain convulsive agents such as picro- about 70%homology among subunit subtypes
~xinwhich inhibit chloride channel activity. (125). Despite the enormous number of possi-
ABA receptors, their diversity, and pharma- ble arrays, it seems that a rather limited num-
)logy have been extensively reviewed (123- ber of hetero-oligomeric combinations actu-
18). ally occur in nature and that a functional
Electron microscopy studies have revealed GABA, receptor requires both an a and P and
at, like acetylcholine receptors, the ion one other subunit type. The pentameric as-
lannel of the GABA, receptor is formed by sembly is illustrated in Fig. 5.11.
le pentameric assembly of hetero-oligomeric
ibunits (129); each subunit has four trans- 5.2.1 Barbiturates. Barbiturate interactions
embrane spanning domains and all five sub- with the GABA, receptor complex have been
nits are arranged so that their second trans- reviewed (130,131). Barbiturate effects on re-
embrane domains comprise the ion channel ceptor-mediated chloride ion flux have been
all. Cloning of the subunits from vertebrates extensively studied employing electrophysio-
ss resulted in nearly 20 cDNAs, which have logical techniques either in vivo or in isolated
Sedative-Hypnotics
hance GABA responses at low nanomolar con- was further substantiated by the finding that
centrations and the effects of diazepam were Hido2of the bovine a1 subunit is the major
blocked by the benzodiazepine antagonist, site of photoaffinity labeling by [ 3Hlflunitraz-
flumazenil (Ro 15-1788). The sedative-hyp- epam (148, 149). It was postulated that the
notic, zopiclone, also potentiated GABA re- residue interacts directly with the pendant
sponses, but at high nanomolar concentrations. phenyl group of diazepam and other 5-phenyl
Two distinct sets of BZR, designated as benzodiazepines. Other amino acids within
BZ-I and BZ-I1 (also called 0-1and 0-2, respec- the a1 subunit that may be part of the benzo-
tively), are prevalent in the CNS (142). Pe- diazepine binding site are Gly200,Thr206,and
ripheral type (0-3) BZRs are located in mito- TyrZo9(150, 151). Replacement of Tyr209 by
chondrial membranes and glial cells, but their Ala, Phe, or Gln afforded mutant receptors
relevance to the central action of benzodiaz- that showed moderate to total loss of affinity
epines has not been established. BZ-I recep-
for diazepam or flunitrazepam (Tyr209Phe:2-
tors have abundance in the cerebellum but
to 8-fold decrease, T y ? O g A l a : -40-fold decrease,
paucity in the hippocampus, whereas the con-
verse is true for BZ-I1 receptors; both receptor Ty?OgGln: no detectable affinity). The 6 t y
types are equally expressed in the cerebral cor- for GABA of these Xenopus oocyt~expressed
tex (125). Recombinant studies of GABAAre- receptors was only slightly diminished, but the
ceptor subunits have demonstrated that the ability of flunitrazepam to stimulate GABA-in-
a1 subunit is important for BZ-I receptor duced currents was abolished.
characteristics and that the y2 subunit pro- Phe77and Met130within the y2 subunit have
motes benzodiazepine binding (137). The been shown to be necessary for high affinity
alp2y2 combination was the first GABAAre- binding of andlor modulation by BZR ligands
ceptor subtype to be clearly identified and is (152). Receptors containing a y2Phe7711emuta-
thought to be the most abundant subtype in tion retained high affinity for flunitrazepam
adult mammalian brain (143). Numerous BZR but not for other ligands such as flumazenil
ligands, including benzodiazepines, p-carbo- and methyl p-carboline-3-carboxylate.How-
lines, and imidazopyridines such as zolpidem, ever the mutation prevented allosteric modu-
interact with this receptor subtype. Both a lation of ion channel currents by flunitraz-
and y subunits contribute to the benzodiaz- epam. It was suggested that Phe77serves as a
epine binding site, which seems to be situated contact point for certain BZR ligands, al-
at the interface of these subunits (144, 145). though it is not essential for high affinity bind-
Mapping the benzodiazepine binding site by ing, the energy requirements for which are
site-directed mutagenesis has revealed that al- satisfied by other contact points. But in the
terations of certain amino acids within the N- absence of Phe77, ligands may occupy the
terminal domains of the a and y subunits have binding site in a conformation that is incapa-
significant effects on the affinity and efficacy ble of initiating the allosteric changes leading
of ligands. One such site identified by alanine to channel modulation. A y2Phe77Tyrmodifi-
scanning of the a1 subunit is histidine 101 (rat cation resulted in significant loss of affinity for
numbering) or 102 (humadbovine number- benzodiazpinones bearing a 5-phenyl moiety,
ing) (146). Various other substitutions of the e.g., diazepam, which had a Ki of 3 f l at the
histidine have been examined and the effects mutant receptor versus 12 nM at the wild-type
of several BZR ligands on the mutant a1 sub- receptor (153). High affinity was retained by
units co-expressed with p2 and y2 subunits in BZR ligands lacking the phenyl moiety, thus im-
Xenopus oocytes were evaluated by electro- plying that the tyrosine hydroxyl interferes with
physiological techniques (147). Substitution the phenyl group and that the latter is in close
by Phe, Tyr, and Gln had little effect on the proximity to Phe77when diazepam and like mol-
ability of flunitrazepam to potentiate GABA- ecules bind to the wild-type receptor. Together
induced currents, but other mutations (Lys the cylHislO1, alGlYo0, a 1 T P o 6 , a l T y ? O g ,
and Glu) resulted in a drastic reduction of the y2Phe77, and y2Met130 residues may be in-
flunitrazepam response. The importance of volved in the formation of the binding pocket of
this amino acid residue for agonist recognition BZR ligands (151).
Sedative-Hypnotics
ANXIOLYTIC
SEDATIVE-HYPNOTIC ANTAGONISTS CONVULSANT
ANTICONVULSANT Flumazenil SPASMOGENIC
MUSCLE RELAXANT (Ro 15788) PROMNESIC
AMNESIC ZK 93426 GABA ANTAGONIST
I *
I
t*
PARTIAL AGONISTS
I
PARTIAL INVERSE AGONISTS
P-CCE
Bretazenil
hidazenil Sarmazenil
Abecarnil Ro 19-4603
Me
CI
CI Br
\
0
cle relaxing activity and sedationkypnosis. reviewed (160, 161), and Fig. 5.12 shows ex-
Functional antagonists display no efficacy of amples of a number of such compounds that
their own, but their occupancy of the benzodi- are classified according to their pharmacolog-
azepine binding site can block the action of ical properties.
both agonists and inverse agonists. The latter, The mechanism of action of agents such as
which include some p-carboline and tricyclic DMCM as inverse agonists has been explained
azepine derivatives, exert a negative coopera- in terms of a two-state model for the benzodi-
tive effect on GABA binding, and thus are anx- azepine-GABA receptor complex (162). The
iogenic and proconvulsant. Some compounds model proposes that the complex exists in
with intermediate activities can be classed as equilibrium between interconvertable open
partial agonists or partial antagonists. BZR li- and closed channel conformations. GABA and
gands of various functional types have been BZR agonists such as diazepam exhibit recip-
5 Structure-Activity Relationships
rocal cooperativity in that they both bind se- the peripheral BZR (166) and development of
lectively to the open conformation and allow a three-dimensional pharmacophore model
chloride ion flux. Conversely, an inverse ago- for BZR ligands having anxiolytic activity
nist binds selectively to the closed conforma- (167).
tion and in so doing exhibits negative cooper- Cook and co-workers have published exten-
ativity by allosterically inhibiting GABA sively on their studies of BZR pharmacophore
binding. This rationale of inverse agonism is modeling (168-173). These studies have em-
supported by the observation that GABA re- ployed Comparative Molecular Field Analysis
ceptor stimulation reduces the BZR affinity of (CoMFA) of the structural parameters of a
DMCM (163),and thus it may be assumed that wide range of BZR ligands, many of which
DMCM reciprocally reduces the affinity of were designed and synthesized to probe the
GABA for its receptor. size, shape, and functional group tolerance of
There is evidence that the functional activ- the benzodiazepine binding domain. An exam-
ity of BZR ligands is caused by the interactions ple of a derived model showing the compara-
(hydrogen bonding, hydrophobic interactions) tive binding fit of an agonist (diazepam) and
of their structural moieties with kev " amino an antagonistlpartial inverse agonist (P-CCE)
acid residues that comprise the ligand recog- is depicted in Fig. 5.13.
nition site. The previously mentioned muta- Of particular relevance to sedativebyp-
tions of Hido1 in the a1 subunit have signifi- notic agents is the development of a pharma-
cant effects on binding affinities, as well as cophore for the sedation endpoint (173). Com-
altering the characteristic agonist, inverse ag- plementary behavioral and computational
onist, or antagonist responses to various li- studies of 21 structurally diverse BZR ligands
gands as determined by electrophysiology that influence spontaneous motor activity (a
(147).Introduction of a Hislo1Arg-mutated a1 behavioral indicator of sedation) were con-
subunit into mice has served to demonstrate ducted. A five-component three-dimensional
that certain behavioral actions of benzodiaz- pharmacophore consisting of two proton-ac-
epines are mediated by specific GABA, recep- cepting moieties, a hydrophobic region, a ring
tor subtypes (164). al(Hislo1Arg) transgenic with polar moieties, and an aromatic ring was
mice failed to show the sedative, amnesic, and derived and is represented in Fig. 5.14. The
in part, the anticonvulsant responses to diaz- model was shown to accommodate the ligand
epam, thus indicating that these behaviors are structural requirements in the overlapping
attributable to BZR agonist activation of the portion of the binding sites for agonists, in-
a1 containing GABA, receptor subtypes, verse agonists, and antagonists that impart
which are localized mainly in cortical areas effects on the behavioral sedation endpoint.
and thalamus. In contrast, the anxiolytic, my- Agonists decreased spontaneous motor ac-
orelaxant, motor-impairing, and ethanol-po- tivity, inverse agonists caused an increase,
tentiating effects of diazepam were retained and antagonists, while lacking intrinsic activ-
and must be caused by the drug's interaction ity of their own, blocked the effects of the ag-
with nonmutated GABAAreceptors in the lim- onist, flunitrazepam. The reliability of the
bic system ((u2, a5), in monoaminergic neu- model was evaluated in several ways: ligands
rons (a3), and in motor neurons (a2, a5). The without effect at the sedation endpoint did not
finding that benzodiazepine-induced behav- accommodate the pharmacophore require-
ioral responses are mediated through distinct ments; several BZR ligands that were not used
neuronal circuits has implication for drug de- in pharmacophore development but that were
sign. That is, agonists acting upon a2-,a3-, known to affect sedation satisfied the pharma-
and/or a5- but not on al-containing receptor cophore; and use of the pharmacophore pa-
subtypes could be nonsedative and nonarnne- rameters to search three-dimensional data-
sic anxiolytics. bases resulted in identification of additional
Numerous pharmacophore models of the BZR ligands known to have effects on the sed-
BZR have been proposed, and a number of
-
ative endpoint. The model may thus be useful
these were reviewed about a decade ago (165). for the design of novel chemotypes having sed-
More recent examples include the mapping of ative/hypnotic activity.
Sedative-Hypnotics
polar moities
Distances in angstroms
D(l-2) = 4.9 f 0.9 1
D(l-3) = 5.0 _+ 1.3 oF2
DL
D(l-4)= l . 8 f 1.0
D(l-5) = 4.8 ?r 0.9
+
D(2-3) 6.1 2.0
D(2-4) = 3.3 f1.5
+
D(2-5) = 4.0 0.8
\ 'N1 \ /
D(3-4)
D(3-5) = 5.0 +
8.1 f 1.3
0.7
D(4-5) = 3.5 f 0.7 Zolpidem
mg) increased sleep propensity and advanced than the mt, receptor (190). Subtype-selec-
sleep termination in the subjects without af- tive compounds may prove to be of value in
fecting the EEG patterns in either REM or elucidating the relative importance of the
non-REM sleep (198). receptor subtypes in the pharmacology of
Other structural permutations of the naph- melatonin.
thalene chemotype, as exemplified by (16-181, Whether any synthetic melatonergic ago-
also retain potent melatonin receptor binding. nists will ever become approved drugs for the
The monomethoxy derivative (16) is less po- treatment of sleep disorders is a matter of
tent (Ki = 2.7 nM) in binding to ovine pars speculation. A major obstacle they face is that
tuberalis receptors than its regioisomer (151, the naturally occurring hormone is available
but addition of a second methoxy group in- on an over-the-counter basis in many coun-
creases the affinity (Ki = 0.7 nM) of (17) by an tries including the United States; thus, any
order of magnitude (199). Quinoline (18) is
synthetic drug would have to have some dis-
about as potent (K, = 5.9 nM)as melatonin in
tinct advantage over melatonin itself. Melato-
binding to human mt, receptors expressed in
CHO cells (200). The phenylene derivative nin receptors have been identified in the vas-
(19) (201), in which the arnide-containing culature and their activation by melatonin
side-chain is conformationally constrained, results in a vasoconstrictor response (181).
and (20) (202), in which the alkoxy group is Thus, melatonin could be contraindicated in
incorporated into a dihydropyran ring, also individuals, particularly the elderly, who have
exhibit potent binding with Ki values of 0.7 cardiovascular conditions. Perhaps a receptor
and 0.1 nM,respectively, at the receptors in subtype-selective agent would maintain the
chicken brain and ovine pars tuberalis. The beneficial effects of melatonin on sleep with-
tetralin derivative (21) is a MT,-selective li- out melatonin's vasoconstrictor properties
gand with 20-fold higher affinity for the MT, and other pharmacological effects.
Sedative-Hypnotics
0 0
Ki for FAAH inhibition = 1 nM 9 nM 17 nM
6.2 Oleamide (2.8 mg) into rats caused sleep analogous to its
In the mid-1990s a collaborative team of IP administration (5 or 10 mglkg), thus indi-
chemists and biologists at the Scripps Re- cating a direct action in the brain. These ef-
search Institute reported on the isolation and fects were found to be compound-specific be-
structural and pharmacological characteriza- cause a number of close structural analogs
tion of a substance that accumulated under including the trans isomer, compounds with
conditions of sleep deprivation in cats (202- the cis double bond migrated to other posi-
205). Clues to the structure of the material, tions within the 18 carbon chain, a saturated
which was obtained in only trace quantities analog, and the corresponding carboxylic acid
from cat cerebrospinal fluid, were afforded by were all weakly effective or ineffective at in-
mass spectometry, which revealed an empiri- ducing sleep.
cal formula of C,,H,,NO and a lipid fragmen- Studies of the degradation and regulation
tation pattern. Subsequent comparison of the of oleamide revealed that it was hydrolyzed to
isolated material with a large number of syn- oleic acid and ammonia by the action of a.
thetic compounds having the correct formula membrane-bound enzyme, which based on the
and degree of unsaturation established its inhibition of its activity, seemed to be a serine
structure as 9(Z)-octadecanamide(22). or cysteine protease. Isolation and sequencing
of the protein led to the cloning of its cDNA
and expression in COS-7 cells. The expressed
enzyme was found to not only hydrolyze ole-
amide but a number of other fatty acid amides
and was thus designated as fatty acid amide
hydrolase or FAAH (206).
A number of inhibitors of FAAH have been
This material, referred to by the trivial synthesized (2071, and the more potent of
name, oleamide, proved to be of interest owing these are depicted in Fig. 5.16. The most po-
to the fact that the synthetic compound was tent compounds contain a highly electrophilic
found to induce sleep in rodents and cats. Var- carbonyl as part of either an a-ketoester, a-ke-
ious measures of sleep parameters after ole- toamide of trifluoromethyl ketone moiety,
amide administration showed the sleep to be structural features common to serinelcysteine
of a physiological quality. The compound was protease inhibitors. Several of these FAAH in-
found to dose-dependently increase the total hibitors caused sleep enhancement and lower-
time of slow wave sleep at the expense of wak- ing of body temperature in rats that were com-
ing and to lower body temperature, which is parable with those induced by oleamide. Based
characteristic of physiological sleep. Further- on these findings, it has been suggested that
more intraventricular injection of oleamide FAAH may represent a therapeutic target for
7 Things to Come
the discovery of sleep aids that potentiate the New opportunities for sleep therapy may
effects of oleamide by blocking its degrada- be forthcoming from recent discoveries per-
tion; some inhibitors (e.g., a-ketoamides) taining to the hypocretinlorexin ligand-recep-
might be dual acting agents that mimic the tor system. The hypocretinlorexin (Hcrt/Ox)
action of oleamide as well as attenuating its gene, described in 1998, encodes two neu-
metabolism (208). Of course, as with any class ropeptides, hypocretin-1 (Hcrtl) and hypocre-
of enzyme inhibitors, the potential clinical use tin-2 (Hcrt2), which are called orexin-A(0x-A)
of FAAH inhibitors is contingent on their se- and orexin-B (Ox-B), respectively (209, 210).
lectivity, because indiscriminate interaction Although the cell bodies in which the hypocre-
with other proteases could result in undesir- tins are made are restricted to the perifornical
able side effects. and dorsal and lateral hypothalamic areas,
A detailed review of oleamide, including a
they send projections to multiple neuronal
discussion of its possible modes of action such
systems throughout the brain including those
as serotonin receptor modulation and gap
junction inhibition, is available (208). containing neurons responsible for mainte-
nance of the waking state (211). mRNAs for
7 THINGS TO COME hypocretin receptors are differentially dis-
tributed in the brain with the highest levels
Pharmaceutical company research on seda- of HcrtlR mRNA occurring in ventromedial
tive-hypnotics rose and peaked within the sev- hypothalamic nuclei and locus coeruleus,
eral decades after the introduction of the ben- whereas Hcrt2R mRNA are found predomi-
zodiazepines and has waned since then nantly in hypothalamic paraventicular nuclei
because CNS-related drug discovery efforts and layer VI of the cortex (212). The hypocre-
have focused instead on improved therapy for tinlorexin ligand-receptor system and its im-
such disorders as depression and schizophre- plications for sleep and sleep disorders have
nia and the unmet medical need of stroke, been reviewed, and a model for the involve-
Alzheimer's disease, and other neurodegen- ment of the neuropeptides in arousal state
erative maladies. However, given the imper- control has been proposed (213). The model
fections of available drugs and the high inci- suggests that a balanced sleep-wake cycle may
dence of sleep disorders among the ever- depend on modulation by hypocretinlorexin
growing elderly population, more efficacious cell activation of monoaminergic neuronal
and safer agents are certainly needed. The populations in the locus coeruleus (noradren-
current stable of drugs typically act as CNS ergic), dorsal raphe nucleus (serotonerigic),
depressants that do not promote physiological and ventral tegmental area (dopaminergic),as
sleep and that may cause cognitive and mem- well as of cholinergic cells in the basal fore-
ory impairment, motor skills impairment (es- brain. In support of such a model are findings
pecially when ingested along with alcohol), that a mutation of the Hcrt2R gene resulting
and have potential abuse liability. Where in nonfunctional receptors causes narcolepsy
then, should we look for better agents, agents in dogs and that hypocretin knockout mice ex-
that may be better classified as sleep normal- hibit "behavioral arrest" and significantly in-
izers rather than sedative-hypnotics? Use of creased levels of REM and non-REM sleep. If
pharmacophore models such as that shown in removal of the hypocretinlorexin ligand-re-
Fig. 5.14 could conceivably lead to the design ceptor system results in a condition marked by
and synthesis of patentably-novel compounds, excessive and uncontrollable sleep (i.e., narco-
but their pharmacological properties would lepsy), then overactivity of this system may
most likely resemble those of the known seda- cause excessive arousal and impaired sleep. It
tive-hypnotics from whose structural parame- will be of interest to see if further research in
ters the models are constructed. As discussed this exciting area and the identification of
in previous sections, development of both mel- small non-peptide molecules capable of selec-
atonin receptor agonists and FAAH inhibitors tively blocking hypocretinlorexin receptors
could prove to be fruitful approaches, al- will lead to novel agents for the effective and
though both have some caveats. safe treatment of sleep disorders.
Sedative-Hypnotics
http://www.well.com/user/woaJfsseda.
htm: facts on sedative-hypnotics including
The following are a number of selected web- abuse potential
sites that pertain to sleep disorders and/or sed- http://www.hc-sc.gc.ca/hppb/alcohol-
ative-hypnotics. otherdrugs/pube/straight/sedative.html:com-
http://www.methodisth~~pitals.org/services/ monly abused sedative-hypnotics
dmgnostics/sleep/sleep4.html:dmgnosis and ther- http://www.aafp.org/afp/20000501/2763.
apy of sleep disorders html: abuse of and intoxication by psycho-
http://www.todoc.com/sleep/sleep.html: tropic drugs including sedative-hypnotics
sleep disorders and their treatment http://www.acnp.org/G4/GN401000173/
http://blueprint.bluecrossmn.com/article/ CH169.html: mechanism of action and phar-
iac/100547893:sleep disorders and their treat- macology of barbiturates
ment http://www.csusm.edu/DandB/Sedatives.
http://www.aafp.org/afp/20000501/2763. html: behavioral effects and mechanism of ac-
html: abuse of and intoxication by psycho- tion of barbiturates
tropic drugs including sedative-hypnotics http://www.sonatasleep.corn/healthpro/
http://www.acnp.org/G4/GN401000173/ about-sonatalabout -sonata.htm1: facts about
CH169.html: mechanism of action and phar- zaleplon (Sonata )
macology of barbiturates http://www.laurushealth.com/Library/
http://www.aafp.org/afp/20000501/2763. HealthGuide/DrugGuide~howTopic.asp?
html: abuse of and intoxication by psycho- topicid=9482&sequence =1: facts about zol-
tropic drugs including sedative-hypnotics pidem
http://www.acnp.org/G4/GN401000173/ http://www.sonatasleep.comlhealthpro/pi/
CH169.html: mechanism of action and phar- pi.htm: drug information on zaleplon (Sonata)
macology of barbiturates http://www.healthyplace.com/medications/
http://www.neuronic.com/neuronics/ triazolamhtm: pharmacology, indications,
sleepand2.htm: abstracts of clinical studies on contraindications, etc., of triazolam (Halcion)
sleep and sleep disorders
http://www.extendedcare.com/library/
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CHAPTER SIX
KENNETH R. SCOTT
School of Pharmacy
Howard University
Washington, DC
Contents
1 Introduction, 264
2 Clinical Applications, 265
2.1 Current Drugs, 265
2.2 Side Effects, Adverse Effects, Drug
Interactions/Contraindications,265
2.3 Absorption, Distribution, Metabolism, and
i Elimination, 273
2.3.1 Hydantoins, 273
2.3.2 Iminostilbenes, 275
2.3.3 Aliphatic Acid, 275
2.3.4 Benzodiazepines, 282
PAitarl ,
,- , d d J. Abraham
nnnn
h.7 5.5 Valproate, 310
ISBN 0-471-27401-1 O 2003 John Wiley & Sons, Inc. 6 Recent Developments, 311
263
Anticonvulsants
/I. ON^
fP\ ONa
1 NH2
Ethotoin, (lc) Fosphenytoin, (Id) Carbamazepine, (2)
L _I n
Valproic acid, (3a) Divalproex sodium, (3b)
C2H5
Ethosuximide, (5a) Methsuximide, (5b) Phensuximide, (5c)
cially when initiating or stopping either drug. hepatotoxicity especially in children <2 years
Likewise, carbamazepine exerts a variable ef- old who are on multiple therapy; (2) teratoge-
fect on phenytoin levels; conversely, carbam- nicity that includes neural tube defects; and
azepine serum levels may also be decreased. (3) life-threatening pancreatitis.
Folate deficiency has been noted with long- Diazepam. The side effects of diazepam for
term phenytoin therapy, given that folate is a intravenous administration are similar to
cofactor in the metabolism of phenytoin those of phenytoin when administered by this
through hydroxylation (see metabolism). Sim- route, that is, cardiovascular collapse when
ilarly, influenza virus vaccine may increase, administered too rapidly.
decrease, or have no effect on the total serum Clonazepam. Clonazepam interacts with
phenytoin concentrations (18). phenytoin with the resultant decrease in
Carbamazepine. The adverse effects with plasma levels of the benzodiazepine.
carbamazepine include the potential to pro- Clobazam and Nitrazepam. Two new inves-
duce aplastic anemia and agranulocytosis; tigational benzodiazepines appear to be safe
however, it should be noted that the majority and effective as anticonvulsants. However,
of patients presenting with leukopenia have these agents have long elimination half-lives
not progressed to these more serious blood that increase the potential not only for drug
dyscrasias. Initial pretreatment hematologi- accumulation but also for residual side effects.
cal testing should be undertaken with all pa- Ethosuximide. This succinimide is rela-
tients with carbamazepine. Drug interactions tively safe from side effects and drug interac-
are focused on the 3A4 isoform of the P450 tions.
enzyme; inhibitors increase carbamazepine Phenobarbital. The older barbiturate is to
levels, whereas inducers produce the opposite be used with caution in patients with patent
effect. Special note should be taken with the liver or respiratory symptoms. Phenobarbital
carbamazepine-isoniazid interaction. Isonia- can cause fetal damage when administered to
zid inhibits carbamazepine metabolism, pregnant women.
whereas carbamazepine may increase isonia- Primidone. As noted from the structure
zid metabolism to hepatoxic products (18). and biotransforrnation pathway, primidone is
Valproic Acid. An adjunctive agent, val- closely related to phenobarbital; thus patients
proic acid bears three warning statements: (1) who are hypersensitive to phenobarbital should
Anticonvulsants
0
H
)-.Z
NH2 0
Vigabatrin, (8) Gabapentin, (9) Felbamate, (10)
0
,,(3°xcH
4 0 CH3 N H2
e0
0'
CH3
H ~ C 0
CH3
Tiagabine, (14) Topiramate, (15) Levetiracetam, (16)
Figure 6.2. "Second-generation"anticonvulsants.
not be given this agent. Because the parent from Sweden disclosed a strong relationship
compound is metabolized to two active metab- between visual field effects (VFDs) and viga-
olites (see Section 2.3), patients with impaired batrin treatment (19). These VFDs were re-
liver function should be carefully monitored. lated to the duration and total dose of the
Trimethadione. This agent contains the agent. Further, the VFDs were irreversible
warning statement that, because of the poten- and, in a significant number of patients, pro-
tial to produce fetal malformations and seri- gressive.
ous side effects, trimethadione should be used Gabapentin. This agent is used as an ad-
only when other less toxic drugs have been junctive agent and is relatively safe from side
found ineffective in controlling petit ma1 sei- effects in nonpregnant adult patients. There
zures. was noted a small decrease in gabapentin ex-
Vigabatrin. A new investigational adjunc- cretion with concurrent cimetidine adminis-
tive agent in the United States, little is known tration, which was not clinically significant. It
about its side effects, drug interactions, or is recommended that gabapentin be taken at
contraindications. However, a recent article least 2 h after antacid administration.
2 Clinical Applications
Table 6.2 Anticonvulsant Effect of First- and Second-Generation Agents Against Different
Types of Seizures in Human Epilepsy"
Clinical Efficacy
Generalized Seizures
Partial
Agent Seizures Tonic-Clonic Absence Myoclonic
First-generation
Carbamazepine + + NE NE
Phenytoin + + NE NE
Phenobarbital + + NE +
Primidone + + + +
Valproate + + + +
Benzodiazepines + + + +
Ethosuximide NE NE + 2
Second-generation
Lamotrighe + + + +
Topiramate + + ? +
Oxcarbazepine + ? NE NE
Felbamate + + & +
Vigabatrin + ? NE NE
Tiagabine + ? ? NE
Gabapentin + ? NE NE
"Effect is indicated by the following: +, effective; ?, inconsistent data; NE, not effective; ?, no data available (or found).
Data are from Ref. 2. For a complete description of the seizure types, refer to previous edition (Ref. 8).
2.3 Absorption, Distribution, Metabolism, ment of the metabolite in urine can be used to
and Elimination determine the rate of metabolism, patient
compliance, or bioavailability (18).
Table 6.5 provides the pharmacokinetic pa-
rameters noted for the listed anticonvulsants, Mephenytoin. Chemically, mephenytoin is
which include, where possible, peak plasma 3-methyl-5-ethyl-5-phenylhydantoin (1.b)
levels, half-life, volume of distribution, and (Fig. 6.1) and is dispensed as the racemate.
plasma protein binding. Additional explana- The R and S forms undergo stereoselective
tions as noted in the table are provided as fol- oxidative biotransformation. The S-mepheny-
lows. toin undergoes rapidpara-hydroxylation (Fig.
6.3), whereas the R enantiomer is slowly de-
2.3.1 Hydantoins methylated to the active N-desmethyl metab-
Phenytoin. Phenytoin (1)is slowly absorbed olite (5-ethyl-5-phenylhydantoin, nirvanol),
from the small intestine. The rate, extent, and which is more potent than the parent com-
bioavailability vary because of the manufac- pound (21). The metabolism of mephenytoin
turer's formulation process. Intramuscular appears to be through cytochrome P450, spe-
injection tends to precipitate at the site of in- cifically through the CYP2 family (24). The
jection, resulting in erratic plasma levels; toxicity of this metabolite has limited its use
these levels are significantly lower than those (8).
obtained by the oral route. Phenytoin is me- Ethotoin. Chemically, 3-ethyl-5-phenylhy-
tabolized in the liver to inactive hydroxylated dantoin, ethotoin (lc) undergoes two bio-
metabolites (see Fig. 6.3) (20). For a complete transformation pathways leading to inactive
discussion, the reader is referred to the earlier products: p-hydroxylation [pathway (111 and
edition of this chapter (8).The metabolism of deethylation [pathway (2)l. This product has
phenytoin is capacity limited and shows satu- relatively low potency compared to that of
rability. Because the elimination of the p-hy- phenytoin. Like phenytoin, ethotoin displays
droxy glucuronide metabolite is rate limited saturable metabolism with respect to the for-
by its formation from phenytoin, measure- mation of the two metabolites (18).
Table 6.3 New Antiepileptic Medications: Pediatric Indications and Effectivenessa
Seizure Type or Epilepsy Syndrome
Agent (Initial
Availability) CPS PGS Abs JME LGS
Felbamate (1993) Monotherapy, ENL ENL ENL Adjunctive > 2 years ENL
Adjunctive > 14
years
N Gabapentin (1994) Adjunctive > 3 years ENL NE NE ? ?
4'
P Lamotrigine (1995) ENL ENL ENL ENL Adjunctive > 2 years ?
Topiramate (1996) Adjunctive > 2 years Adjunctive > 2 years ENL ENL ENL ENL
Tiagabine (1997) Adjunctive > 12 years ? NE NE ? ENL
Levetiracetam (1999) ENL ? ? ? ? ?
Oxcarbazepine (2000) Adjunctive 4-16 years ? ? ? ? ?
Zonisamide (2000) ENL ENL ENL ? ENL ENL
"CPS, complex partial seizures; PGS, primary generalized seizures; Abs, absence; JME, juvenile myoclonic epilepsy; LGS, Lennox-Gastaut syndrome; IS, infantile spasms; ENL,
effective, not labeled (although no indication exists, studies support its use in pediatric patients); NE, not effective; ?, not studied (no well-performed pediatric studies exist for this
indication). Data are from Ref. 17.
2 Clinical Applications
Anticonvulsant
kent Side Effects Adverse Effects Drua Interactions Contraindications
Phenytoin Drowsiness, dizziness, IV administration: Increased effects: Do not use in sinus
or blurred vision cardiovascular collapse," a. Inhibition of metabolism with bradycardia, sinoatrial block,
hypotension allopurinol, amiodarone, second- and third-degree AV
Oral administration: CNS: benzodiazepines, chloramphenicol, block, or in patients with
nystagrnus, ataxia, cimetidine, disulfiram, ethanol, Adams-Stokes syndrome.
slurred speech fluconazole, isoniazid, metronidazole,
Dermatologic: rash miconazole, omeprazole, phenacemide,
Endocrine: diabetes succinimides, sulfonamides,
insipidus, trimethoprim, valproic acida
hyperglycemia b. Plasma protein displacement with
Others: Gingival salicylates," tricyclic antidepressants,
hyperplasia," valproic acida
thrombocytopenia, c. Unknown mechanism:
agranulocytosis, chlorpheniramine, ibuprofen,
tinnitus, diplopia phenothiazines
Decreased effects:
a. Increased metabolism with
barbiturates," carbamazepine,"
diazoxide, ethanol, rifampin,
theophylline
b. Decreased absorption with antacids,
charcoal, sucralfate
c. Unknown mechanism: antineoplastics,
folic acid," influenza virus vaccine,"
loxapine, nitrofurantoin, pyridoxine
Carbamazepine Drowsiness, dizziness, Hematologic: aplastic Increased effects (P450 3A4 inhibitors): History of bone marrow
or blurred vision anemia, leukopenia, cimetidine, danazol, diltiazem, depression; hypersensitivity
agranulocytosis, bone erythromycin, troleandomycin, to carbamazepine and
marrow depression clarithromycin, fluoxetine, isoniazid, tricyclic antidepressants;
CNS: dizziness, niacinamide, nicotinamide, concomitant use of
drowsiness, propoxyphene, ketoconazole, monoamine oxidase (MAO)
unsteadiness itraconazole, verapami1,and valproate inhibitors. Discontinue MA0
Other: nausea, vomiting Decreased effects (P450 3A4 inducers): inhibitors for 214 days
cisplatin, doxorubicin, felbamate, before carbamazepine
rifampin, phenobarbital, phenytoin, administration.
primidone, theophylline
Valproic acid Dizziness, suicide CNS: asthenia, Increased effects with chlorpromazine, Hepatic disease;
ideation, somnolence, dizziness, cimetidine, erythromycin, felbamate, hypersensitivity to valproate;
hyperammonemia, tremor salicylates pregnancy (FDA category D);
nausea GI: nausea, vomiting, Decreased effects with rifampin, children < 2 years (especially
abdominal pain carbamazepine, cholestyramine, those on multiple
Hematologic: phenytoin, lamotrigine, phenobarbital antiwnvulsant therapy,
thrombocytopenia those with congenital
Respiratory: infection metabolic disorders, those
Other: alopecia, headache with severe seizure
disorders, and those with
organic brain disease);
panmeatitis.
Clorazepate Drowsiness, ataxia, Hypersensitivity to
dipotassium and confusion benzodiazepines.
Diazepam Drowsiness, ataxia, IV administration: Hypersensitivity to
and confusion cardiovascular collapse benzodiazepines; psychoses;
CNS: sedation and acute narrow-angle
sleepiness glaucoma; children < 6
h)
years; lactation; concomitant
V alcohol administration.
V
Clonazepam Drowsiness, ataxia, Decreased effects with phenytoin Hypersensitivity to
and confusion benzodiazepines; clinical or
biochemical evidence of
significant liver disease.
Clobazam Drowsiness, hangover None reported Hypersensitivity to
effects, dizziness, benzodiazepines.
weakness, and
lightheadedness
Nitrazepam Fatigue, dizziness, None reported Hypersensitivity to
lightheadedness, benzodiazepines.
drowsiness,
lethargy, mental
confusion, ataxia
Ethosuximide Drowsiness, ataxia GI: nausea, vomiting Ethosuximide increases phenytoin levels Hypersensitivity to
Ethosuximide decreases primidone and succinimides.
phenobarbital levels
Table 6.4 (Continued)
Anticonvulsant
kent Side Effects Adverse Effects Drug Interactions Contraindications
Phenobarbital Drowsiness, ataxia Somnolence Increased effect with MA0 inhibitors, Barbiturate sensitivity;
valproic acid alcohol; pregnancy (FDA
Decreased effect with chloramphenicol, category Dl;manifest or
rifampin latent porphyria; severe
respiratory disease when
dyspnea or obstruction is
evident; nephritic patients.
Primidone Drowsiness, dizziness, CNS: ataxia and vertigo Increased effect with isoniazid, Lactation; pregnancy;
GI upset, GI: nausea, anorexia, nicotinamide, phenytoin porphyria; hypersensitivity
vomiting Decreased effect with carbamazepine, to phenobarbital.
ethosuximide
Trimethadione Drowsiness, dizziness, Systemic lupus Hypersensitivity to
sore throat, blurred erythematosus; skin oxazolidinediones; pregnancy
vision rash (leading to (FDA category D);
exfoliative dermatitis or photosensitivity.
severe erythema
multiforme); fatal
aplastic anemia; fatal
nephrosis has occurred
Vigabatrin None reported in 75% Somnolence, fatigue Decreases phenytoin levels Dose-dependent field effects."
of patients tested (adults); agitation,
insomnia (children)
Gabapentin Drowsiness, dizziness, Somnolence, dizziness, Increased effect with cimetidine Hypersensitivity to the drug;
headache, viral ataxia, fatigue, and Decreased effect with antacids patients > 12 years of age;
infection, nausea, nystagrnus pregnancy (FDA category C).
vomiting
Felbamate Lymphadenopathy, Aplastic anemia, liver Decreased effect with phenytoin, Hypersensitivity to the drug or
leukopenia, failure, carcinogenic (in phenobarbital, carbamazepine other carbamates; avoid
agitation, animal studies)," prolonged exposure to
tachycardia sunlight or sunlamps; may
cause photosensitivity;
history of blood dyscrasias or
hepatic dysfunction;
pregnancy (FDA category C).
Lamotrigine Dizziness, diplopia, Rash, dizziness, headache Increased effect with folate inhibitors, Hypersensitivity to the drug;
ataxia, blurred valproic acid dizziness; pregnancy (FDA
vision," nausea, and Decreased effect with acetaminophen, category C); children < 16
vomiting primidone, phenobarbital, phenytoin, years; renal andlor hepatic
carbamazepine function impaired patients.
Oxcarbazepine Psychomotor slowing; Cross sensitivity from Decreased effect with (P450 3A4 Hypersensitivity to the drug
dizziness and carbamazepine, inducers): carbamazepine, phenytoin, and to carbamazepine,
somnolence hyponatremia and phenobarbital, also decreased with alcohol. Recommend
verapamil and valproic acid additional nonhormonal
forms of contraception.
Zonisamide Somnolence, anorexia, Pruritus, vomiting, Decreased effect with (P450 3A4 Hypersensitivity to
dizziness, headache, amblyopia, tinnitus, inducers): carbamazepine, phenytoin, sulfonamides or zonisamide;
nausea, agitation1 asthenia, kidney stones and phenobarbital skin rash; pregnancy (FDA
irritability category C); sudden back
pain; abdominal pain; or
blood in the urine." Not
approved for pediatric use.
Tiagabine Generalized weakness Dizziness, somnolence, None reported Hypersensitivity to the drug;
depression, confusion, pregnancy; lactation.
asthenia
Topiramate Psychomotor slowing; Fatigue, headache, injury, Increases the effect of alcohol and CNS Hypersensitivity to the drug;
somnolence anxiety, rash, depressants, carbonic anhydrase use in children has not been
palpitation, kidney inhibitors established.
stones Decreases the effect of oral
contraceptives, digoxin
Levetiracetam Dizziness Somnolence, asthenia, None reported Hypersensitivity to the drug;
infection, and dizzinessa pregnancy (FDA category C).
"For a full discussion, see Section 2.2. Data are from Ref. 18.
Table 6.5 Pharmacokinetic Properties of Antiepileptic Agentsa
Volume of
Generic Name (USP or Peak Plasma Levels Distribution Protein
Nonproprietary Name) Achieved (h) tm (h) w& Wkg) Metabolism/Excretion Binding (%)
Phenytoin sodium, Extended, (la) Liverkidney; <5% of 87-93
the unchanged drug
remaining
Phenytoin, Prompt, (la)
Mephenytoin, (lb) Liverkidney; -42% of No data
unchanged drug
remaining
Desmethyl metabolite (active) 150-200
Ethotoin, (lc) 2 3-9 Liverkidney No data
Carbamazepine, (2) 4-5 (tablets); -1.5 25-65 (initial); Liverkidney; 76
(suspension); -3- 3-24 (repeated metabolites are found
12 (extended administration) in the urine (72%)
release) and the feces (28%)
Epoxide metabolite (active)
Oxcarbazepine, (12) Liverkidney; >95%
found in the
Monohydroxy (MHD) metabolite urine, constituting 4 % 40
(active) of unchanged drug;
-80% as MHD, or
conjugates
Valproic acid, (3a) Liverjkidney 80-94
Clorazepate dipotassium, (4a) Liverkidney 97
Desmethyl metabolite (active) 55-100
Diazepam, (4c) 20-50
Clonazepam, (4b) 18-60
Nitrazepam, (4e) 2
Clobazam, (4d) 18
Desmethyl metabolite (active) -77
Ethosuximide, (5a) 30 (children);60 Liverkidney; 25% 0
(adults) excreted unchanged
in urine
Methsuximide, (5b) Liver; <1% excreted No data
Desmethyl metabolite (active) unchanged in urine
Phenobarbital, (6) 0.42-0.73 Liver; 25%excreted 40-60
unchanged in urine
Primidone, (7) 5-15 (primidone); Liverkidney; -40%is 20-25
53-140 excreted unchanged;
(phenobarbital); metabolized to
10-18 ( P E W ) phenobarbital and
P E W (both active)
Trimethadione, (17) 11-16
Desmethyl metabolite (active) 6-13 days
Gabapentin, (9) 5-7 0.65-1.04 Not appreciably <3
metabolized; excreted
in urine unchanged
Felbamate, (10) 0.84-0.76 Liverkidney; 40-50% 22-25
unchanged in urine;
40%as unidentified
metabolites and
t
4
conjugates
2 Lamotrigine, (11) 0.9-1.3 Liverkidney; 94% -55
excreted in urine as
conjugates, 2%in
feces
Zonisamide, (12) -63 (plasma); 105 -1.45 Liver; kidney -40%
(erythrocytes)
Tiagabine, (14) -45 min 5.4-8.0 Liver; kidney 96
Topiramate, (15) -2 21 Liverkidney; 70%of 13-17
unchanged drug in
urine
Levetiracetarn, (16) Liverkidney; 66%
unchanged drug
"Data are from Ref.18.
Anticonvulsants
activity after a relatively large dose of val- transformation to the 7-amino (43 and 7-acet-
proate. Levels of (E)-2-eneVPA in the cerebro- amido (4)derivatives. Abrupt withdrawal of
spinal fluid (CSF) (34) and the brain (35) in the drug has led to worsening of seizures with
humans were determined to be too low to pro- or without additional psychic symptoms such
vide effective anticonvulsant protection. as dysphoria, restlessness, or autonomic signs
Valproic acid is rapidly distributed and the (42).
plasma protein binding is concentration de- Nitrazepam. As indicated previously, ni-
pendent (18). As previously noted, valproic trazepam (4e), is similar in structure to clon-
acid is extensively metabolized, primarily in azepam. Absorption after oral administration
the liver, with about 30-50% of the drug ex- occurred within 1 h; however, in some cases
creted as the glucuronide (phase I1 metabo- relatively slow absorption (up to 4 h) has been
lism) in the urine, about 30-40% by the phase reported (43-48). Rectal administration pro-
I mitochondrial P-oxidation pathway, and vided more rapid absorption (median peak
about 10-20% by microsomal cytochrome time 18 min versus 38 min orally) (48). Al-
P450-mediated hydroxylation/dehydrogena- though a good correlation was apparent be-
tion of the side chain that provides the major tween the volume of distribution and elimina-
phase I metabolites (36). The metabolites of tion half-life, when comparing the young,
valproic acid have been thought to be the elderly, female, and male subjects, the differ-
cause of a rare, but fatal hepatotoxicity (35). ences observed in volume of distribution re-
The synthetic (El-2,Cdiene VPA has been lated to sex, age, and body weight seem to de-
shown to induce the same hepatic microve- pend on the relative proportion of body
sicular steatosis seen in patients, in chronic adipose tissue (49). The metabolic pattern of
administration studies in rats (36). The ulti- nitrazeparn is similar to that of clonazepam,
mate causative factorb) of hepatoxicity of val- with the principal formation of the inactive
proic acid currently remain undefined (28,29). 7-amino (41) and 7-acetamido (4m) com-
pounds.
2.3.4 Benzodiazepines Clobazam. Clobazam (4d) differs from the
Clorazepate Dipotassium. Clorazepate di- previous benzodiazepines, given that it is a
potassium is a prodrug that is rapidly and al- 1,5-benzodiazepine rather than a 1,4-deriva-
most quantitatively converted into the active tive. It was demonstrated that N-desmethyl-
decarboxylated analog, N-desmethyldiazepam clobazam (4k) possessed anticonvulsant activ-
[nordazepam (401 (37-40). ity (50, 51).
Diazepam. Like clorazepate, diazepam is
converted into nordazepam (40 by N-demeth- 2.3.5 Succinimides
ylation (Fig. 6.3) and to oxazepam (4g). Direct Ethosuximide. Ethosuximide (5a)is rapidly
hydroxylation produces N-methyl-oxazepam absorbed when administered orally (18). The
(4h).The low concentrations of (4g) and (4h) drug is extensively metabolized principally to
preclude their role as active anticonvulsants the inactive diasteromeric 2-(1-hydroxyethy1)-
(41). Unlike clorazepate, diazepam is bioac- 2-methylsuccinimide (5d) (Fig. 6.3) (52) and
tive. The transformation of diazepam to nor- the inactive 2-hydroxyethyl isostere. About
dazepam is less complete, in that 62-73% of 20% of the drug is excreted unchanged in the
diazepam is excreted in the urine and about urine.
10% is found in the feces (41). Diazepam is Methsuximide. Like ethosuximide, meth-
rapidly absorbed when taken orally (30-90 suximide (5b)is rapidly absorbed. The drug is
min) or rectally (10-60 min). also rapidly N-demethylated to N-desmethyl-
Clonazepam. Clonazepam, chemically 5-(2- methsuximide (5d), the active metabolite (53).
chloropheny1)-1,3-dihydro-7-nitro-2H-1,4- Less than 1% of unchanged methsuximide is
benzo-diazepin-2-one (4b),is closely related to found in the urine.
nitrazepam (4e), differing only at position 5
with the o-chloro substituent. Only 0.5% of 2.3.6 Barbiturate and 2-Desoxybarbiturate
the original drug is recovered unchanged in Phenobarbital. Phenobarbital (5-ethyl-5-
the urine after 24 h, indicating extensive bio- phenylbarbituric acid, 6) is relatively insolu-
2 Clinical Applications
S-(-)-5-(pHydroxyphenyl)
Phenytoin, (1) -5phenylhydantoin, Major (inactive)
I
Phenytoin sodium, (la)
R-isomeric Pathway
S-isomeric Pathway
OH
pHydroxymephenytoin Desmethylmephenytoin,
(inactive) Nirvanol (active)
Figure 6.3. Metabolism of anticonvulsants.
ble in water (1g i n 1000 mL), but readily sol- decreases resorption in the nephron and in-
uble as the sodium salt (1g i n 1 mL) (54). The creases clearance, whereas increasing urinary
drug is metabolized principally to the p- pH also increases excretion (59).
hydroxy metabolite (6a)(55). Absorption of Primidone. Primidone (2-desoxyphenobar-
the sodium salt is rapid and relatively com- bital, 7 ) is readily absorbed from the gastroin-
plete (80-100%)(56,57).It is distributed to all testinal tract. As seen in Fig. 6.3, primidone
body tissues (58). The elimination of pheno- undergoes two principal biotransformation
barbital follows fmt-order kinetics, and thus pathways: (1) C , oxidation to form pheno-
is independent of concentration (59). Al- barbital (6) and (2) C , ring cleavage to form
though the average half-life is not influenced phenylethylmalonamide (PEMA, 7a), both
by the route of administration, the rate of active. It has been shown that primidone is,
urine flow and urinary pH do influence the in fact, active and not a prodrug of pheno-
elimination rate (59). Increasing urine flow barbital (60).
Anticonvulsants
0 4
NH2 NH2
Carbamazepine, (2) Carbamazepine-10 , l l -epoxide
(active)
C02H
3-ene-VPA
(E)-and(Z)-isomers 4-Hydroxy-VPA 2-Propylsuccinic
acid
H02C
/'
2-Propylglutaric
acid
Figure 6.3. (Continued.) (a) P-oxidation; (b) P450-dependent desaturation; (c) P450-dependent
w-hydroxylation; (d) P450-dependent (o-1)-hydroxylation;(e) P450-dependent (o-2)-hydroxylation).
The broken arrows indicate a metabolic route in which the details are not yet confirmed. (After Ref. 27.)
lower in non-whites than that in whites, but teins, resulting in an eightfold higher concen-
was not affected by gender (18). tration in red blood cells. It is excreted
primarily in the urine as:(1) unchanged drug
2.3.1 1 Oxcarbazepine. Oxcarbazepine (12) (35%); (2) inactive N-acetyl zonisamide (13a)
was previously discussed[see Carbamazepine (2)]. (15%); and (3) as the 0-glucuronide of the
ring-opened 2-sulfamoyl-acetyl phenol (SMAF',
2.3.1 2 Sulfonamide 13b)(50%) (18).
Zonisamide. Zonisamide(13)is chemically
1,2-benzisoxazole-3-methanesulfonamide.It 2.3.1 3 Nipecotic Acid Analog
should be noted that zonisamide binds exten- Tiagabine. Tiagabine, chemically (R)-(-)-
sively to erythrocytes as well as plasma pro- 1-[4,4-bis(3-methyl-thieny1)-3-butenyll-3-
i
i 2 Clinical Applications
H H3C H3C
\
CI -N
\
Clorazepate dipotassium, (4a) Diazepam, (4c) 3-Hydroxydiazepam
(N-Methyloxazepam), (4h)
- 0 H /N-Demethylatim H
Methsuximide, (5b)
Phenobarbital, (6)
OH
gHydroxyphenobarbitaI, (6a)
H
Phenobarbital, (6)
(active)
\
Primidone, (7)
Phenylethylmalonamide
(PEMA), (7a) (active)
H3C
CH3 H
Trimethadione, (17) Diimethadione, (17a)
--
*
<
*<
*
B
0
)-NH~
0
3-Carbamoyloxy-2-phenyl-
propionic acid, (10d)
C02H
4
N-2 glucuronide, (1l a )
Lamotrigine, (11)
A'
0 0 0 CH3
2-Sulfamoylacetyl phenol Zonisamide, (13) N-acetylzonisamide, (13a)
(SMAP),(13b) 0
.C02H
piperidinecarboxylic acid hydrochloride (14), pathways have been identified in humans: (1)
is rapidly and nearly completely absorbed thiophene ring oxidation, forming inactive iso-
(95%), with an absolute bioavailability of mers of 50x0 tiagabine (4-5% of the dose)
about 90% (18). The metabolism of tiagabine (14a);and (2) carboxylic acid glucuronidation
has not been fully elucidated, although two (69). Of the administered dose, 25% was ex-
3 Physiology and Pharmacology
NH2
OH
3-Hydroxy levetiracetam, (16b)
Figure 6.3. (Continued.)
creted in the urine, with 60% (- 14% of the dine acetamide (16). The drug is rapidly ab-
dose) identified as the isomers of (14a). The sorbed, with peak plasma concentrations oc-
remaining dose was recovered in the feces 3-5 curring in about 1 h after oral dosing (18).The
days after dosing (69). Two major, as yet un- oral bioavailability of the tablets is 100%. Le-
identified metabolites were present in the fe- vetiracetam produces one major metabolite,
ces, and accounted for >60% of the fecal resi- levetiracetam carboxylic acid (16a) (Fig. 6.3)
due. The mean half-life of the drug in healthy in humans. The inactive carboxylic acid (16a)
volunteers was independent of the adminis- constitutes 24% of the dose. Two minor inac-
tered dose, indicating linear elimination ki- tive metabolites, the diastereomeric 3-hy-
netics (69). droxy levetiracetam (16b) (2%of the dose) and
the ring-opened butanamide (16c) (1%of the
2.3.1 4 Sulfamate dose) were also isolated. There was no enan-
Topiramate. Topiramate, chemically 2,3: tiomeric interconversion of levetiracetam or
4,5-bis-0-(l-methylethylidene)-/3-~-fructopyr-its major metabolite (18). The plasma half-life
anose sulfamate (15), is rapidly absorbed. The of the drug in adults is 7 5 1 h and is elimi-
relative bioavailability of the tablet is about nated by renal excretion of unchanged drug
80% of that of the solution (18).Metabolism of (66%).The mechanism of excretion is glomer-
the drug is not extensive and -70% of the ad- ular filtration, with subsequent partial tubu-
ministered dose is found in the urine. The me- lar reabsorption; the carboxylic acid metabo-
tabolites (Fig. 6.3) result from w-oxidation, lite (16a) is excreted by glomerular filtration
that is, di- (15a) and mono-hydroxymethyl and active tubular secretion.
(15c) derivatives of the isopropylidene car-
bons and ring cleavage (15b)as well as glucu-
ronidation (81). None of the metabolites pos- 3 PHYSIOLOGY AND PHARMACOLOGY
sessed significant activity. There is evidence of
renal tubular reabsorption of topiramate. As stated in the previous edition (8), seizures
in humans and laboratory animals result in
2.3.1 5 Pyrrolidinone Acetamide rapid voltage changes in their EEG patterns.
Levetiracetam. Levetiracetam is a single These changes are accompanied by an extra-
enantiomer, (-)-(S)-a-ethyl-2-0x0-1-pyrroli- cellular depolarizing shift caused by a large
Anticonvulsants
excitatory postsynaptic potential. The exact slow off-rate caused by- anticonvulsants that
biochemical mechanisms leading to these dis- act as sodium channel blocking agents pro-
charges and the resultant epileptic attack are vides an accumulated block after repeated de-
still unknown. Several events, however, are polarization (termed use-dependency). Thus,
known to occur. The EEG changes relate to in seizures, the sodium channel-blocking
the opening of specific ion channels in the neu- agents are effective only if the depolarization
ronal membrane. At the onset of the hyper- lasts for at least 5 s. These agents normally do
synchronous discharge, the extracellular Ca2+ not interfere with the normal action potential
concentration falls and the extracellular K+ or excitatory synaptic potentials that typically
concentration rises (3). Further, the excessive last less than 200 ms (86).
neuronal discharge may release large amounts As noted in Tables 6.6 and 6.7, several first-
of excitatory neurotransmitters at synapses generation and second-generation agents act
that may result in an avalanche of stimula- by blockade of voltage-dependent Na+ chan-
tion. The current proposed cellular mecha- nels; however, there are problems in explain-
nisms by which the anticonvulsants exert ing the clinical and experimental facts con-
their effect are indicated for the "first-gener- cerning these agents. As an example,
ation" and "second-generation" agents in Ta- carbamazepine and phenytoin are listed as
bles 6.5 and 6.6, respectively. These mecha- Na+ channel-blocking agents (80); however,
nisms are summarized below. in the clinic, an epileptic patient found to be
3.1 Ion Channels resistant to one of these agents may respond
favorably to alternative treatment with the
As noted in Tables 6.6 and 6.7, there is evi- other of the two drugs, pointing out that these
dence that some classes of the older and newer agents may act by more than one mechanism
anticonvulsants interact with voltage-depen- (87). Furthermore, in a subgroup of rats found
dent sodium channels (82-85). Voltage-gated resistant to phenytoin, they have responded
sodium channels are responsible for the gen- well to carbamazepine (88, 89). Of the newer
eration of action potentials of nerve fibers agents, lamotrigine, as noted in Table 6.1, is a
through fast, selective transport of sodium broad-acting anticonvulsant acting against a
ions across the cell membrane, leading to the variety of seizure types; however, as noted in
rapid depolarization of the cell network (85). Table 6.7, it does not effect either GABAergic
The Hodgkins and Huxley model for the func- potentiation or blockade of thalamic T-type
tion of the sodium channel postulates that Ca2+ channels, thus providing further doubt
they exist in at least three different states, as of these agents acting by a single mechanism.
shown in Fig. 6.4(86). These states are: (1) the This problem was caused by the classical
resting (closed) or nonconducting state; (2) method of animal testing of anticonvulsants
the activation state resulting from changes in for generalized tonic-clonic seizures [i.e., the
the resting potential of the channel, which in- maximal electroshock seizure (MES) test].
creases the ability of the channel to inwardly This method was developed by Goodman and
conduct Na+ across the cell membrane until coworkers in 1944 and is still employed today
an action potential is elicited; and (3) this open (8,90-96). The MES test has been found to be
channel state exists for a short period and particularly sensitive to Nat channel blockers
closes rapidly to the inactivated state, which (97).
terminates inward flow of Na+ and the result-
ing voltage change. The reactivation to the
3.2 GABAergic Mechanisms
resting state is membrane potential depen-
dent, given that repeated depolarizations de- GABA, y-or 4-aminobutyric acid (19),formed
lay the transformation back to the resting by the decarboxylation reaction shown in Fig.
state (86). 6.5, is present within a large proportion of the
Drugs that interact with sodium channels central nervous system, where it is the major
to block ion flux cause the channels to inacti- inhibitory neurotransmitter controlling syn-
vate to a greater degree and with smaller de- aptic transmission and neuronal excitability
polarizations than normal (86). The relatively (98, 99).
Table 6.6 Proposed Cellular Mechanisms of the "First-Generation" Agentsa
- -
/[ 2:;ed 17 Inactivation
Fast
Depolarized
4
/ Slow
Inactivation
( +'
Closed but
can be Inactivated v
activated Polarized
u
Figure 6.4. Relationship between open, closed, and resting state of Naf channels. (After Ref. 86.)
There are at present three known classes bles 6.6 and 6.7, the older agents (i.e., pheno-
of GABA receptors, GABAA, GABA,, and barbital, valproate, and the benzodiazepines)
GAB&, with distinctive binding properties act through this mechanism, whereas the
and different functional responses to GABA, newer agents (i.e., topiramate, felbamate, vi-
although each is involved with inhibition of gabatrin, tiagabine, and gabapentin) are also
the CNS (100). All GABA receptors are found GABAergic agonists, although by different
as pre- and postsynaptic receptors, and as au- mechanisms (105, 106). Topiramate, in addi-
toreceptors (100). tion to potentiating GABA, also prolongs inac-
tivation of sodium channels. Felbamate, also a
3.2.1 CABA, Receptors. Of the three, the GABA agonist, like topiramate, also blocks
postsynaptic receptors are responsible for in- the (RS)-2-amino-3-(3-hydroxy-5-methyl-4-
hibiting neuronal excitability (101, 102). isoxazolyl)propionic acid (AMPA, 25) as well
GABA, receptors (Table 6.8) are major GABA
receptors that are linked to chloride channels
and are activated by isoguvacine (21) (Fig.
6.6), modulated by barbiturates and the ben-
zodiazepines, and antagonized by bicuculline
(101, 102). This receptor is termed a hetero-
oligomeric complex and is composed of at least
four types of multiple allosterically interact-
ing binding sites (GABA, benzodiazepine, bar-
biturate, and picrotoxin sites), together with
an intrinsic chloride ion channel (103). Each
of the allosteric binding sites is thought to be as the N-methyl-D-aspartate (NMDA, 26) re-
physically distinct, and can be occupied simul- ceptors (these latter compounds are discussed
taneously to induce their individual pharma- later) (107).
cological effects through allosteric interaction. Vigabatrin acts by blocking GABA-T, the
It is established that the GABA, receptor com- enzyme responsible for the breakdown of
plex plays a significant role in the action of GABA (Fig. 6.5); tiagabine acts by inhibit-
anticonvulsant agents (104). As noted in Ta- ing the reuptake of the neurotransmitter,
H2N LC02H
Glutarnic acid, (18)
-GAD
-CO,
--
H2N -CO~H
GABA, (19)
GABA-T
-----'
-NHq ~U
H C02H
Succinic sernialdehyde
(20)
Figure 6.5. Major pathway for the synthesis and degradation of GABA. GABA, y-aminobutyricacid;
GAD, glutamic acid decarboxylase; GABA-T, GABA transaminase.
296 Anticonvulsants
@C
, -H
COO
Vigabatrin, (8)
Figure 6.7. Proposed mechanism of action of vigabatrin (8)(after Ref. 175). B, base in enzyme active
site; Nu, nucleophilic residue in enzyme active site.
Excitatory nerve
terminal
1
Stimulus Carbamazepine
Phenytoin
Valproate
~amotri~ine
Gabapentin(?)
I
Felbamate (?)
Oxcarbazepine
Zonisamide
J
Glutamate
/ Glycine
Glutamate / 1 /
Postsynaptic
Zn2+ neuron
Mg2+
Felbamate (?)
Zonisamide
NMDA
receptor
Figure 6.8. Possible sites of interaction of antiepileptic drugs on glutamate-mediated transmission
(after Ref. 174). The NMDA receptor is associated with an ion channel permeable to Na+ and Ca2+,
and is associated with a number of modulatory sites, including a strychnine-insensitive glycine-
binding site. Glycine is an absolute requirement for the receptor-channel complex to enter the open
state.
recently been found a specific binding site for sant activity, whereas the R-(-) enantiomer
a [3H]gabapentin (182). It is localized in (36b)was less active in each evaluation (185).
discrete areas in the brain of rats that are as- These data support the conclusion that there
sociated with excitatory input (183, 184). is an association between the ability of gabap-
S-(+)-3-Isobutyl-GABA (364 displaces entin to interact with its binding site and the
[3H]gabapentin binding and has anticonvul- anticonvulsant effects (174).
Anticonvulsants
(37a)Tryptophan
(37) Indole
(37b)Serotonin
alcohol (45a) and its (10R)-enantiomer (45b), statistically significant different IC,, of 138 t
both appearing in plasma (220) and urine 32 a, compared to that of carbamazepine
(221) in an approximately 4:l ratio. The first (210 + 15 and provided 95.6 t 2.6% inhi-
study (222) evaluated the esters of the mono- bition of sodium uptake at 300 $4 (carbamaz-
epine 64.8 2 3.0% at 300 pM). In a follow-up
study, they indicated that acetates (45) appear
to be preferable to oxcarbazepine because they
provide a clearer metabolic pathway (223).
Therapeutically, acetate (45a) was found to be
more appropriate as the R-( -)-isomer, which
has a greater propensity to undergo inactiva-
tion to the trans-diol (45c) (222). The bio-
transformation of the esters is shown in Fig.
6.9. The predominant pathway is indicated
with the heavy arrow.
The quadracyclic (*)-5-aminocarbonyl-
10,ll-dihydro-5H-dibenzo[a,d]cyclohepten-
5,lO-imine(ADCI, 47), results from the fusion
of two active anticonvulsant compounds, MK
801 (dizocilpine, 46), a potent noncompetitive
NMDA antagonist (224, 225) and carbamaz-
epine (2). The compound acts as a selective,
low affinity channel blocker of the NMDA re-
ceptor and also possesses Naf channel-block-
ing activity (226). ADCI is devoid of the ten-
dency to cause behavioral impairment as MK
801. ADCI is a racemate, although the (+)-
enantiomer displays a four- to fivefold greater
potency at the NMDA receptor and a greater
than twofold potency for seizure models inan-
imals. There was no enantioselectivity in the
hydroxy derivatives and it noted that the ace- Nat channel evaluation, however. The (+)-
tates (R = COCH,) were the most active com- enantiomer (SGB-017)is currently in Phase I1
pounds. Of further interest was the fact that of clinical development.
the acetate of (45a) was active in its own right
and not a prodrug in the true sense. Compar- 5.3 Barbiturates
ative data are provided in Table 6.12. As
noted, whereas carbamazepine was the most Of renewed interest is the emergence of etero-
active, the acetate of (45a) was as active as barb (N,Nr-dimethoxymethyl phenobarbital,
oxcarbazepine. In addition, blockade of volt- 6b) (200). This agent possesses attenuated
age-sensitive sodium channels was also evalu- sedative and hypnotic properties compared
ated, revealing that the acetate of (45a) had a to those of phenobarbital (6). Although
Table 6.12 Comparative Data of Carbamazepine (2), Oxcarbazepine (12), and Acetate of 45a
and 45ba
Compound % Protection MES (mglkg) TD5,( m g k ) Protective Indexb
Carbamazepine, (2) *
100 0.0 *
3.4 0.1 *
27.4 0.1 8.1
Oxcarbazepine, (12) 68.3 5 20.3 6.1 t 0.1 40.1 t 1.2 6.6
Acetate of (4Sa) 100 t 0.0 6.3 t 0.1 78.6 t 4.2 12.5
Acetate of (45b) 5.9 t 1.8 18.0 t 0.1 134.9 t 3.5 7.5
"Values are means 2 SEM of 5-8 ratslgroup.
bProtediveindex = TD,,,IED,o.
5 Structure-Activity Relationships
0
NH2
(45a) acetate; S(+) (45b) acetate; R(-)
Oxcarbazepine (12)
Figure 6.9. Proposed metabolism of acetates o)foxcarbazepine (12). Dark arrows are the preferred
pathway.
(6) Phenobarbital
5 Structure-Activity Relationships
tives developed by Kohn and coworkers (236, aqueous media and readily diffuses through
237) and Paruszewski (238,239). Harkoseride biological membranes. It is thus rapidly ab-
shows excellent anticonvulsant activity in sev- sorbed and extensively distributed through-
eral animal models, including two models of out body water. It is not bound to plasma pro-
status epilepticus. It also provides neuropro- teins, but is extensively metabolized, with
tective effects in rat models of focal ischemia about 50% excreted in the urine of rats as the
(198). It is currently undergoing phase I1 clin- w (i.e., 4-hydroxy), and w-1 (i.e., 3-hydroxy)
ical evaluation. It was found to be rapidly and oxidation products.
completely absorbed, was less than 1% plasma The drug has successfully completed Phase
bound, and possessed a half-life of about I clinical trials (198).
12 h. The drug is eliminated primarily by
renal excretion and the metabolites have not 6.5 NW-1015
been identified. Preliminary data indicate
Chemically, NW-1015 is (S)(+)-2-[4-(3-fluoro-
that harkoseride does not affect the blood
benzyloxy)benzylamino]propanamide (57)
levels of carbamazepine, phenytoin, or val-
proate (198).
anoic acid (36a).The mechanism of action is protective potential, trials have also been
unknown, but it is likely to differ from that of conducted for other indications, including
other anticonvulsants. Pregabalin does not Parkinson's disease and Huntington's dis-
appear to have any direct action at Na+ and ease. A phase 111study in a monotherapy trial
Ca2+ channels, and it does not seem to affect with carbamazepine, however, indicated that
transmitter responses to glutamate, NMDA, the efficacy of remacemide was inferior to that
or GABA. Additionally, it does not change of carbamazepine (198).
neurotransmitter uptake e . , glutamate,
GABA, monoamine, adenosine, cholinergic, or 6.8 Retigabine (D-23129)
opiate receptors). Pregabalin, however, in- Chemically, retigabine (D-23129) is ethyl
creases GABA content in neuronal tissues,
N-(2-amino-4-(4-fluorobenz1amino)phenyl-
binds to the a218 subunit of the Ca2+channels, carbamate (59), and is structurally unrelated
and enhances glutamic acid decarboxylase ac-
tivity (185).Pregabalin is not significantly me-
tabolized in humans. Studies in healthy volun-
teers indicate the drug has a 90% oral
bioavailability and is not bound to plasma pro-
teins. In clinical studies, pregabalin provided
positive results against partial seizures. Addi-
tional studies are planned to evaluate its use
as monotherapy and in pediatric patients
(198).
. . -
.
Hydrogen j bonds
bonds
1 1
Proximal ring
I
Distal ring
Hydrogen bonding area bonding area bonding area
Figure 6.10. Postulated interaction of compound (65)at a binding site. (After Ref. 251.)
I
CI
N 3
1 : HAD
HAD
Figure 6.12. Selected anticonvulsants for the development of a pharmacophore model. The essen-
tial structural elements are indicated by dotted rectangles. 1 = phenytoin; 2 = carbamazepine; 11 =
lamotrigine; 13 = zonisamide; 60 = rufinamide; inset, 58 = remacemide. R, hydrophobic unit; D,
electron-donor group; HAD, hydrogen donor/acceptor unit. (After Ref. 281.)
Anticonvulsants
Features of commonalitv " include: ( 1 ) at More data in animal models with focal epi-
least one aryl ring (R);(2)one electron donor lepsy are needed to determine long-term appli-
atom (D);and (3)a second donor atom in close cability (198).
proximity to the N-H group, forming a hydro-
gen bond acceptor/donor (HAD). In the struc- 7.8 A New Causative Agent for Epilepsy
tures listed in Fig. 6.12, the moieties are indi-
A recent report indicated that cryptogenic ep-
cated with either a bracket or a dotted
ilepsy, the group of epilepsy syndromes for
rectangle. Through molecular modeling tech-
which an etiology is unknown, consisting of
niques, each structure was minimized and su-
about 20% of all epilepsy syndromes, may be
perimposed (281). The deviation was <0.7 A.
caused by Toxoplasm gondii (299). A statisti-
In the case of remacemide (581, it contains the
cally significant elevation of T. gondii antibod-
HAD function as the amide, but not at the
ies was found compared to that of controls,
correct position. Taking the carbonyl oxygen
suggesting that T. gondii infection with brain
as the donor atom (D), the hydrogen-bond
cysts may be a cause of the disease.
function would be represented by the m i n e
function, which was different in the previous
compounds. This model is not all inclusive and 8 WEB SITE ADDRESSES AND
deviated from that proposed by Brouillette et RECOMMENDED READING FOR FURTHER
al. (209, 293-295), in that the orientation of INFORMATION
the aromatic ring was not in a specific confor-
mation (the rings are rotated in relation to the 8.1 For Information on Anticonvulsant
R-D-HAD plane by 10-40"). Needless to say, Evaluations
research in this area is sorely needed. This
problem is complicated by the fact that the 0 NINDS ADD Program Webpage: http://
three-dimensional structure of the sodium www.ninds.nih.gov/asp.htm
channel is unknown, and as stated by Madge
et al. (75), it is likely to be many years before 8.2 For Information on Epilepsy
high resolution structures are available for
these channels. 0 Epilepsy Foundation of America Webpage:
7.7 Porcine Embryonic GABAergic Cell http://www.e fa.org/
Transplants 0 Society for Neuroscience Webpage: http://
www.sfn.org/
Historically, transplantation of fetal neurons
American Epilepsy Society: http://www.
and glia have been demonstrated to survive,
aesnet.org/
integrate, and reduce functional deficits in
animal models of Parkinson's disease and University of Pennsylvania: http://www.
Huntington's disease (296, 297). In addition, med.upenn.edu/health/hi -files/neurology/
surgical implants in humans have been suc- epilepsy/ep-seizures-.html
cessfully performed in pilot studies (298). An
FDA-approved safety and feasibility study of 8.3 References on Animal Procedures
transplantation of embryonic porcine lateral
ganglionic eminence cells (NeuroCellTMFE, 0 The Neuronal Microenvironment (Neuro-
Diacrin, Inc.) into epileptogenic tissue in pa- methods Series, Vol. 91, A. A. Boulton, G. B.
tients with surgically amenable temporal or Baker, and W. Walz. Humana Press, To-
frontal lobe onset seizures was initiated (198). towa, NJ, 1988.
Before transplantation, the cells were treated 0 Check the previous edition of this chapter
with anti-MHC I monoclonal antibodies, thus also.
removing the need for treatment with the im-
munosuppressant cyclosporine. Preliminary 8.4 Sodium Channels
evaluation in three patients with medically re-
fractory partial-onset seizures have shown 0 The study reported by Madge et al. (75) is
that the procedure is safe and well tolerated. the most current on the topic.
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References 327
Narcotic Analgesics
JANE V. ALDRICH
SANDRA C. VIGIL-CRUZ
Department of Medicinal Chemistry
School of Pharmacy
University of Kansas
Lawrence, Kansas
Contents
1 Introduction, 331
2 Clinical Use of Agents, 332
2.1 Current Drugs on the Market, 333
2.2 Side Effects, Adverse Effects, Drug
Interactions/Contraindications,333
2.2.1 Central Side Effects, 335
2.2.1.1 Respiratory Depression, 335
2.2.1.2 Tolerance, Dependence, and
Addiction Liability, 336
2.2.1.3Sedation and Cognitive
Impairment, 337
2.2.1.4 Nausea and Vomiting, 337
2.2.2 Other Side Effects, 337
2.2.2.1 Constipation, 337
2.2.2.2 Itching, 338
2.2.3 Contraindications,338
2.2.4 Drug Interactions, 338
2.2.4.1Interactions with Cytochrome
P,,, Enzymes, 338
2.3 Absorption, Distribution, Metabolism,
Elimination, 339
2.3.1 Absorption and Distribution, 339
2.3.2 Metabolism and Elimination of
Morphine and Derivatives, 340
2.3.3 Metabolism and Elimination of Other
Opioid Agents, 341
3 Physiology and Pharmacology, 341
3.1 Opioid Effects in the Central Nervous
System and the Periphery, 341
3.2 Multiple Opioid Receptor Types, 341
3.2.1 Discovery of Multiple Opioid Receptor
Types and Current Nomenclature, 341
3.2.2 Signal Transduction Mechanisms, 342
Burger's Medicinal Chemistry and Drug Discovery 3.2.3 Characterization of Opioid Receptors,
Sixth Edition, Volume 6: Nervous System Agents 343
Edited by Donald J. Abraham 3.2.3.1 Ligands Used to Characterize
ISBN 0-471-27401-1 0 2003 John Wiley & Sons, Inc. Opioid Receptors, 343
Narcotic Analgesics
and synthesis of agonists and antagonists se- American Academy of Pain MedicirLe and the
lective for the different receptor types (Sec- American Pain Society also advocate the pru-
tions 5.3, 5.9, and 5.10), the design and syn- dent use of narcotic analgesics for the treat-
thesis of opioid peptide analogs (Section 61, ment of chronic pain (15,16). The widespread
and the recent characterization of the related use of opioids in chronic, nonmalignant pain,
opioid-receptor like (ORL1) receptor and its however, is still somewhat controversial be-
endogenous ligand orphanin FQ/nociceptin cause of the lack of substantial evidence from
(Section 7.2). Before describing the different long-term controlled studies demonstrating
classes of nonpeptide analgesics and opioid effectiveness in this setting (17). The clinical
peptides, the clinical use of analgesic agents use of opioids in different types of pain and in
(Section 2), opioid receptors, and the methods different clinical settings has been reviewed in
used to characterize the opioid activity of com-
detail in a recent book (18).
pounds (Section 3) are discussed.
The World Health Organization (WHO) in-
troduced a three-tiered approach for the treat-
2 CLINICAL USE OF AGENTS ment of cancer pain (19) that also serves as a
model for the management of acute and
Opioid analgesics are indispensable drugs in chronic pain. In this model, the first tier con-
the management of cancer pain (141, and the sists of acetaminophen (APAP) or a nonsteroi-
2 Clinical Use of Agents
dal anti-inflammatory agent [e.g., aspirin Pentazocine is a prototype for the mixed ago-
(MA)or ibuprofen] for mild to moderate pain. nistlantagonist class and acts as an agonist at
If the pain persists or increases, then treat- K opioid receptors generating analgesia (see
ment progresses to the second tier, where a Section 3.2); its antagonist activity at p opioid
narcotic analgesic is added to the regimen. receptors significantly decreases or eliminates
Frequently, this is accomplished by use of a the potential for respiratory depression and
combination product such as ASA plus codeine addiction liability generated through p recep-
that combines an opioid with a nonnarcotic tor activation. The mixed agonistslantago-
analgesic. The third tier is reached when the nists shown in Table 7.3 find limited clinical
pain escalates from moderate to severe. At this utility, despite the analgesia resulting from
level the opioid may be used as a single agent, activating K opioid receptors. The analgesic ef-
given that opioids do not have a ceiling to their fect produced reaches a maximum despite in-
analgesic effect as do the nonnarcotic analge- creased drug dose (analgesic ceiling). Further,
sics such as ASA and MAP (20). Adjuvant these drugs exhibit a different side-effect pro-
drugs, such as tricyclic antidepressants or an- file, including dysphoria and hallucinations,
ticonvulsants, may be added to opioid therapy which also appears to be a result of K-agonist
as a means to enhance the efficacy of opioids activity (23).
for pain relief (17, 21). Opioid agonists also have an antitussive ef-
For continuous pain, analgesic agents are fect attributed to the depression of the cough
generally prescribed for use on a regular, reflex. Thus some opioids, typically codeine or
around-the-clock basis by use of a long-acting one of its derivatives, are used for their anti-
analog. For acute pain or pain after surgery, tussive activity, predominantly in combina-
often an immediate-release, short-acting opi- tion products. The antitussive effect is in part
oid is used. In addition, short-acting opioids the result of the interaction with opioid recep-
with rapid onset are used for "rescue" doses tors at the cough center in the brain (23). The
when breakthrough pain is problematic (21, dose required for antitussive activity, how-
22). ever, is lower than that required for analgesia;
the opioid receptors involved in blocking the
2.1 Current Drugs on the Market
cough reflex are less sensitive to naloxone
The structures for the most commonly used than those responsible for analgesia (23). .
clinical agents are shown in Fig. 7.1. Some of Opioid antagonists (Table 7.4), predomi-
the opioid agonists used clinically (Table 7.1) nantly naloxone, are used clinically to reverse
such as morphine may be used as the sole the effects of opiates in overdose or postoper-
agent for analgesia. Because of their rapid on- ative sedation. Naltrexone, which has oral bio-
set and short duration of action, fentanyl and availability, is used for the treatment of nar-
other 4-anilidopiperidines have been used ex- cotic addiction and alcohol dependence. As
tensively as adjuncts to anesthesia, whereas discussed below (Section 2.2.2.1), peripherally
methadone and its analog levomethadyl ace- selective antagonists are being evaluated for
tate (LAAM) are used as maintenance agents treatment of constipation and other gastroin-
for individuals who are addicted to narcotics. testinal side effects associated with opioid ag-
Other agents such as loperamide or diphe- onist use.
noxylate are used primarily for their consti- 2.2 Side Effects, Adverse Effects, Drug
pating side effect to treat diarrhea. Some Interactions/Contraindications
drugs are used extensively in combination
products (Table 7.2) for treatment of pain. In addition to analgesia, clinically used opioids
Most of the clinically used agents are agonists display a plethera of biological effects. The
at p opioid receptors. In contrast, mixed ago- most common sideladverse effects involve al-
nists/antagonists generally interact with two terations of the nervous, respiratory, gastro-
distinct opioid receptors to provide analgesic intestional, and integument systems (see Refs.
activity while exhibiting decreased potential 23, 25 and references cited therein for more
for serious side effects such as respiratory de- detailed discussions). The most serious side
pression and addiction (see Section 2.2 below). effects associated with the majority of opioid
Table 7.1 Opioid Agonists Used Clinically"
Equal
. Aanalgesic
Dose (mg)b
Chemical Class Generic Name Trade Name (manufacturer) Route of Administration
-
4,5p-Epoxymorphinans Morphine (1) Available as generic p.o., i.v., i.m., s.c., rectal
Codeine (2) Available as generic p.o., i.v., i.m., s.c.
Hydromorphone (5) Dilaudid (Knoll), also available as generic p.o., i.v., i.m., s.c., rectal
Hydrocodonec
Oxymorphone Numorphan (Endo Laboratories) i.v., i.m., s.c., rectal
Oxycodone (6) OxyContin (Purdue Pharma LP), also p.0.
available as generic
Morphinans Levorphanol Levo-Dromoran (ICN), also available as
generic
Phenylpiperidines Meperidine (7) Demerol (Sanofi-Synthelabo) also available as
generic
Lomotil (Searle), also available as generic
Motofen (Carnrick)
Imodium A-D (McNeil-CPC), also available as
generic
Sublimaze (Taylor) i.v.
Fentanyl Oralet (Abbott) Lozenges
Actiq (Abbott) Lozenges on a stick
Duragesic (Janssen) Transdermal patches
Alfentanil Alfenta (Taylor) i.v. with individualized dosing
Remifentanil Ultiva (Abbott) i.v. with individualized dosing
Sufentanil Sulfentanil citrate (ESI Lederle), Sufenta i.v. with individualized dosing
(Taylor)
Acyclic analgesics Methadone (9) Available as generic i.m., s.c., p.0. with
individualized dosing
Levomethadyl Orlaam (Roxane) p.0. with individualized
acetate dosing
Propoxyphene (10) Darvon-N (napsylate) (Eli Lilly), available a s p.0.
generic (HC1)
"Agents currently marketed in the United States. Unless otherwise noted these drugs are Class-I1 controlled substances. From Ref. 24.
bBased on short-term use for acute pain.
"Used only as an antitussive produd.
dRectaladministration.
'Class V narcotic available only by prescription for treatment of diarrhea.
fClass IV narcotic available only by prescription for treatment of diarrhea.
gA noncontrolled substance available both by prescription and over the counter for treatment of diarrhea.
hi.v. dose.
2 Clinical Use of Agents 335
Table 7.2 Examples of Narcotic Agonists Currently Marketed as Oral Combination Products
for Painasb
Trade or Common Name Nonnarcotic
(manufacturer) Narcotic Component Component
APAP with codeine (generic) Codeine MAP
ASA with codeine (generic) Codeine ASA
DHC Plus (Purdue Frederick) Dihydrocodeine APAP, caffeinec
Synalgos-DC (Wyeth-Ayerst) Dihydrocodeine ASA, caffeine
Vicodin (Knoll) Hydrocodone APAP
APAP with hydrocodone (generic)
Alor 51500 (Atley) Hydrocodone ASA
Vicoprofen (Knoll) Hydrocodone Ibuprofen
Percocet (DuPont) Oxycodone APAP
N A P with oxycodone (generic)
Percodan (Du Pont) Oxycodone ASA
I,
ASA with oxycodone (generic)
Mepergan Fortis (Wyeth-Ayerst) Meperidine Promethazined
Darvocet-N (Eli Lilly) Propoxyphene sapsylate APAP
I,
APAP with propoxyphene (generic)
Wygesic (Wyeth-Ayerst) Propoxyphene HCl APAP
APAP with propoxyphene (generic) ,
Darvon Propoxyphene HC1 ASA, caffeinec
"Products currently marketed in the United States. From Ref. 24.
%odeine, hydrocodone, and hydromorphone are used in combination products as antitussives.
"May be beneficial in vascular headaches.
dUsed for sedative effect.
analgesics are respiratory depression, addic- ing in one of the most serious adverse effects
tion liability, and constipation that are associ- (22). Respiratory depression is caused at least
ated with their agonist activity at p opioid re- in part by interaction of opioids with the respi-
ceptors. ratory center in the brain stem, causing a de-
creased response to carbon dioxide and thus
2.2.1 Central Side Effects depression of breathing rate (23). Respiratory
2.2.1.1 Respiratory Depression. Mu opi- depression can occur at doses far lower than
oids used for analgesia slow breathing, result- those that affect consciousness and increases
tive antagonists inhibit dopamine release (311, digestion in the small intestine and decrease
producing aversion rather than motivation in peristaltic waves in the large intestine, re-
(32). sulting in the retention of bowel contents.
2.2.1.3 Sedation and Cognitive Impairment. This is compounded by the enhanced tone of
The initiation or dose escalation of narcotic the anal sphincter and the reduction of the
analgesics may cause drowsiness and impair reflex relaxation in response to rectal disten-
cognitive function. Tolerance usually develops sion. Tolerance does not usually develop to
fairly quickly to these side effects, but other this side effect, and the patient on long-term
medications that induce somnolence will opioid therapy remains chronically consti-
produce an additive effect when taken con- pated.
comitantly. If sedation remains problematic, Patients are generally started prophylacti-
in order to achieve adequate analgesia a psy- cally on a regimen including a laxative such as
chostimulant such as caffeine, dexamphet- bisacodyl or senna that increases bowel motil-
amine, or methylphenidate may be added to ity plus a stool softener like docusate (20,221.
counteract the side effect (22). In patients refractory to laxatives, oral nalox-
2.2.1.4 Nausea and Vomiting. The most one (22) has been successfully used as a ther-
bothersome and unpleasant side effects for pa- apeutic alternative for constipation without
tients receiving opioids for pain are often the loss of analgesia (37). Because of its central
nausea and vomiting that have been associ- activity, however, higher doses of naloxone
ated with all clinically used p agonists. Emesis can decrease the analgesic effectiveness of the
predominantly results from direct stimulation opiate and precipitate opioid withdrawal in
of the chemoreceptor trigger zone, yet the de- some patients (37). Peripherally selective an-
gree of effect depends on the individual (23). tagonists offer the advantage of reversing the
Nausea and vomiting related to narcotic anal- gastrointestinal and other peripheral side ef-
gesics occur in 10-40% of patients (33). Toler- fects of narcotic analgesics without the poten-
ance often develops to these side effects, how- tial for decreasing their central analgesic ac-
ever, and they often vanish with long-term use tivity. Two peripherally selective antagonists,
(22). Nausea and vomiting can be treated by the quaternary derivative of naltrexone
use of a variety of drugs such as transdermal N-methylnaltrexone bromide (methylnaltrex-
scopolamine, hydroxyzine, or a phenothiazine one, see Section 5.3.1) and the phenylpipe~i-
for movement-induced nausea (201, or meto- dine alvimopan (ADL 8-2698, LY246736; see
clopramide or cisapride for patients with nau- Section 5.7. I), are undergoing clinical trials
sea and vomiting stemming from delayed gas- for opioid-induced constipation (34-36, 38).
tric emptying. If the nausea and vomiting After both i.v. and oral administration meth-
persist, steroid therapy with dexamethasone ylnaltrexone reverses the opioid-induced de-
may be initiated or a 5-HT, antagonist such as lay in GI transit ( 3 4 3 6 ) and is effective in
ondansetron may be used (22). Another alter- individuals receiving chronic opioid treatment
native is to try a different opioid, given that (methadone users) as well as in healthy volun-
there is significant individual variability in teers (34,36). In clinical trials oral alvimopan
this side effect (20). reverses the delay in GI transit after the ad-
ministration of exogenous opioids to both opi-
2.2.2 Other Side Effects oid naive individuals and patients receiving
2.2.2.1 Constipation. The most common chronic opioid treatment (both pain patients
side effect of long-term narcotic analgesia is and individuals taking methadone for opioid
constipation plus other gastrointestinal (GI) addiction) (38, 39); in addition it has been
effects collectively referred to as opioid bowel shown to speed the recovery of bowel function
dysfunction. The frequency of these side ef- after abdominal surgery (40).
fects is very high [40-50% or more in patients The constipating effect of orally adminis-
receiving opioids (34-36)] and can become the tered opiates can be used for the treatment of
limiting factor in opioid use. These effects are diarrhea, as with camphorated tincture of
mediated predominantly by p receptors in the opium (Paregoric or Parepectolin, which is a
bowel (23).The effects begin with delayed food paragoric plus kaolin as an adsorbent and pec-
Narcotic Analgesics
tin as a demulcent) (24). Two phenylpiperi- may induce or exacerbate asthmatic attacks;
dine derivatives are used solely as antidiar- hence, fentanyl may be a better choice in asth-
rheal agents. Diphenoxylate, which is a matic patients (23). Other relative contraindi-
congener of meperidine, is available only in cations to the use of narcotics also exist with
combination with atropine, which has anti- respect to the potential for drug abuse (17).
spasmodic activity in the intestine. At thera- Although a history of substance abuse does
peutic doses diphenoxylate does not show any not definitely preclude the use of opioids, it
central nervous system (CNS) effects, but at does necessitate careful vigilance. If the epi-
high doses it displays the typical opioid profile sode of abuse is active or recent, then another
including euphoria. The carboxylic acid me- pain management strategy may be prudent.
tabolite, difenoxylic acid (Motofen; Table 7.1) Consideration of the social network also re-
has activity similar to that of the parent (23). quires consideration, especially if the patient
Unlike diphenoxylate, the second opioid used lives with a substance abuser or has a home
for diarrhea, loperamide, does not exhibit life conducive to enabling abuse.
pleasurable CNS effects even at large doses
(23); loperamide is a substrate for P-glycopro- 2.2.4 Drug Interactions. The pharmacolog-
tein in the blood-brain barrier, which excludes ical activity of opioids can be affected by a
this drug from the CNS (41,421. number of other drugs, including amphet-
2.2.2.2 Itching. After administration of amines, antihistamines, antidepressants, and
opioids there may be urticaria at the injection antipsychotics (see Ref. 23). Small doses of
site or generalized itching because of degran- amphetamine significantly enhance the anal-
ulation of mast cells, resulting in histamine gesic activity and euphoric effects of morphine
release. The itching is a common side effect and may counteract sedation. Diphenhydra-
and often one that results in severe patient mine and hydroxyzine are antihistamines that
distress (23). The histamine release may also exhibit modest analgesic activity themselves,
be partially responsible for the pruritus and and hydroxyzine has been shown to enhance
sweating after drug administration as well as the analgesic effects of low doses of narcotic
flushing resulting from blood vessel dilation of analgesics (47).The depressant effects of some
the skin (23). Antihistamines may be used to opioids may be exaggerated and prolonged by
combat the discomfort (20) or patients can be monoamine oxidase inhibitors, tricyclic anti-
switched to either fentanyl or oxymorphone depressants, and phenothiazines; the exact
(43),which do not tend to cause histamine re- mechanism of action is not fully - understood,
lease. Opioid antagonists such as naloxone are but may involve metabolic or neurotransmit-
effective in controlling the pruritus and can be ter alteration (23). The antidepressants des-
used at low doses without loss of pain control ipramine (48) and nefazodone (49) appear to
(34,44); the peripherally selective antagonist enhance morphine-induced analgesia. The
methylnaltrexone exhibits antipruritic effi- phenothiazine antipsychotics can potentiate
cacy without the potential to reverse mor- the analgesic effect of opioids, but also in-
phine analgesia (45). crease respiratory depression and sedation
(23).
2.2.3 Contraindications. Contraindications 2.2.4.1 Interactions with Cytochrome P,,,
include hypersensitivity to opioids, head Enzymes. Drug interactions with opioid anal-
trauma or increased intracranial pressure, se- gesics can also result from their interaction
vere respiratory depression or compromised with cyctochrome P,,, (CYF') isozymes, specif-
respiratory function, and potentially, liver or ically 3A4 and 2D6 (Table 7.5) (50).
renal insufficiency (46). Whether morphine or The CYP3A4 isozyme is responsible for the
other opioids are used depends on the severity metabolism of a large number of endogenous
of the contraindication, and the potential ben- compounds as well as a wide range of drugs
efits must be weighed relative to the risk. Ana- (50). Fentanyl, alfentanil, and sufentanil are
phylactoid reactions have been reported after substrates for CYP3A4, and therefore drugs
morphine or codeine administered i.v., al- that inhibit this enzyme, such as erythromy-
though the reactions are rare (23). Morphine cin, HIV protease inhibitors, or cimetidine,
2 Clinical Use of Agents
Table 7.5 Cytochrome P,,, Isozymes and 2.3 Absorption, Distribution, Metabolism,
Opioid Substrates and Inhibitors Elimination
P450
Isozyme Substrate Inhibitor 2.3.1 Absorption and Distribution. Opioid
analgesics are available for administration by
1A2 Methadone
2D6 Codeine Codeine
a variety of routes, including by subcutaneous
Hydrocodone Methadone (s.c.) and intramuscular (i.m.) injections and
Meperidine Propoxyphene rectal suppositories as well as by oral and in-
Methadone travenous (i.v.1 administration (see Tables
Morphine 7.1,7.3, and 7.4). The least invasive and safest
Propoxyphene route that provides adequate analgesia should
3A4 Alfentanil Dextropropoxyphene be chosen (22). Opioids are absorbed from the
Fentanyl Propoxyphene gastrointestional tract or the rectal mucosa
Sufentanil and are also readily absorbed into the blood-
stream after s . ~ or
. i.m. iniection.
" Some nar-
cotics with increased lipophilicity may be ab-
sorbed through the nasal or buccal mucosa
may result in oversedation or increased respi- (butorphanol nasal spray and fentanyl loz-
ratory depression as well as prolonged dura- enges, respectively). Fentanyl is sufficiently li-
tion of action of the opioid (51). Conversely, pophilic to be absorbed through the skin (fen-
more rapid metabolism of alfentanil or fenta- tanyl transdermal patch) (23,241.
nyl may result when these agents are used in Intravenous administration of opioids re-
combination with rifampin, which is an in- sults in a rapid onset of action. The more li-
ducer of 3A4 (51). pophilic drugs show more rapid onset of action
Genetic polymorphism in CYP2D6 results after S.C.administration because of differing
in varied drug metabolism (52), with the lack rates of absorption and penetration into the
of this isoenzyme affecting between 5 and 10% CNS across the blood-brain barrier (23).. . In-
of Caucasians and 1-3% of African-Americans traspinal administration produces long-last-
and Asians (52). Individuals lacking 2D6 may ing analgesia, but the hydrophilicity of mor-
display a larger response to some drugs and be phine can result in rostral spread of the drug
at greater risk for toxicity because of their in- in the spinal fluid. This may be problematic,
ability to metabolize certain drugs. Several given that respiratory depression can occur up
opioids such as morphine, meperidine, and to 24 h after the last administered dose as a
methadone are metabolized by 2D6 (Table result of the drug reaching the respiratory
7.5), and interactions with drugs that induce control center in the brain (23). Fentanyl and
"
2D6 result in loss of opioid activity. Thus ri- hydromorphone are highly lipophilic and are
fampin, phenytoin, phenobarbital, prima- rapidly absorbed by spinal tissue, and thus the
done, and carbamazepine can all result in a rostral spread is significantly reduced, result-
significant reduction in opioid concentrations ing in a localized analgesic effect (23).
and concomitant use may require increasing The binding of opioids to serum proteins is
the opioid dose. This genetic defect is also of considerable importance, given that it influ-
problematic when metabolism to an active me- ences the distribution of the drug as well as
tabolite occurs. Because CYP2D6 can convert metabolism and excretion (53). The protein
codeine to morphine (see Section 2.3.2 below), binding of morphine is low (35%), moderate
2D6 inhibitors, especially quinidine, can sig- with meperidine (70%), and high for metha-
nificantly diminish the analgesic effects of co- done (90%)(54). The high human plasma pro-
deine (51). Ritonavir and cimetidine also in- tein binding of methadone is well known (55);
hibit metabolism by 2D6 and therefore can the highest binding is to p-globulin I11 (56)
significantly increase the concentration of opi- and albumin (53, 56). The extensive protein
oids metabolized by 2D6 (Table 7.5), including binding of methadone is an important factor
meperidine, methadone, and propoxyphene, because it significantly affects the amount of
which may result in toxicity (51). drug available in the plasma for penetration
Narcotic Analgesics
Both the free and conjugated forms of codeine tors have also been implicated in analgesia,
are excreted in the urine (23). particularly in cases of inflammation (see
Refs. 71-74 for reviews). Readers are referred
2.3.3 Metabolism and Elimination of Other to a comprehensive two-volume series on opi-
Opioid Agents. Meperidine is also metabo- oids (75, 76) plus more recent reviews (18, 77,
lized in the liver. It is either hydrolyzed di- 78) for detailed reviews of opioid pharmacol-
rectly to meperidinic acid or N-demethylated ogy and physiology.
to normeperidine and then hydrolyzed to
normeperidinic acid. The acid forms are con- 3.2 Multiple Opioid Receptor Types
jugated, then excreted (23).
Fentanyl is also hepatically metabolized 3.2.1 Discovery of Multiple Opioid Recep-
and renally excreted. However, the congener tor Types and Current Nomenclature. Our un-
remifentanil is metabolically distinct when derstanding of opioid receptors has expanded
compared to other members in its chemical or considerably from the early assumption of a
pharmacological class. Remifentanil is metab- single opioid binding site to the characteriza-
olized by plasma esterases to remifentanil tion of multiple types of opioid receptors (see
acid, which is approximately 3000-fold less po- Refs. 79-81 for reviews). The initial proposal
tent than the parent opioid (67). of opioid receptors by Beckett and Casy in
Methadone undergoes significant hepatic 1954 (1)assumed a single opioid binding site.
metabolism by N-demethylation and cycliza- Multiple opioid receptors were postulated as
tion to form pyrrolidines and pyrroline (23). early as the 1960s by both Portoghese (82) and
Propoxyphene is also hepatically metabolized Martin (83), but it was behavioral studies in
predominantly by N-demethylation and re- the chronic spinal dog by Martin and cowork-
nally eliminated. The metabolite norpropoxy- ers in the mid-1970s (5,6) that led to the clas-
phene is cardiotoxic and produces arrhyth- sification of multiple opioid receptors. On the
mias and pulmonary edema that have led to basis of the pharmacological profile of a vari-
reports of cardiac arrest and death (59). This ety of opioids, Martin proposed three types of
is especially problematic because of the long opioid receptors, p, K, and a receptors, with
half-life of norpropoxyphene that accumulates morphine (1, Fig. 7.1), ketocyclazocine (13),
with repeated doses of the parent drug. Meth- and SKF-10,047 (N-allylnorcyclazocine, 141,
adone is excreted in the urine but also in the respectively, as the prototypical ligands (Fig.'
bile (23). 7.3). The discovery of the enkephalins led to
the proposal of a distinct opioid receptor type,
the 6 receptor, for these opioid peptides (84).
3 PHYSIOLOGY AND PHARMACOLOGY
The existence of three distinct opioid receptor
types, the p, K, and S receptors, has now been
3.1 Opioid Effects in the Central Nervous
clearly established and these receptors have
System and the Periphery
been cloned (see below). Sigma (m) receptors,
Opioid receptors are found in both the CNS however, are not considered opioid receptors
and in the periphery. In the CNS different because effects associated with this receptor
types of opioid receptors (p, K, and 6 receptors; are not reversed by opioid antagonists such as
see below) exhibit distinct anatomical distri- naloxone (see Ref. 85). Other opioid receptor
butions (see Refs. 68-70 for reviews), and types have also been proposed (see Ref. 86),
there is considerable species variation in both but these receptor types are not universally
relative receptor density and receptor distri- accepted.
bution. Peripheral receptors mediate some ef- During attempts to clone the opioid recep-
fects of opioids, such as inhibition of gut mo- tors (see Section 3.2.4 below), a related recep-
tility, and for a number of years receptors tor with high sequence homology was identi-
from tissues such as the guinea pig ileum fied by several research groups (see Refs.
(GPI) formed the basis of standard bioassays 87-90 for recent reviews). This receptor, re-
used to assess compounds for opioid activity ferred to by Mollereau et al. as opioid-recep-
(see Section 3.2.3.3 below). Peripheral recep- tor-like 1 (ORL1) receptor (91), does not dis-
Narcotic Analgesics
play affinity for classical opioid ligands, tion of opioid and other receptors with a vari-
including naloxone. Although distinctly differ- ety of effector systems, including adenylyl cy-
ent from opioid receptors, the ORLl receptor clase and ion channels. Numerous forms of
interacts with the opioid receptor system in G-proteins have been identified, including Gi
the regulation of analgesia and other physio- and Go that inhibit adenylyl cyclase and G,
logical effects. Details concerning the pharma- that stimulates adenylyl cyclase. Pertussis
cology of the ORLl receptor and its endoge- toxin inhibits Gi and Go by ADP-ribosylation
nous ligand orphanin FQ/nociceptin (OFQIN) of the a-subunit, whereas cholera toxin persis-
(92,93) are discussed below under Recent De- tently activates G,. Thus sensitivity to pertus-
velopments (Section 7.2). sis toxin is an indication of the involvement of
In 1996 the International Union of Phar- Gi (or Go) in the transduction mechanism,
macology (IUPHAR) recommended that OP1, whereas sensitivity to cholera toxin is an indi-
0P2, and 0 P 3 be used as the accepted names cation of involvement of G,.
for 6, K, and p receptors, respectively, (94) to Of the effector systems that have been im-
replace the DOR, KOR, and MOR nomencla- plicated in the transduction mechanisms for
ture typically used in the literature; 0 P 4 was opioid receptors, the best studied is opioid in-
the proposed name for the related ORLl re- hibition of adenylyl cyclase (see Refs. 69,
ceptor. This nomenclature has not gained 97-99 for reviews). Thus binding of an agonist
widespread acceptance, however, and in 2000 to opioid receptors inhibits the activity of ad-
the International Narcotic Research Confer- enylyl cyclase and decreases intracellular
ence (95) recommended a modified nomencla- CAMPin a number of different tissues. Pertus-
ture DOP, KOP, MOP, and NOP for 6, K, p, sis toxin sensitivity of opioid inhibition of ad-
and ORLl receptors, respectively, which is enylyl cyclase has been demonstrated in many
consistent with the nomenclature require- systems, indicating the involvement of either
ments of IUPHAR. Gi or Go in the transduction mechanism. Ago-
nist activation of all three types of cloned opi-
3.2.2 Signal Transduction Mechanisms. oid receptors to inhibit adenyl cyclase has
There is considerable evidence that opioid re- been demonstrated (see Ref. 100 and refer-
ceptors are coupled to G-proteins and produce ences cited therein). There is also some evi-
their effects through these proteins (see Refs. dence that p and S opioid receptors can stim-
96, 97 for reviews of opioid receptors and G- ulate adenylyl cyclase in certain tissues (see
proteins). The structure of cloned opioid re- Refs. 69,97 for reviews). There are conflicting
ceptors is consistent with their belonging to reports on whether K opioid receptors stimu-
this receptor superfamily (see below). G-pro- late or inhibit phosphatidylinositol turnover
teins are heterotrimers, consisting of a, p, and in some tissues (see Ref. 100); S and p recep-
y subunits, which bind guanine nucleotides to tors, however, do not appear to be coupled to
their a-subunit and catalyze the hydrolysis of phosphatidylinositol turnover in neuroblas-
GTP to GDP. G-proteins mediate the interac- toma cell lines NG108-15 and SK-N-SH (101).
3 Physiology and Pharmacology
Opioid receptors can also be coupled to ion (MVD), have been routinely used to assess opi-
channels through G-proteins (see Refs. 69,98, oid activity. Radioligand binding assays for
99, 102 for reviews). All three receptor types each of the opioid receptor types have been
can decrease voltage-dependent Ca+ cur- + instrumental in the identification of selective
rent. The coupling of opioid receptors to cal- opioids. With the cloning of the opioid recep-
cium channels involves a G-protein, and the tors, assays for both opioid receptor affinity
actions of opioids on Ca++current are blocked and efficacy can now be routinely performed
by pertussis toxin, indicating involvement of by use of these cells that express only a single
Gi or Go. Activation of p and 6 receptors can receptor type, greatly simplifying the inter-
also increase Kt conductance. Similar to the pretation of the results of the assays. Each of
results found for calcium channels, potassium these types of assays and their utilization in
channel coupling to opioid receptors appears characterizing compounds for opioid activity
to involve a G-protein and is sensitive to per- are discussed below.
tussis toxin. Considerable evidence suggests 3.2.3.1 Ligands Used to Characterize Opi-
that the effects on ion currents are attributed oid Receptors. Since the identification of mul-
to direct coupling of opioid receptors to ion tiple opioid receptor types, considerable effort
channels through G-proteins and are not re- has focused on developing more selective li-
lated to inhibiton of adenylyl cyclase (see Ref. gands for each of the receptor types (see Figs.
102). 7.4-7.6 and Sections 5.3, 5.9, 5.10, and 6 be-
Agonist binding to opioid receptors also ap- low). Ligands commonly used to study the dif-
pears to activate the extracellular signal regu- ferent receptor types include both nonpep-
lated kinase (ERK) cascade, which consists of tides and peptides (see Table 7.6). Morphinans
three intracellular kinases: a mitogen-acti- such as morphine and the antagonists nalox-
vated protein kinase (MAPK) kinase kinase, a one (16), naltrexone (17), and cyprodime (18)
MAPK kinase, and a MAPK homolog (see Ref. (104) are used to study p receptors (Fig. 7.4);
100). This activation appears to be through naloxone and naltrexone retain significant af-
G,or Go (see Ref. 100 and references cited finity for 6 and K receptors and therefore at
therein) higher concentrations these compounds will
antagonize all three receptor types. Several
3.2.3 Characterization of Opioid Recep- peptides, including the enkephalin analog
tors. Early evaluation of compounds for opioid DAMGO ([D-Ala2,MeNPhe4,glyol]enkephalin,
activity relied on testing for antinociceptive 15),and CTOP and CTAP (19 and 20), soma-
activity in vivo. These pharmacological assays tostatin analogs that antagonize p receptors
are often predictive of analgesic activity in hu- (105), are also used to characterize p recep-
mans, but the activity of compounds observed tors. Early studies of 6 opioid receptors used
in these assays is affected by a variety of fac- the enkephalin analog DADLE ( [ D - A ~ ~ ~ , D -
tors, including the route of administration of Leu5]enkephalin, 21), but several more 6-se-
the compound, the ability of the compound to lective enkephalin analogs, including DSLET
cross the blood-brain barrier into the CNS, the and DTLET ( [ ~ - S e r ~ , L e u ~ , T h r22
~ ] -and
,
2 5 6
susceptability of the compound to metabolism [D-Thr ,Leu ,Thr ]enkephalin, 23) and par-
and pharmacokinetics, the choice of noxious ticularly the cyclic analog DPDPE (cyclo[~-
stimulus, and the animal species and strain Pen2,~-Pen5]enkephalin,24) (Fig. 7.51, are
used for the assay (see Ref. 103). The results of now used routinely to characterize 6 recep-
in vitro assays are not influenced by many of tors; the naturally occurring deltorphins (e.g.,
these factors that complicate in vivo assays. 25), peptides isolated from frog skin (106),ex-
The pharmacological activity of opioids in hibit marked 6-receptor selectivity. The non-
vitro can still be complex, however, because peptide agonists BW373U86 (26),SNC 80 (27)
more than one opioid receptor type is present (107), and TAN 67 (28) (108,109) also exhibit
in many tissues. Opioid receptors are present very high selective for 6 receptors and are used
in a variety of peripheral tissues, and isolated frequently to study these receptors. Delta-re-
tissue preparations, particularly the guinea ceptor antagonists include the peptides ICI
pig ileum (GPI) and mouse vas deferens 174,864 (29) (110), TIPP (Tyr-Tic-Phe-Phe
Narcotic Analgesics
(18) Cyprodime
(42) Etorphine
(Pen = penicillarnine)
X = Orn (19) CTOP
X = Arg (20) CTAP
Agonists
Tyr-D-Ala-Gly-Phe-D-Leu 0
(21) DADLE
(CH3CH2)2N
Tyr-X-Gly-Phe-Leu-Thr
X = D-Ser (22) DSLET -
(24) DPDPE
0 OH
yNpt
HO
( y-~ib-~ib- he-~eu
(Aib = -NHC(CH&CO-)
(29) ICI 174,864
- +H3N 0 N
H
0
N
--
C02-
Agonists
(38) Dynorphin A
Antagonists
OH
R= H (39) norBNl (41) GNTl
R = CH3 (40) BNI
but studies now commonly use the more 6-se- 34) in the presence of unlabeled p- and 6-se-
lective ligand [3H]DPDPE ([3H]-24). Early lective ligands such as DAMGO and DPDPE to
binding studies of K opioid receptors were block p and 6 receptors in the tissue prepara-
hampered by the low K selectivity of available tion. Because the highly K-selective ligand
tritiated ligands and the low levels of K recep- U69,593 ([3H]-36)is now available in tritiated
tors in rat brain (see below). One solution was form (116), it is routinely used in K-receptor
to examine the binding of ligands such as binding assays; the K-selective ligand CI-977
[3H]EKC ([3H]-33)or [3H]bremazocine ([3H]- (37) (117, 118) is also available in tritiated
3 Physiology and Pharmacology 347
Table 7.6 Ligands Commonly Used to Study Different Opioid Receptor Typesa
Receptor Type Agonists Antagonists
P Morphine (1) (Naloxone, 16)
DAMGO (15) (Naltrexone, 17)
Cyprodime (18)
CTOP, CTAP (19,20)
6 DPDPE (24) TIPP (30)
DSLET, DTLET (22,231 TIPP[+I (31)
Deltorphins (25) ICI 174,864 (29)
(DADLE, 21) Naltrindole (32)
BW373U86 (26)
SNC 80 (27)
TAN 67 (28)
K U50,488 (35), U69,593 (36) norBNI (39)
CI-977 (37) GNTI (41)
(EKC, 33) (bremazocine, 34)
Dynorphin A (38) and derivatives
"Compounds with limited selectivity for a given receptor type are listed in parentheses (see Table 7.8 for affinities and
opioid activities of these agents). See Figs. 7.1 and 7.4-7.6 for structures of these agents.
form. The affinity of a variety of opioids in ing assays. In contrast the rabbit cerebellum
radioligand binding assays for p, 8, and K re- contains predominantly (>70%) p opioid re-
ceptors are given in Table 7.8. ceptors (127). Species differences also exist for
Prior to cloning of the opioid receptors, af- S receptors, with mouse brain exhibiting sub-
finity for these receptors was most commonly stantially higher 6-receptor density than rat
determined by the use of homogenates or brain (128). NG108-15 cells contain only 6 re-
membrane fractions from rat, guinea pig, or ceptors and therefore have been used in radio-
mouse brain, which contain all three types of ligand binding assays for these receptors
opioid receptors. The relative amounts of dif- (129). With the cloning of opioid receptors
ferent opioid receptor types vary between spe- (Section 3.2.4 below), binding assays are now
cies, however, particularly for K receptors. In typically performed using these cells that ex-
rat brain K opioid receptors constitute only press only a single receptor type.
about 10-15% of the total number of opioid 3.2.3.3 In Vitro Assays for Efficacy. Until
receptor sites (1241, whereas in species such as the cloning of the opioid receptors isolated tis-
guinea pig they represent approximately 30% sue preparations, particularly smooth muscle
of the total opioid receptor population (125). preparations, were used extensively to charac-
Over 80% of the opioid receptors in the guinea terize opioids (see Refs. 131, 132 for reviews).
pig cerebellum are K receptors (126), so this The electrically stimulated GPI myenteric
tissue was frequently used in K-receptor bind- plexus-longitudinal muscle and the MVD
Table 7.7 Commercially Available Tritiated Opioid Receptor Ligandsa
Receptor Type
Nonselective
P 6 K (or low selectivity)
Morphine (1) DPDPE (24) U69,593 (36) EKC (33)
DAMGO (15) [D-Ala21deltorphinI1 (25) CI-977 (37) Diprenorphine (43Ib
I1
[~-Ala~,IIe~~~]deltorphin (Bremazocine, 34) Naloxone (16Ib
Naltrindole (32Ib
(DADLE, 21)
"Compounds with limited selectivity for a given receptor type are listed in parentheses. Ligands commercially available
from PerkinElmer Life Sciences, Amersham Biosciences, or Tocris. Iodinated derivatives of @-endorphinand Met(0) en-
kephalin are also available.
bAntagonists
348 Narcotic Analgesics
Table 7.8 Opioid Receptor Affinities and Opioid Activity in the GPI and MVD of a Variety
of Opioid Ligands Commonly Used to Study Opioid Receptorsa
a. Agonists Ki (nM) KiRatio Ic50(nM)
Agonist P 6 K P/~/K GPI MVD
Nonselective agonists
Etorphine (42) 1.0 0.56
(-1-EKC (33) 1.0 5.5
Agonists preferentially interacting with p receptors
Morphine (1) 1.8 90
Meperidine (7) 385 4,350
Fentanyl(8) 7.0 150
Methadone (9) 4.5 15
DAMGO (15) 1.9 345
Agonists preferentially interacting with 6 receptors
BW373U86 (26)" 46d 0.92d
SNC 80 (27)" 2,470d l.Od
TAN 67 (28)' 2,320 1.1
DPDPE (24) 710 2.7
DSLET (22) 39 1.8
DADLE (21) 14 2.1
[D-Ala2]deltorphin11(25) 2,450 0.71
Agonists preferentially interacting with K receptors
(-)-Bremazocine (34) 0.62 0.78
U50,488 (35) 435 9,200
U69,593 (36) 2,350 19,700
CI-977 (37) 100 1,040
[D-ProlO]Dyn A-(1-11) 0.56 2.3
b. Antagonists Ki (nM) KiRatio K, (nM)
Antagonist P 6 K P/~/K p, (GPI) K (GPI) 6 (m)
Nonselective antagonists
Naloxone (16) 1.8 23 4.8 1/13/2.7
Naltrexone (17) 1.1 6.6 8.5 1/6.0/7.7
Diprenorphine (43) 0.24 1.0 0.14 1.7/7.1/1
Antagonists preferentially interacting with p receptors
Cyprodime (18) 9.4 356 176 1/38/19
CTOP (19) 1.7 >1,000 >1,000 1/>590/>590
Antagonists preferentially interacting with 6 receptors
Naltrindole (32) 11 0.12 18 92/1/150
ICI 174,864 (29) 29,600 190 65,400 155/1/345
TIPP (30Y 1,720 1.2 ND 1,43011
TIPP[JI] (3Uk 3,230 0.31 ND 10,500/1
Antagonists preferentially interacting with K receptors
NorBNI (39) 14 10 0.34 41/29/1
GNTI (41)' 22 46 0.18 125125711
"Data from Ref. 130 except where otherwise indicated.
bAntagonistat p and 6 receptors in the rat and hamster vas deferens, respectively.
"From Ref. 107.
dIC50.
'From Ref. 109.
fND, not determined.
BDetermined in the MVD.
hAgonist in the GPI (IC,, = 1.4 nM).
'NA, not active at indicated dose.
&om Ref. 111.
'From Ref. 112.
lFrom Ref. 122.
3 Physiology and Pharmacology
preparations have been the tissues used most cies have been used to examine the activity of
extensively. The predominant effect of opioids potential analgesics. Animal models for pain
is to inhibit smooth muscle contraction, gen- include models of acute pain (e.g., hot plate,
erally by inhibiting the release of neurotrans- tail flick, paw pressure, and writhing assays),
mitters; in the GPI and MVD acetylcholine persistent pain (e.g., the formalin test),
and norepinephrine, respectively, are the neu- chronic pain (e.g., adjuvant-induced arthritis),
rotransmitters effected (see Ref. 132). In con- and neuropathic pain (e.g., nerve ligation) (see
trast to radioligand binding assays, which Table 2.1 of Ref. 144). The different types of
measure only opioid affinity, these bioassays noxious stimuli commonly used in these an-
provide information on the intrinsic activity of tinociceptive assays include heat (e.g., hot -
the compounds tested. The activity of com- plate and tail flick assays), pressure (e.g., tail
pounds in these assays can be complex, how- pinch and paw pressure assays), chemical
ever, because both tissues contain more than (writhing or abdominal constriction assay and
one opioid receptor type. The GPI contains the formalin test), and electrical (tail shock
both p and K receptors, with little if any func- vocalization) stimuli. Among the most com-
tional 6 receptors, whereas the MVD contains monly used assays are the hot plate, tail flick,
all three opioid receptor types. The activity of and writhing assays. In the hot plate test, the
a variety of opioids in these tissues are given in latency to various behavioral responses (e.g.,
Table 7.8. GPI and MVD preparations en- forepaw or hindpaw licking) is measured when
riched in a single receptor population have the animal is placed on a hot plate, typically
been prepared by use of the affinity labels set at 55"C, whereas the tail flick assay mea-
p-chlornaltrexamine (p-CNA) and p-funal- sures the time for the animal to flick its tail
trexamine (p-FNA) (133, 134) (see Section away from radiant heat focused on the tail. In
5.11). Vas deferens from other species have the writhing or abdominal constriction assay
been used to characterize opioids and appear the animal is injected intraperitoneally (i.p.)
to contain predominantly a single receptor with an irritant (typically phenylquinone or
population. The rabbit vas deferens appears to acetic acid) and the dose of a test compound
contain only K receptors (1351, whereas the required to abolish the writhing syndrome de-
hamster vas deferens contains only 6 recep- termined. On the basis of a comparison of the
tors (136). In the rat vas deferens P-endorphin potencies of standard opioids in several pain
is a potent inhibitor, which led to the proposal models, it was concluded that the mouse ab-
that this tissue contains an additional type of dominal constriction assay (use of 0.4% acetic
opioid receptor, the E receptor (137-139). acid) was the most sensitive to opioids,
With the cloning of the opioid receptors, in whereas the mouse hot plate test (at 55°C) was
vitro functional assays that used these recep- the least sensitive; the rat tail flick test was
tors have been developed. These assays have intermediate (see Ref. 144). For a detailed dis-
used measurement of effects on signal trans- cussion of these antinociceptive assays, read-
duction systems to determine the efficacy and ers are referred to excellent recent reviews
potency of compounds being studied. Because (144, 145) that describe these procedures and
opioid receptors are G-protein-coupled recep- discuss methodological issues and recent
tors, measurement of the stimulation of bind- developments.
ing of the radiolabeled nonhydrolyzable ana- Activation of all three types of opioid recep-
log of GTP [35SlGTPyS(140, 141) following tors (p, 6, and K) can produce antinociception,
interaction of a compound with the receptor but there are significant differences in the ef-
can be used to determine the efficacy and po- fects of activating different receptor types, de-
tency. Inhibition of adenylyl cyclase has also pending on the noxious stimulus used and the
been used as a functional assay to evaluate the animal species (see Refs. 103,146 for excellent
efficacy and potency of opioid ligands at opioid reviews; see particularly Table 1 of Ref. 103
receptors (142,143). for a summary of supraspinal opioid receptor
3.2.3.4 in Vivo Evaluation of opioids. A va- involvement in antinociceptive assays). Al-
riety of antinociceptive assays that use differ- though p agonists are active against all types
ent noxious stimuli and different animal spe- of noxious stimuli, the activity of K agonists
350 Narcotic Analgesics
Figure 7.7. Schematic diagram of the protein structure of the three opioid receptors. Rectangles
indicate transmembrane helices and numbers indicate the percentage identical residues among the
three opioid receptors in that segment (from Ref. 100).
depends on the type of stimulus. Kappa ago- type of cloned opioid receptor and in knockout
nists are active against chemical and pressure mice lacking individual opioid receptors have
stimuli, but they are inactive against electrical confirmed the involvement of the cloned re-
stimulation and their efficacy against thermal ceptors in analgesia mediated by p, 6, and K
stimuli is dependent on the intensity (147). receptors (see Refs. 149,153,154 for reviews).
Delta agonists may be active against all four The opioid receptors belong to the family of
types of stimuli in mouse, depending on the G-protein-coupled receptors. On the basis of
route of administration (see Ref. 103), but the model for this family of receptors, the re-
there are species differences. It has been re- ceptors contain extracellular regions includ-
ported that while S agonists are effective in ing the N-terminus and extracellular loops,
both the tail flick and hot plate assays in mice, seven putative transmembrane (TM) regions,,
in rats they are active in the hot plate, but not and intracellular regions including the C-ter-
tail flick assays (148). minus and intracellular loops (Fig. 7.7). Com-
parison of the sequences (155) indicates the
3.2.4 Opioid Receptor Structure and Molec- highest sequence homology between the K, p,
ular Biology. The first successful cloning of and S receptors in TM2, TM3, and TM7 (Fig.
opioid receptors used a cDNA library from 7.7). TM2 and TM3 each contain a conserved
NG108-15 cells to clone S receptors (8,9).Af- Asp residue; the conserved Asp in TM3 is
ter expression of the cDNA library in COS thought to interact with protonated amine
cells, the cells were screened for L3H1 S-ligand groups on opioid ligands (see below). There
binding and the clone isolated. Yasuda et al. are also high similarities in the intracellular
subsequently reported cloning of both mouse loops; the third intracellular loop is thought to
brain K and 6 receptors while trying to isolate be involved in interactions with G-proteins.
cDNAs encoding somatostatin receptors (111, The second and third extracellular loop, TM1,
and Chen et al. reported cloning of rat p recep- and TM4-6 are less conserved. The largest
tor (10). Since then the cloning of all three structural diversity occurs in the extracellular
opioid receptor types from several different N-terminus. Potential sites for possible post-
species, including human, have been reported translational modification have been identi-
(see Refs. 79,80,94,149-152 for reviews). The fied on the receptors. Two potential glycosyla-
human p, 8, and K receptors exhibit 91-95% tion sites are located in the N-terminal
sequence homology to the corresponding type sequence. Two possible sites for protein ki-
of receptor from rat and mouse (79). Studies nase C phosphorylation are located in the C-
with antisense oligodeoxynucleotides to each terminus plus a third site is found in the third
3 Physiology and Pharmacology
larity between the opioid and ORLl receptors ceptor (1821, arylacetamides to the K receptor
(see Section 7.2) has been clearly demonstrated (182, 183, 186), fentanyl analogs at the f i re-
by the ability to convert the ORLl receptor to an ceptor (182, 1871, and piperazine and piperi-
opioid receptor with the mutation of only four dine derivatives at 6 receptors (182, 188).
residues in TM6 and TM7 (176). Site-directed Modeling of the flexible opioid and opioid-like
mutagenesis has also been used to examine pos- peptides bound to their receptors is more chal-
sible points of attachment of the affinity label lenging, and therefore reports describing their
P-FNA (177) (see Section 5.11.1 below). receptor-binding modes have been more lim-
. and site-directed
Thus chimeric rece~tors ited. Models have been developed for the bind-
mutagenesis have provided tremendous in- ing of the conformationally constrained 6-se-
sight into receptor-structure and receptor-li- lective peptides JOM-13 (Tyr-cyclo[~-Cys-
gand interactions. However, the data from Phe-D-PenlOH) and DPDPE to 6 opioid
these studies must be intermeted with cau- receptors (182), and the extracellular loops
tion, particularly in cases involving loss of have been incorporated into computational
function (157). Changes in the primary se- models of K receptors (181) and ORLl recep-
quence of a receptor could have significant ef- tors (1891, so that possible binding modes of
fects on the protein secondary or tertiary Dyn A-(1-10) and OFQN-(1-13) to these re-
structure, which in turn could affect the af- ceptors could be examined. With the recent
finities of various ligands. Thus whether ob- report of the X-ray structure of rhodopsin
served changes in ligand affinity are attrib- (2.8-A resolution) (178), additional reports of
utable to the direct effect of the mutations or computational models of opioid receptors de-
indirect effects that result from altering the rived from this structure are beginning to ap-
tertiary structure of the protein is not pear in the literature (see Refs. 156, 190).
known. 3.2.4.3 Receptor Subtypes, Splice Variants,
These studies demonstrate the comdexi- - and Receptor Dimerization. Before cloning of
ties of ligand binding to opioid receptors and the opioid receptors, subtypes of each of the
indicate that more research is required to three opioid receptors were proposed on the
more fully understand, at the molecular level, basis of evidence from pharmacological assays
the interactions of different opioid ligands (see Refs. 69, 103 for excellent reviews), al-
with their receptors. though opioid receptors identified by cloning
3.2.4.2 Computational Models of Opioid have consistently represented only a single
Receptors. ~ e c a u s eG-protein-coupled recep- subtype for each receptor type. Currently,
tors are transmembrane proteins, until quite there is still considerable debate on the exis-
recently (178) structural information on these tence and nature of some of these receptor
receptors was limited to low resolution (6-9 A) subtypes.
structures from electron microscopy studies of There is considerable evidence from both
rhodopsins.
* With the deduced amino acid se- functional assays and binding studies support-
quences of the opioid receptors available, com- ing the existence of two types of 6 opioid recep-
putational models of the three-dimensional tors (see Refs. 69,191,192). A key factor in the
structures of opioid receptors were developed characterization of these &receptor subtypes
based initially on these low resolution struc- was the availability of ligands selective for
tures (166, 179-188). The rigid structures of each of the proposed subtypes (Fig. 7.8). The
many nonpeptide opioid ligands decreases the 6, subtype was characterized by its preferen-
possible degrees of conformational freedom, tial stimulation by the enkephalin analog
decreasing the complexity of docking these DPDPE (24) and DADLE (21) and selective
compounds to opioid receptor models, and antagonism by the naltrindole analog BNTX
therefore several of the reports have described (7-benzilidenenaltrexone, 44) (193) and the
possible binding modes for these compounds. affinity label (see below) DALCE (45) (194).
These include tifluadom-like benzodiazepine The 6, subtype was distinguished by its stim-
ligands at K opioid receptors (1791, naltrexone- ulation by [~-Ala~]deltor~hin 11 (25) and
derived antagonists at K and 6 receptors ( N O ) , DSLET (22) and antagonism by the benzofu-
morphinans including morphine to the p re- ran naltrindole analog naltriben (46) (195)
3 Physiology and Pharmacology
Tyr-D-Ala-Gly-Phe-Leu-Cys
(45) DALCE
(44) BNTX
(49) Naloxonazine
(48) Naloxazone OH
and the irreversible antagonist naltrindole ible ligands (see Section 5.11) FIT (198) or
isothiocyanate (NTII, 47) (196). Delta-recep- (+)-transSUPERFIT (199) to selectively acy-
tor subtypes were also proposed by Rothman late a,, receptors (200) and used the p-recep-
and coworkers which were differentiated by tor affinity label BIT (198) to deplete the mem-
whether they were associated with a p-6 re- branes of S, receptors (201). On the basis of
ceptor complex (S,,) or were not associated binding and pharmacological studies Roth-
with such a complex (S,,,) (see Ref. 197 for a man and coworkers proposed that the S,, and
review). These researchers used the irrevers- the S,, receptors corresponded to the 6, and 6,
Narcotic Analgesics
receptors, respectively (202, 203). Pharmaco- ria, are decreased in these animals). Mice de-
logical characterization of the cloned 6 opioid ficient in each of the opioid receptors and
receptor is consistent with classification as the animals lacking all three opioid receptors have
S,subtype (2041,and antisense oligonucleotides been examined for residual K, receptor bind-
differentially affect the two subtypes (205). In ingsites (216);all of the residual non-K, recep-
&receptor knockout mice binding of both [,HI- tor labeling could be accounted for by p and 6
DPDPE and [3H]-[~-Ala2]deltorphin I1 is ab- receptors, and the triple receptor deficient
sent (206), indicating that the proposed sub- mice exhibited no residual binding of
types are not due to different gene products; [3H]bremazocine, indicating that no other
however, selective 6 agonists still retain anal- gene product was involved in binding this opi-
gesic potency, suggesting the existence of a oid ligand. A third K-receptor subtype, K,, has
second 6-like analgesic system (206). also been proposed on the basis of studies that
Multiple types of p opioid receptors have used naloxone benzoylhydrazone, Nal(Bzo)H
been proposed by Pasternak and coworkers. (50) (217).
Two subtypes of p receptors, the p, site, which Recently, an alternative explanation for
was suggested to be a common high affinity opioid receptor subtypes, receptor dimeriza-
site for both nonpeptide opioids and opioid tion, has appeared in the literature that could
peptides, and the p2site, which was proposed explain why different gene products have not
to correspond to the "traditional" p-binding been identified for the proposed receptor sub-
site (2071, were initially postulated by these types in spite of the evidence from pharmaco-
researchers (see Refs. 69,208). The hydrazone logical assays for their existence. The details
derivative of naloxone, naloxazone (48) (209), of these studies are discussed under Recent
and its dimeric azine derivative naloxonazine Developments (Section 7.1) below.
(49) (210) have been used to study the puta-
3.3 Physiology of Non-p Opioid Receptors
tive p, receptors (see Ref. 691, which is
thought to represent about 20% of the specific Because of the side effects associated with an-
binding to rat brain membranes (208). Follow- algesics that interact with p opioid receptors,
ing the cloning of the opioid receptors, Paster- there has been considerable interest in devel-
nak and coworkers characterized multiple oping opioid ligands that interact with other
splice variants of the p receptor (see Refs. 211, opioid receptors. In addition to analgesic activ-
212 for reviews). ity, there are a number of other potential ther-
Three or more subtypes of K receptors have apeutic applications of ligands, both agonists
been postulated. In the early 1980s several and antagonists, for non-p opioid receptors.
groups reported differences between the bind-
ing of [,HIEKC and [3H]diprenorphine or 3.3.1 Delta Receptors. There has been con-
[,H1etorphine (after blockade of p and 6 recep- siderable interest in 6 opioid agonists because
tors), suggesting that there might be K-recep- they exhibit antinociceptive effects without
tor subtypes; there was considerable debate, the side effects associated with p opioid recep-
however, concerning the nature of these tor agonists. Antinociceptive activity was first
different binding sites (see Refs. 69, 213). demonstrated with 6-selective opioid peptides
Subsequently, the K-selective arylacetamides (see Ref. 218 for a review), and more recently
U50,488 and U69,593 helped to clarifjr the def- with nonpeptidic 6-selective agonists (see
inition of different K-receptor subtypes, and Refs. 219-222 for reviews). Of particular in-
two populations of binding sites, K , versus K,, terest is the activity of 6 agonists in inflamma-
were differentiated on the basis of their sensi- tory and neuropathic pain (220). Delta opioid
tivity and insensitivity, respectively, to these receptors also modulate p opioid receptors
arylacetamides (214). The cloned K receptors and, as discussed earlier, one classification of 6
appear to be the K, subtype on the basis of opioid receptor subtypes was based on their
pharmacological characterization (204) and association with p opioid receptors. There is
the analgesic activity of U50,488 is abolished now considerable evidence that interaction
in K-receptor knockout mice (215) (other ef- between the two receptor types can alter the
fects of U50,488, hypolocomotion and dyspho- activity of p opioid agonists. Delta agonists
3 Physiology and Pharmacology
can potentiate the analgesic activity of p ago- ences cited therein). There is considerable ev-
nists (223,224) and 6 antagonists can decrease idence that endogenous opioids and opioid
the development of tolerance and dependence receptors play important roles in the abuse of
to morphine (225, 226). Although the 6-selec- alcohol (235,236), and the opioid receptor an-
tive antagonist naltrindole can block the de- tagonist naltrexone has been used to treat al-
velopment of tolerance to the analgesic effects coholism. The involvement of 6 opioid recep-
of morphine (225), it does not block the bene- tors in alcohol abuse, however, remains
ficial development of tolerance to the respira- unclear. Delta-selective receptor antagonists
tory depressant effects of morphine (227). have been examined for their ability to de-
Delta ligands can also reverse the respiratory crease alcohol consumption in animal models,
depression caused by p agonists, and interest- but the results have been mixed, with some
ingly, 6 agonists as well as antagonists were studies reporting decreased alcohol consump-
reported to have similar effects on respiration tion, whereas others observed no effects (see
(228). Thus 6 receptor ligands, particularly 6 Refs. 222,236 for reviews). One possible expla-
antagonists, could have important therapeutic nation for this discrepancy could be differ-
applications to minimize the deleterious ef- ences in the genetic backgrounds of the ani-
fects of morphine. These findings have mals studied.
prompted the search for compounds that ex- Delta receptors are also involved in a num-
hibit both p agonist and 6 antagonist activity, ber of other biological effects, including both
and peptidic ligands with this mixed activity centrally and peripherally mediated effects
have been reported by Schiller to produce po- (see Refs. 219,220,222 for reviews). Delta ago-
tent analgesic effects without physical depen- nists are effective in learned helplessness ani-
dency and less tolerance than that of mor- mal models, suggesting a potential therapeu-
phine (229). Nonpeptide ligands with mixed tic application in depression (237).Convulsant
activity have also recently been reported (230) effects have been observed with nonpeptide 6
(see Section 5.3.3 below). agonists [particularly BW373U86 (see Section
Delta receptors may also modulate re- 5.10.11, however, which could prevent their
sponses to substances of abuse other than opi- clinical use unless these adverse effects can be
oids, includingcocaine, amphetamines, and al- separated from their desired action (e.g., an-
cohol (219, 220, 222). Several 6 agonists have tinociceptive activity; see Ref. 238). Opioids,
been reported to at least partially discriminate including 6 receptor ligands, have immuno-
for cocaine (see Ref. 222). There are numerous modulatory effects (239). Delta agonists have
reports that 6-receptor antagonists can atten- been reported to stimulate immune responses,
uate a number of the effects of cocaine (see whereas 6 antagonists can cause immunosup-
Refs. 219,220,222), and an antisense oligode- pression (see Refs. 219, 220). The immuno-
oxynucleotide to 6 opioid receptors blocks co- modulatory effects are still present in 6 recep-
caine-induced place preference in mice (231). tor knockout mice (240), however, indicating
Thus 6 antagonists could potentially be used that these effects are not mediated by these
in the treatment of cocaine abuse. One study, receptors.
however, found that the 6-receptor antagonist
naltrindole can potentiate the lethal effects of 3.3.2 Kappa Receptors. Like p and 6 recep-
cocaine; this was found only after intracister- tors, activation of K opioid receptors can
nal administration and not after i.v. adminis- produce antinociceptive effects. However, the
tration, so it is not clear whether this would effectiveness of K opioid agonists as antinoci-
limit the application of 6-receptor antagonists ceptive agents varies in different types of pain
in treatment of cocaine abuse (232). Similar (see Ref. 146),and K agonists are less effective
results have been found for 6 agonists and an- in thermal antinociceptive assays involving
tagonists in methamphetamine-induced place more intense stimuli.
preference (see Ref. 222); one important find- Kappa agonists can also produce antinoci-
ing was that the 6 agonist DADLE can protect ceptive effects in inflammatory pain through
against the neuronal damage caused by meth- interaction with peripheral K opioid receptors.
amphetamine (see Refs. 233, 234 and refer- Kappa agonists have been shown to have anti-
Narcotic Analgesics
arthritic activity (241, 2421, and to produce model, a K-receptor antagonist could be useful
antinociception in capsaicin-induced thermal to normalize this imbalance and treat opioid
allodynia through interaction with peripheral addiction (249).
K receptors (243, 244). Because many painful The effects of K opioid agonists on dopa-
conditions are associated with inflammation, mine levels also has implications for the
there is significant potential for the applica- treatment of cocaine abuse. Cocaine blocks
tion of K agonists as peripheral analgesics. Pe- reuptake of dopamine, and considerable evi-
ripheral K opioid receptors also appear to be dence suggests that cocaine's reinforcing effects
involved in visceral pain and the activity of K are mediated by these increases in extracellular
agonists is enhanced in the presence of chronic dopamine (see Refs. 250, 251 and references
inflammation of the viscera (245, 246), sug- cited therein). Because K agonists can decrease
gesting potential therapeutic applications of dopamine levels, they can ad as h d i o n a l an-
tagonists of cocaine (250, 251). Several K ago-
peripherally selective agents without the side
nists have been shown to decrease cocaine self-
effects (e.g., dysphoria) associated with many
administration (250-254; but see Ref. 255) and
centrally acting K agonists. K agonists can also attenuate many of the behav-
In many cases activation of central K opioid ioral effects of cocaine (see Ref. 250).
receptors opposes the activity of p opioid re- Central K opioid receptors also mediate
ceptors (see Ref. 247 for a recent review). Sev- other effects that could be therapeutically
eral studies have reported that K agonists at beneficial. Kappa agonists exhibit neuropro-
doses that do not affect the baseline pain tective effects in stroke (256);these effects are
threshold can antagonize the analgesic activ- observed in experimental models even several
ity of p opioid agonists (see Ref. 247). The hours after blood vessel occlusion (see Ref. 257
K-opioid peptide dynorphin A-(1-13) has been and references cited therein), which is partic-
reported to improve the memory impairment ularly promising for the potential therapeutic
induced by the p agonist DAMGO (248). use of K agonists in stroke. Dynorphin im-
Kappa agonists also reduce tolerance to mor- proves memory dysfunction in an animal
phine in a variety of antinociceptive tests, and model of amnesia (see Ref. 247). Kappa opioid
dynorphin can inhibit morphine withdrawal agonists have also been shown to cause down-
symptoms in morphine-dependent animals regulation of HIV expression in human micro-
(see Ref. 247 and references cited therein). glial cells (258) and CD4+ lymphocytes (259),
Kappa agonists generally lack reinforcing ef- which raises the intriguing possibility of using
K ligands as adjuvant therapy in HIV-infected
fects and can abolish the reinforcing effects of
morphine (see Ref. 247). This effect of K ago- individuals; whether this will have clinical ap-
plicability remains to be determined.
nists on morphine reward may be attributed
to the opposing modulation of the mesolimbic
dopamine system by p and K opioid receptors, 3.4 Endogenous Opioid Peptides
where p agonists increase dopamine levels During the mid-1970s the search for endoge-
while K agonists decrease dopamine levels. nous ligands for opioid receptors led to the
Kappa agonists also generally have opposite discovery of peptides with opiate-like activity.
subjective effects from p opioid agonists (dys- The first opioid peptides reported were the
phoria for K agonists versus euphoria for p pentapeptides leucine and methionine en-
agonists). Chronic administration of mor- kephalin (3and 4) (7), followed shortly there-
phine and other drugs with positive reinforc- after by dynorphin A (38) (260,261) and @en-
ing effects increases brain levels of the endog- dorphin (51)(262). Because these peptides are
enous K opioid peptide dynorphin (see Ref. 249 structurally distinct from the alkaloid opiates,
and references cited therein). It has been pro- the term opioid was introduced to describe all
posed (249) that this produces an imbalance in compounds, both nonpeptide and peptide,
abstinent p opioid-dependent individuals and with opiate-like activity. These mammalian
dysphoric mood states, which can result in the opioid peptides share a common N-terminal
desire to take p opioid agonists to normalize tetrapeptide sequence, but differ in their C-
mood. Thus on the basis of this " K overdrive" terminal residues (Fig. 7.9). They also differ in
3 Physiology and Pharmacology
Proenkephalin peptides
Leu-Enkephalin (3) Tyr-Gly-Gly-Phe-Leu
Met-Enkephalin (4) Tyr-Gly-Gly-Phe-Met
Met-enkephalin-Arge-Phe7 Tyr-Gly-Gly-Phe-Met-Arg-Phe
Met-enkephalin-Arg6-Phe7-Leu8 Tyr-Gly-Gly-Phe-Met-Arg-Gly-Leu
Prodynorphin peptides
Dynorphin B Tyr-Gly-Gly-Phe-Leu-Arg-Arg-GIn-Phe-Lys-Val-Val-Thr
a-Neoendorphin Tyr-Gly-Gly-Phe-Leu-Arg-Lys-Tyr-Arg-Pro-Lys
p-Neoendorphin Tyr-Gly-Gly-Phe-Leu-Arg-Lys-Tyr-Arg-Pro
Pro-opiornelanocorlin peptides
&-endorphin (51) Tyr-Gly-Gly-Phe-Met-Thr-Ser-GIu-Lys-Ser-GIn-Thr-Pro-Leu-Val-Thr-Leu-
Phe-Lys-Asn-Ala-lle-lle-Lys-Asn-Ala-Tyr-Lys-Lys-Gly-Glu
Endornorphins
Orphanin FW
Nociceptin (54) Phe-Gly-Gly-Phe-Thr-Gly-Ala-Arg-Lys-Ser-Ala-Arg-Lys-Leu-Ala-Asn-GIn
Orphanin FQ 2 Phe-Ser-Glu-Phe-Met-Arg-Gln-Tyr-Leu-Val-Leu-Ser-Met-GIn-Ser-Ser-Gln
Nocistatin Pro-Glu-Pro-Gly-Met-Glu-Glu-Ala-Gly-Glu-Met-Glu-Gln-Lys-Gln-Leu-Gln
(human)
Figure 7.9. Mammalian opioid peptides and orphanin FQ/nociceptin.
the receptor types with which they preferen- functioned as "address" components to direct
tially interact. Although the enkephalins ex- the peptides to particular opioid receptors.
hibit some preference for interacting with S Recently, a new class of opioid peptides, the
receptors (Table 7.9), the dynorphins prefer- endomorphins (52 and 53, Fig. 7.9), were dis-
entially interact with K receptors; p-endor- covered (264) that do not share the classical
phin possesses high affinity for both p and 6 "message" sequence with other mammalian
receptors. This led Goldstein to apply the opioid peptides. In contrast to other mamma-
"message-address" concept (see also Section lian opioid peptides, the endomorphins show
6.3) to the opioid peptides (263). In Goldstein's high selectivity for their preferential receptor,
proposal the common N-terminal tetrapeptide the receptor (Table 7.9). Since their discov-
sequence constituted the "message" sequence ery the pharmacology of these new mamma-
responsible for activating opioid receptors, lian opioid peptides has been studied exten-
whereas the unique C-terminal sequences sively (see Ref. 265 for a detailed review).
358 Narcotic Analgesics
Table 7.9 Opioid Receptor Affinities and Opioid Activity in the GPI and MVD of Endogenous
Opioid Peptidesasb
Ki (nM) KiRatio IC5, (nM)
Peptide P 6 K ~ 1 6 1 ~ GPI MVD
Proenkephalin peptides
Leu-enkephalin (3)
Met-enkephalin (4)
Met-enkephalin-kg6-Phe7
Met-enkephalin-Arg6-Gly7-Leus
Prodynorphin peptides
Dynorphin A (38)
Dynorphin A-(1-8)
Dynorphin B
p-Neoendorphin
a-Neoendorphin
POMC peptide
&-Endorphin (51)
Endomorphins"
E n d o m o r p h -1 (52)
Endomorphin-2 (53)
"Data from Ref. 130 except where otherwise indicated.
bSeeSection 7.2 for data on orphanin FQlnociceptin.
'Data from Ref. 264.
After the identification of the ORLl recep- dues along with one copy of Leu-enkephalin.
tor, two groups isolated a 17-residue peptide In addition, extended Met-enkephalin deriva-
(54, Fig. 7.9) as the endogenous ligand for this tives Met-enkephalin-kg6-Phe7 and Met-en-
receptor (92,93). This peptide was referred to kephalin-kg6-Gly7-Leu8and longer peptides
as orphanin FQ by one group because it was (e.g., peptides E and F and BAM-20) are ob-
the ligand for the orphan receptor (FQ are the tained from proenkephalin A. The dynor-
N- and C-terminal residues, respectively, of phins, which contain a number of basic resi-
the peptide) (92) and named nociceptin by the dues in the C-terminus, are derived from
other group, since in the initial studies this prodynorphin (also called proenkephalin B;
peptide was reported to produce hyperalgesia see Ref. 269 for a review). In addition to dynor-
(93).Although the N-terminal tetrapeptide se- phins A and B, longer dynorphins (e.g., dynor-
quence of orphanin FQInociceptin (OFQ/N) is phin 32, which contains both dynorphin A and
similar to that of the classical opioid peptides, B) and a- and p-neoendorphins are obtained
the presence of Phe rather than Tyr in posi- from prodynorphin B. The precursor protein
tion 1 is an important factor in the peptide's of the endomorphins has yet to be identified.
high selectivity for ORLl over opioid receptors The OFQ/N precursor protein prepronocicep-
(see Section 7.2). tin (93) has been characterized and also con-
The classical mammalian opioid peptides tains additional biologically active peptides re-
are derived from three distinct precursor pro- lated to OFQ/N, orphanidnociceptin 2 (270)
teins (Fig. 7.10) (see Refs. 75,266 for reviews). and nocistatin (271) (Fig. 7.10).
Processing of the precursor proteins occurs at
pairs of basic residues. p-Endorphin is derived
from proopiomelanocortin (POMC), along 4 HISTORY: IDENTIFICATION OF
with ACTH, a-MSH, and p-lipotropin (see Ref. MORPHINE AND EARLY ANALOGS
267 for a review). The enkephalins are derived
from proenkephalin A (see Ref. 268 for a re- The effects of opium have been known for
view). This protein contains four copies of thousands of years, with written references
Met-enkephalin flanked by pairs of basic resi- dating as far back as the third century B.C.
listory: Identification of Morphine and Early Analogs 359
Proorphanin
Orphanin
a-neoendorphin
Prodynorphin Dynorphin A
I l l I
~ ~ n b r ~ hB' i n
Proenkephalin Peptide F Octapeptide Heptapeptide
POMC
II
~rphinewas first isolated from opium in somniferum in 1832. By the mid-1800s the pu-
15 by Sertiirner, who named it after Mor- rified alkaloids morphine and codeine began to
?us,the Greek god of sleep and dreams. The be used in place of crude opium preparations
nethyl ether codeine (2, Fig. 7.1) was subse- for medicinal purposes (23). Because of its
3ntly isolated from the poppy plant Papaver complex structure, it took more than a century
(-)-Morphine(1)
Cavity
H
Flat surface
Binding site
OH Figure 7.11. Beckett and Casy model
for opioid receptor binding (adapted
(+)-Morphine from Ref. 1).
Narcotic Analgesics
-
Rigid opiates
H3C
I
OH OH OH
TTH3'\
Morphine Morphinans Benzomorphans
H~C/
N
%
Flexible opiates
N /- ~C'
H H3c\N$
H3C /
'\
0
CH3
\ \
Axial Equatorial
phenylpiperidines phenylpiperidines Methadone
compounds such as morphine to flexible acy- Opium contains over 50 alkaloids that fall into
clic analgesics such as methadone and the opi- one of two chemical classes: the phenanthrene
oid peptides. The majority of the classical non- class, including morphine and related deriva-
peptide opioids fall into one of five chemical tives; and the benzylisoquinoline alkaloids,
classes: the 4,5a-epoxymorphinans, the mor- such as papaverine (58,Fig. 7.14; see Ref. 291
phinans, the benzomorphans, phenylpiperi- for a complete listing of alkaloids present in
dine derivatives, and acyclic analgesics. Al- opium). In addition to morphine, which on av-
though not related in many cases to how the erage accounts for 10-20% of opium, other re-
opioid activity of these different chemical
classes was originally identified nor to how
these compounds are prepared, these different
chemical classes can be related conceptually
by a systematic dismantling of morphine (Fig.
7.13). As rings present in morphine are elimi-
nated, conformational flexibility increases and
changes in SAR occur. The effects on confor-
mation and SAR are discussed for each of -0CH3
these chemical classes in turn below, followed (58) Papaverine
by descriptions of agonists selective for K and S
receptors and of affinity labels, which interact
with opioid receptors in a nonequilibrium
manner.
lated alkaloids to morphine found in opium 5.2.2 Morphine Derivatives. The synthesis
include codeine (2) (0.5%) and thebaine (59) of 3,6-diacetylmorphine (heroin, 55) in the
(0.3%).Whereas the latter is inactive as an late 1800s (280) marked the beginning of syn-
analgesic, it is a key synthetic intermediate for thetic efforts to modify the structure of mor-
the preparation of several potent analgesics phine, and since then numerous structural
(see Section 5.4 below); although present in modifications have been made to morphine
low amounts in P. sornniferum, it is the prin- (see Refs. 299, 300 for detailed reviews). Al-
cipal alkaloid found in another species of though modifications have been made to all
poppy, Papaver bracteaturn (292). portions of the molecule, the focus has been in
One interesting finding has been the detec- three regions: the phenol at position 3, the C
tion of morphine and related opiate alkaloids ring, and the basic nitrogen. The phenol, al-
in vertebrates, including in a variety of mam- though not critical for activity, enhances opi-
malian tissues (see Refs. 58, 293-295 for re- oid receptor affinity (301).Masking of the phe-
views; see also Ref. 296 for a review of isolation nolic hydroxyl group generally decreases
techniaues). The conversion of codeine and opioid activity. Methylation of the 3-phenol of
thebaine, which are known intermediates in morphine gives codeine (2, Fig. 7.1), which has
the biosynthetic pathway in P. somniferum, to approximately 10-20% the potency of mor-
morphine in several tissues from rat supports phine. As noted in Section 2.3.2, a small but
-
the conclusion that the momhine found in an- significant percentage (-10%)of codeine is 0-.
imals is of endogenous origin. Endogenous demethylated to morphine, accounting for its
morphine has been postulated to be involved analgesic activity. Heroin, on the other hand,
in neural and immune regulation (see Ref. is approximately twice as potent as morphine.
295), but the levels detected in mammalian Heroin penetrates the blood-brain barrier
tissues are low [low pmol/g (295)l compared to very rapidly [68% uptake of heroin versus un-
those of the endogenous opioid peptide levels measurable uptake of morphine 15 s after ca-
(2971, so what physiological roles such endog- rotid injection into rats (302)], and this may
enous opiate alkaloids play are still unclear. partially account for its greater potency. Her-
The conformation of morphine was deter- oin is rapidly deacetylated to give 6-acetylmor-
mined from X-ray studies (see Ref. 298 for a phine, which has potency similar to that of
review). The overall shape of the molecule is a morphine. As discussed in Section 2.3.2, the
three-dimensional "T," with rings A, B, and E 6-glucuronide metabolite of morphine (12) is
forming the vertical portion of the "T" and about twice as potent as morphine (58) and
rings C and D forming the top of the "T"(Fig. may account for a significant portion of mor-
7.15). The piperdine (D) ring is in the energet- phine's analgesic activity, especially with
ically favored chair conformation, but the C chronic use (59).
ring of morphine is in a boat conformation, Much of the early synthetic efforts focused
which places the 6a-hydroxylin the equatorial on modifications of the C ring. Changing the
position (see Ref. 298). (+)-Morphine, the en- B-C ring juncture in morphine or codeine from
antiomer of the naturally occurring (-)-mor- cis to trans decreases potency 2- to 10-fold
phine, has been synthesized (see Refs. 283, (303). This is understandable because such a
299) and is devoid of analgesic activity. change causes severe distortion in the C ring
5 Structure-Activity Relationships of Nonpeptide Opioid Ligands
OyFOH
OH
(66) Nalbuphine
Figure 7.16. C-ring derivatives of morphine. Structures of hydromorphone (5) and oxycodone (6)
from Fig. 7.1 are included for comparison. Nalmefene (64) is an antagonist, TRK-820 (65) is a
K-selectiveagonist, and nalbuphine (66) is a mixed agonist/antagonist.
of trans-morphine. Reduction of the C,-C, ring is in the chair conformation (see Fig. 7.16
double bond of morphine and codeine yields and Ref. 298). Numerous additional modifica-
dihydromorphine (60, Fig. 7.16) and dihydro- tions have been made to the 6 position of 4,5a-
codeine (61), respectively, which have similar epoxymorphinans (see Refs. 299, 300). These
to slightly increased (15-20%) potencies com- include analogs that can bind irreversibly to
pared to those of their parent compounds. Ox- opioid receptors and selective antagonists in
idation of the C, hydroxyl in morphine de- which additional rings are attached at the 6
creases analgesic potency, but when this same and 7 positions; these types of opiates are dis-
modification is made to dihydromorphine or cussed in Sections 5.11 and 5.3, respectively,
dihydrocodeine to give hydromorphone ( 5 ) later in the chapter. Zwitterionic groups have
and hydrocodone (62), respectively, opioid po- also been attached at the 6 position to produce
tency is enhanced. This modification also al- peripherally selective derivatives with de-
ters the conformation of the C ring of the opi- creased ability to penetrate the CNS (304). A
ate (see Fig. 7.16). Although the C ring of 14p-hydroxylgroup can be introduced into the
morphine and other derivatives containing a opiate skeleton by oxidation of thebaine (see
6a-alcohol (60 and 61) is in a boat conforma- Refs. 299, 300) and this modification can en-
tion, in oxymorphone (63)and the antagonist hance potency. The 14P-hydroxylatedderiva-
derivative naloxone (16) (see below), the C tives oxyrnorphone (63)and oxycodone (6) are
Narcotic Analgesics
potent analgesics, with oxymorphone exhibit- The N-ally1 derivative of oxymorphone, nalox-
ing approximately 5-10 times the potency of one (16, Fig. 7.4), was the first example of a
morphine. The analgesic activities of these pure opioid antagonist essentially devoid of
morphine derivatives, along with a variety of agonist activity, and is 7-10 times as potent an
other opioids, are compared in Table 7.1 in antagonist as nalorphine (see Ref. 305). Nal-
Section 2.1 above. oxone has a short duration of action and is
used for the treatment of narcotic overdoses
5.3 N-Substituted 4,5a-Epoxymorphinans: (see Section 2.1). Although naloxone exhibits
Opioid Antagonists some preference for p receptors, it also antag-
onizes Sand K receptors as well (see Table 7.8),
5.3.1 introduction. A critical determinant and sensitivity to antagonism by naloxone is
of the type of activity observed for morphine routinely used as a criterion for opioid recep-
derivatives is the identity of the nitrogen sub- tor involvement in an observed response. The
stituent, and variation in this group yields N-CPM derivative naltrexone (17) is a pure
compounds ranging in activity from pure ago- antagonist that is more potent than naloxone,
nists to pure antagonists. Removal of the N- has a longer duration of action, and is orally
methyl of morphine to give normorphine de- active, making it more useful for the treat-
creases potency (the relative potency of ment of former opiate addicts. The two antag-
normorphine, however, is dependent on both onists have different metabolic routes, which
the route of administration and the species accounts for the differences in duration of ac-
examined; see Ref. 305). Although some nitro- tion and doses required (see Section 2.3.2 and
gen substituents such as phenethyl enhance Ref. 305). The 6-methylene analog of naltrex-
agonist activity, other nitrogen substituents, one, nalmefene (64), is also used clinically as
notably propyl, allyl, cyclopropylmethyl (CPM), an antagonist (see Table 7.4) to reverse the
and cyclobutylmethyl (CBM), impart antago- effect of opioids and has a longer duration of
nist activity to the compounds. The replace- action than naltrexone (24). Interestingly, at
ment of the N-methyl group of morphine and cloned p opioid receptors nalmefene is an in-
codeine by an allyl group yields N-allylnor- verse agonist, whereas naloxone and naltrex-
morphine (nalorphine, 56), and N-allylnorco- one are neutral antagonists
- (the latter two
deine (57, Fig. 7.12), respectively. Although compounds display inverse agonist activity4f
the first narcotic antagonist N-allylnorcodeine the cells were pretreated with morphine)
was prepared in 1915 (2811, it was the demon- (308). The 6p-fluoro analog of naltrexone, cyclo-
stration of the antagonist activity of nalor- foxy, has been prepared in tritiated form (309).
phine in the 1940s that stimulated the exam- Recently, the 6P-substituted naltrexamine
ination of other N-substituted morphine derivative TRK-820 (65) was reported to be a
analogs for antagonist activity (see Ref. 305). K-selective agonist [Ki = 3.5 nM, Ki(dp)ratio
Nalorphine is inactive as an analgesic in ani- = 15; see Table 7.10 below], which acts as a
mals, but it is an effective analgesic in humans full agonist at K receptors, partial agonist at p
(306). Although it was thought that mixed ag- receptors, and a weak antagonist at ORLl re-
onist/antagonists such as nalorphine were ceptors (see Section 7.2 below) (310,311). The
agonists at K receptors and antagonists at p N-CBM analog of naltrexone is a mixed ago-
receptors, these drugs may be more accurately nistlantagonist, and the 6a-alcohol derivative,
described as partial agonists at both K and p nalbuphine (661, is used clinically (see Table
receptors (see Ref. 307). Nalorphine produces 7.3). Examination of diastereomeric pairs of
intense psychotomimetic effects and dyspho- nalorphine and naloxone quaternary ammo-
ria, which precluded its clinical use as an an- nium salts (which have chiral nitrogens)
algesic. However, it was used as an antidote found that the diastereomers with the N-ally1
for opioid overdose until the introduction of group equatorial were the more potent antag-
naloxone in the late 1960s. onists (312, 313).
Modification of the N-substituent of the po- The quaternary derivatives of several nar-
tent morphine analog oxymorphone has led to cotic antagonists have been used to explore
several compounds with antagonist activity. the central versus peripheral actions of opi-
5 Structure-Activity Relationships of Nonpeptide Opioid Ligands 365
oids. These compounds generally have much tained from triethylene glycol (322,323). Both
lower affinity for opioid receptors and antago- the monovalent analog with a single pharma-
nist potency (1-12%) relative to that of the cophore and an analog with a longer spacer
corresponding tertiary antagonists (314), and were much less potent K antagonists, suggest-
in some cases (e.g., N-methylnaloxone) the ing that TENA contained the appropriate
quaternary antagonist or an active metabolite spacer length for K-receptor affinity and selec-
appears to enter the CNS after peripheral ad- tivity. Subsequently, glycyl units were used in
ministration (see Ref. 314). The peripheral se- the spacer, making it possible to easily vary the
lectivity of the quaternary antagonist meth- chain length of the spacer; a central succinyl (X
ylnaltrexone (315) appears to be greater than = -Glyn-COCH,CH,CO-Glyn- in 67) (324)or fu-
that of N-methylnaloxone or N-methylnalor- maryl group (-Glyn- COCH4HCO-Glyn- in
phine (316), most likely because of the higher 67) (325) were used in the spacer. Although
resistance of methylnaltrexone to N-demeth- the optimum length of the spacer for interac-
ylation (317). The extent of this metabolic tion with p receptors was similar in the two
pathway varies among species; in humans no series (two glycyl units, i.e., n = 2, in each half
appreciable N-demethylation has been ob- of the spacer), the optimum length of the
served, which probably has a significant im- spacer for interaction with K receptors was dif-
pact on the peripheral selectivity of this drug. ferent in the two series (325). In the series
Methylnaltrexone is currently being exam- with the succinyl spacer, the shortest spacer
ined in clinical trials for the treatment of opi- (with no glycyl units, i.e., n = 0) yielded the
oid-induced constipation (see Section 2.2.2.1). most potent K antagonist, whereas in the se-
Although the above agents have been use- ries with the more rigid fumaryl spacer a
ful pharmacological tools as well as therapeu- much longer spacer (two glycyl units, n = 2, in
tic agents, these compounds preferentially an- each half) was required for maximal K-antag-
tagonize p opioid receptors. Antagonists onist activitv.
"
selective for all three opioid receptor types are Incorporation of a rigid pyrrole spacer led
valuable tools in understanding both the phar- to the synthesis of the K-selective antagonists
macological effects of opioid agonists and the norbinaltorphimine (norBNI, 39, Fig. 7.6) and
physiological effects of the endogonous opioid binaltorphimine (40) (119, 120). These com-
peptides. In the past decade Portoghese and pounds were much more selective for antago-
coworkers have synthesized numerous nal- nism of K receptors than TENA (1201, and
trexone derivatives with additional groups norBNI has been routinely used to determine
and ring systems attached to the C ring. De- whether K opioid receptors are involved in an
pending on the modifications made, analogs observed activity. A more detailed under-
selective for both K and S receptors have been standing of how these compounds interact with
prepared (see Refs. 289,318-321 for reviews). opioid receptors has come from examining their
structure-activity relationships and molecular
5.3.2 Kappa-Receptor Selective Antagonists. modeling. The monovalent ligand with only the
Portoghese and coworkers used the bivalent pyrrole ring attached (73) is not selective for K
ligand approach to design K-selective ligands. receptors, suggesting that the pyrrole ring func-
In this design strategy it was envisioned that
two pharmacophores could bridge two neigh-
boring opioid recognition sites if they were
connected by an appropriate spacer. This
should then lead to substantial increases in
potency because of the close proximity of the
second pharmacophore to the neighboring site
and hence a large increase in the local concen-
tration of the pharmacophore (see Ref. 319).
The first K-selective antagonist TENA (68,
Fig. 7.17) was prepared by connecting two nal-
trexamine pharmacophores with a spacer ob-
Narcotic Analgesics
N-X-N
H H
OH OH
(67)
(68) X = -(CH2CH20)2CH2CH2-
(TENA)
Figure 7.17. K-Receptor selective antagonists and related compounds. The structures of norBNI
(39), binaltorphimine (401, and GNTI (41) are shown in Fig. 7.6.
tions as a spacer and does not contribute to K one pharmacophore is required for interaction
selectivity (326).This conclusion is supported by with K receptors (328).This suggested that the
comparison of different spacers; whereas the second half of the bivalent ligand might be
thiophene analog of norBNI in which a sulfur mimicking the basic C-terminal "address" se-
replaces the indole nitrogen, which has a very quence of dynorphin, which imparts K-recep-
similar structure, retains K-receptor selectivity, tor affinity to the peptide, and that simpler
a pyran derivative with a very different spacer derivatives could be used in place of the second
loses K selectivity (327). pharmacophore. A simplified analog of norBNI
While the K antagonists described earlier that does not contain the second aromatic ring
were designed using the bivalent ligand ap- (70)is twice as selective as norBNI for K recep-
proach, subsequent studies revealed that tors (329).The basicity of the second nitrogen
these ligands do not appear to bridge two opi- is important for K-receptor selectivity in both
oid binding sites, but that a basic amino group norBNI analogs (330) and the simplified ana-
in the second pharmacophore interacts with a log (70) (329). Examination of the binding of
subsite on the K opioid receptor. Thus the norBNI to mutated K receptors (175)suggests
meso isomer of norBNI (69) . . was also found to that this nitrogen interacts with G ~ at the u ~ ~
be a potent K antagonist, indicating that only top of TM6 on the receptor; a significant (70-
5 Structure-Activity Relationships of Nonpeptide Opioid Ligands 367
fold) increase in affinity for the preceptor mu- to the synthesis of naltrindole (NTI, 32, Fig.
tated at the corresponding position ([K303E]) 7.5) (113,338),the first nonpeptide 8-selective
(331) is consistent with this conclusion. A antagonist. In addition to being 8 selective,
computational model of norBNI bound to K this naltrexone analog is about 500 times as
receptors has been developed ( N O ) , and in this potent as the 8-selective peptide antagonist
model the second nitrogen forms an ion pair ICI 174,864 (29, Fig. 7.5) (338). In a computa-
with G ~ u ~With
' ~ . the recent proposal that opi- tional model of NTI bound to 8 opioid recep-
oid receptors may exist as dimers (see Section tors ( N O ) , the indole moiety is directed toward
7.1 below), however, Portoghese is revisiting a hydrophobic pocket formed by residues from
these bivalent ligands and how they may in- TM6 and TM7, which includes zg9
two residues
teract with dimeric receptors (321). (TrpZs4 in TM6 and Leu in TM7 of the
These results led to the preparation of an- mouse 6 receptor) unique to the 8opioid recep-
alogs of the 8-selective antagonist naltrindole tor. Site-directed mutagenesis studies of these
(see below) with basic alkylamidino groups positions in 8, p, and K receptors were consis-
(71) (122, 332, 333) and guanidinium groups tent with these positions contributing to the
(GNTI, 41) (121-123) attached to the indole &receptor affinity and selectivity of NTI (334).
ring as K-selective antagonists. Like norBNI, Substitution of Ala in the position correspond-
the decrease in binding affinity of GNTI to ing to Leu2" in p and K receptors increased
[E297K] mutated K receptors and increase in the affinity for NTI by 21- and 96-fold, respec-
afinity for the corresponding [K303El mu- tively (334). Substitution of Trp284in the 8
tated p receptors and the [W284E] mutated 8 receptor had a smaller effect, with mutation to
receptors suggests that the guanidinium Glu (334) and Lys (166) (the residues found in
group of GNTI interacts with G ~ on uK opi-~ ~the corresponding
~ position in K and p recep-
oid receptors (121, 334). Interestingly, shift- tors, respectively) decreasing the affinity of
ing the position of the guanidinium group NTI 9- and 1Bfold [mutation to Ala decreased
from the 5' position in GNTI to the 6' position NTI affinity fivefold (16611.
(72) results in a potent K agonist (335). Fluorescent derivatives of naltrindole have
been prepared. The fluorescent analog in
5.3.3 Delta-Receptor Selective Antagonists which fluorescein was attached through a tet-
and Related Compounds. Portoghese and co- raglycine spacer to 7'-amino-NTI has been
workers used the "message-address" concept prepared (339); its fluorescence is blocked by
to design naltrexone derivatives selective for 6 NTI, indicating specific binding to 8 receptors.
receptors (see Ref. 336). The "message-ad- Recently, a fluorogenic "reporter affinity la-
dress" concept, which was originally described bel" derivative of naltrindole, PNTI, was pre-
by Schwyzer for ACTH (337), was applied to pared from 7'-amino-NTI that, in contrast to
the opioid peptide dynorphin by Chavkin and its reversible counterpart, is an agonist in the
Goldstein (263). In this model the "message" MVD (340) (see Section 5.11 below).
consists of the amino acids in the peptide that Naltrindole analogs containing other het-
are responsible for activating the receptor and erocyclic spacers have also been examined.
producing a biological response, whereas the The benzofuran analog naltriben (NTB, 46,
"address" component is the portion of the mol- Fig. 7.8) is also a 8-selective antagonist (195);
ecule that enhances affinity for a given recep- as discussed earlier in Section 3.2.4.3, subse-
tor type. Portoghese postulated that in the en- quent studies indicated that this analog is a
dogenous enkephalins the Tyrl residue selective 8, antagonist (341, 342).
functions as the "message" and the phenyl The aromatic ring in the "address" portion
ring of Phe4 functions as part of both the of naltrindole and its analogs is important for
"message" and the "address"; the intervening antagonist activity at 8 receptors. The tetra-
Gly2-Gly3 then functions as a spacer. In nal- hydroindole derivate is much less potent, as
trexone analogs, the naltrexamine moiety of are several 6-aryl derivatives (195). The 7(E)-
these antagonists functioned as the <<mes- benzylidine analog of naltrexone, 7-benzyli-
sage" portion and a phenyl ring was attached denenaltrexone (BNTX, 44, Fig. 7.81, how-
to the pyrrole spacer as the "address." This led ever, is a potent 8 antagonist (193,342). It has
Narcotic Analgesics
100-fold greater affinity for [3H]DPDPEsites ceptor affinity, in several cases [e.g., the N-2-
(6,) than for DSLET (6,) sites, and therefore is methylallyl naltrindole analog (351)], the
a selective 6, antagonist (193); it is also a se- 3-methyl ether of naltrindole (352), and the
lective antagonist for 6, receptors in the corresponding ring-opened 4-hydroxy-3-me-
mouse spinal cord in antinociceptive assays thoxyindolemorphinan (353), the selectivity
(342). Substitution on the phenyl ring of for 6 receptors increased. Replacement of the
BNTX with either o-methoxy or o-chloro re- cyclopropylmethyl (CPM) group by the
sulted in analogs with increased antagonist 2-methylallyl in NTB and SoRI 9409 (see be-
potency and 6-receptor selectivity in smooth low), but not BNTX or SIOM (see below), re-
muscle assays, but in vivo in the tail flick assay sulted in compounds that retained reasonable
these analogs exhibited similar selectivity but 6-receptor affinity and selectivity (354). The
lower potency than BNTX (343). Both the E- 14-alkoxy derivatives, with or without a
and corresponding 2-isomers of a series of aryl 5-methyl group, also exhibited high 6-receptor
analogs of BNTX have been prepared (344), selectivity in the MVD assay (355,356). Inter-
and the 2-isomers were found to have higher estingly, the N-cyclohexylethyl derivative (76)
6-receptor selectivity; the (2)-1-naphthyl de- derivative is a p-selective agonist (357). Addi-
rivative exhibited the highest 6-receptor affin- tional analogs of naltrindole have been re-
ity (Ki = 0.7 versus 6.2 nit4 for BNTX) and ported in the patent literature (see Refs. 219,
selectivity for 6 over p receptors. 220 for reviews).
-
The relative ~osition of the "address" aro- Fragmentation of the indolomorphinan
matic ring in these analogs is important. structure of NTI by removal of the 4,5a-epoxy
Those analogs with the phenyl ring in the and 10-methylene groups resulted in a series
same region of space as the phenyl ring of the of octahydroisoquinolines (see Refs. 219,358).
indole in NTI are active as 6 antagonists (345); The compounds that are the analogs of NTI
a large decrease in activity was observed for with a five-membered ring spacer [SB
indole regioisomers of NTI that have the aro- 205588B (78) and SB 206848 (79, Fig. 7.18)
matic ring in a different relative position are 6-receptor antagonists, whereas com-
(346). Attachment of the phenyl ring directly pounds with a six-membered ring as the
to the 7 position of the morphinan system re- spacer are 6 agonists (see Section 5.10.2 be-
sulted in analogs with decreased 6 receptor low). Interestingly, the related compound (84)
potency and selectivity (347). peripherally se-
lective naltrindole analogs were prepared by
conjugating amino acids to 7'-carboxynaltrin-
dole (74, Fig. 7.18); these derivatives are S-se-
lective antagonists in smooth muscle assays
and 6,-selective antagonists in vivo (348).
Benzylation of the indole nitrogen of NTI, by
contrast, results in a selective and long-lasting
6, antagonist (75) (349). Attachment of fluo-
resceinearnine through a thiourea linkage to
the para position of the benzyl ring of N-ben-
zylnaltindole resulted in an analog with po-
tent antagonist activity in vitro. The inability
to block the fluorescence by several &selective
ligands, including NTI, however, suggested
that the fluorescent analog exhibits high non-
specific binding, possibly because of the high without the pyrrole ring spacer and the cis
lipophilicity of the fluorophore (350). configuration of the ring juncture is an antag-
A number of other naltrindole analogs have onist at p and K receptors (359).
been synthesized, including numerous ones by As discussed earlier in Section 3.3.1, 6-re-
Rice and coworkers. Although most of the ceptor antagonists can decrease the develop-
modifications examined decreased 6 opioid re- ment of tolerance and dependence to mor-
5 Structure-Activity Relationships of Nonpeptide Opioid Ligands
''a
R = CH3 (81) SlOM
R = CPM (82) SlNTX
Figure 7.18. Delta receptor antagonists and agonists related to naltrindole (32).Other &selective
ligands, including naltrindole, are shown in Figs. 7.5 and 7.8.
phine, and therefore there is considerable tached to a pyridine rather than to an indole
interest in the development of compounds ex- ring (230), is a 8-receptor antagonist, but un-
hibiting both 8 antagonist activity and p ago- expectedly also exhibits p opioid agonist activ-
nist activity. Recently, Rice and coworkers ity in the GPI assay (230); interestingly, the
identified naltrindole analogs with p agonist corresponding derivative without the chlorine
activity as well as &receptor antagonist activ- is a preceptor antagonist. Further examina-
ity (230, 360). The 7'-phenoxy naltrindole de- tion of SoRI 9409 in nociceptive assays indi-
rivative exhibits weak (IC,, = 450 nM) ago- cated that the compound is a weak partial ag-
nist activity in the GPI, while retaining potent onist in the high intensity (55°C)tail flick test
6-receptor antagonist activity (K,= 0.25 nM) and weak full agonist in the acetic acid writh-
in the MVD (360). The naltrindole analog ing assay (230,361); studies in vivo were also
SoRI 9409 (SO), bearing a phenyl ring at- consistent with activity as a mixed partial p
Narcotic Analgesics
0CH3
(59) Thebaine endoetheno
derivatives
P
0'
. Figure 7.19. Diels-Alder reaction to
give 6,14-endoetheno derivatives of
thebaine (59)and the structures of bu-
R = CH3 (43) Diprenorphine (86) BU48 prenorphine (85) and BU48 (86). The
structure of diprenorphine (43) is in-
R = C(CH3)3 (85) Buprenorphine cluded for comparison.
gesic, over 1000-fold more potent than mor- unique pharmacological profile offers several
phine, which has been widely used to clinical advantages; it causes less severe respi-
immobilize animals, including large game an- ratory depression than full agonists and has
imals. It exhibits high affinity for all three opi- less abuse potential. It can suppress witli-
oid receptor types (see Table 7.8), and there- drawal symptoms in addicted individuals un-
fore f3H1etorphine has been used as a dergoing withdrawal from opiates and thus
universal tritiated ligand for opioid receptors has been used in the maintenance of these pa-
(see Section 3.2.3.2). The N-cyclopropyl- tients. Because of its partial agonist activity,
methyl 6,14-ethano derivatives diprenorphine however, it can also precipiate withdrawal
(43) and buprenorphine (85, Fig. 7.19), which symptoms in those addicted to opiates. The
differ only in the identity of one of the alkyl 18,19-dehydro derivative of buprenorphine
groups attached to C,, (see Refs. 374, 375 for HS-599 exhibits higher affinity, selectivity,
structural studies of these compounds), ex- and potency at p receptors than does the par-
hibit antagonist and partial agonist activities, ent compound (377). A series of buprenor-
respectively. Diprenorphine also has high af- phine analogs were prepared in which C,, was
finity for all three opioid receptor types and its constrained in a five-membered ring to assess
tritiated form has been used as a universal the influence of the orientation of the C,, hy-
tritiated ligand for opioid receptors (see Sec- droxyl on activity; although the configuration
tion 3.2.3.2); it antagonizes p, 6, and K ligands of this hydroxyl did not affect the binding af-
in the MVD (see Table 7.8). Buprenorphine, finity, it did influence K-receptorefficacy and
which is used clinically, is a potent partial ag- potency (378). One of these novel ring-con-
onist at preceptors with antagonist (or partial strained analogs BU48 (86) exhibits an un-
agonist) activity at K receptors (376).This very usual pattern of pharmacological activity, pro-
lipophilic agent dissociates slowly from opioid ducing 6 opioid receptor-mediated convulsions
receptors, and its complex pharmacology is but not S receptor-mediated antinociception
not completely understood (see Ref. 307). Its (238).
Narcotic Analgesics
R/
3 7 / 6 1,
N $
Q
,: R
/ /
#
\
2'
'0 \ 8
Figure 7.21. Numbering systems for benzo-
3' 9 morphans based on benzomorphan and benzazo-
cine nomenclature (used in Chemical Abstracts).
(92) Benzazocine numbering The more common benzomorphan numbering is
6,7-Benzomorphan numbering (Chemical Abstracts) used in this chapter.
As is the case for morphine derivatives, 14- carbons as that of morphine (i.e., 1R,5R; see
hydroxylation yields potent analogs. The N- Refs. 283, 388), although in some cases the
cyclobutylmethyl derivative with a 14P-hy- dextro isomers retain weak activity.
droxyl group, butorphanol (91) is one of the The majority of the benzomorphan deriva-
mixed agonists/antagonists used clinically in tives were prepared before the classification of
the United States (Table 7.3); in addition to a multiple opioid receptors and were character-
parental formulation, a nasal spray formula- ized by the use of in vivo assays. In the classi-
tion of this drug was introduced in 1992. Be- fication of multiple opioid receptors Martin
cause 4,14-dimethoxy-N-methylmorphinan-6- and coworkers (5, 6) used the benzomorphan
one proved to be a very potent agonist, ketocyclazocine (13,Fig. 7.3) as the prototyp-
Schmidhammer et al. prepared the corre- ical ligand to define K receptors (see Section
sponding N-CPM derivative cyprodime (18, 3.2.1 above), and subsequent characterization
Fig. 7.4) (104). Cyprodime is a pure preceptor indicated that a variety of benzomorphans had
antagonist without any agonist activity (1041, high affinity for K receptors. Several of these
which has enhanced preceptor selectivity benzomorphans, both agonists and antago-
compared to that of naloxone and naltrexone nists, were important ligands in early studies
(see Table 7.8). of K receptors. The selectivity of these benzo-
morphans for K receptors is generally low,
5.6 Benzomorphans
however (see Table 7.10). and much more se-
Further structural simplification by elimina- lective agonists and antagonists [i.e., the aryl-
tion of the C ring of the morphinan structure acetamide agonists (Section 5.9) and antago-
yields the benzomorphans (see Fig. 7.211, nists norBNI and GNTI (Section 5.3.2)l are
which are also known as benzazocines. now available for studying K receptors.
Although the benzomorphan ring system A variety of benzomorphans have been pre-
was first synthesized in 1947 by Barltrop pared with various combinations of alkyl sub-
(386),it was the synthesis of 2,5-dimethylben- stituents (methyl, ethyl, propyl) at the 5 and 9
zomorphan (92, R = R' = CH,) by May and positions (see Refs. 283,388). The alkyl group
Murphy (387) that began the investigation at the 9 positioncan be oriented either a, in
into the synthesis and pharmacology of this which the substituents in the 5 and 9 positions
structural family. As is the case for the mor- are cis, or p, in which these substituents are
phinans, the benzomorphans are prepared trans (see 92, Fig. 7.21). The synthetically mi-
synthetically and therefore are obtained as ra- nor p isomers, which have the opposite stere-
cemic mixtures. A number of these racemates ochemistry from that of the corresponding po-
have been resolved and the activities of the sition in morphine (C,), are more potent than
individual isomers examined (see Refs. 283, the a isomers as antinociceptive agents (see
388: for reviews. also see Ref. 389). The active Refs. 283, 388). Attachment of a 3-alkanone
isomers are the'levo isomers, which have the side chain at the 9/3 position of metazocine
same absolute configuration at the bridgehead (94) yielded a series of potent compounds
374 Narcotic Analgesics
Table 7.10 Opioid Receptor Affinities and Opioid Activity in the GPI and MVD of
Benzomorphan Derivatives and TRK-820"
-
which range from pure agonists to pure antag- fluences the relative agonist versus antagonist
onists, depending on the length of the side activities of these compounds, with the orien-
chain (390). One of these derivatives, Win tation of this group affecting antagonist po-
44,441 (93, Fig. 7.22) is a potent K-receptor tency less than agonist potency (see Ref. 283).
antagonist that has been used to characterize A number of these derivatives exhibit dys-
K receptors; the active isomer is the levo iso- phoric and psychotomimetic effects, limiting
mer, Win 44,441-3 (391). It is also a potent their clinical usefulness. Thus the N-ally1 de-
antagonist at p receptors, however, and does rivative N-allylnormetazocine (SKF 10,047,
not exhibit selectivity for K over p receptors 14, Fig. 7.3) was the prototypical ligand used
(see Table 7.10). In the benzomorphan deriv- by Martin and coworkers (5,6)to characterize
atives introduction of a 9p-hydroxyl in the 9a- u receptors and exhibits psychotomimetic arid
methylbenzomorphans, which corresponds to dysophoric effects in humans (see Ref. 393);
the 14-hydroxyl in oxymorphone and nalox- based on animal studies, these adverse effects
one, does not enhance the agonist activity in appear to reside in the dextro isomer (394).
the N-methyl derivatives (see Ref. 283), but in The N-dimethylallyl derivative pentazocine
benzomorphans with other substituents on (95), however, produces considerably less dys-
the nitrogen [e.g., cyclopropylmethyl or di- phoria than does cyclazocine (96) (307) and is
methylallyl (see below)], a 9P-hydroxyl en- the only benzomorphan used clinically (see
hances antagonist potency 3- to 10-fold (392). Table 7.3). The N-CPM analog cyclazocine
As in the morphinan series, a variety of (96) is a potent mixed opioid agonistlantago-
substituents on the nitrogen have been exam- nist, but its considerable psychotomimetic ef-
ined (see Refs. 283,388,389). Metazocine (941, fects prevent its clinical use (see Ref. 393). As
with an N-methyl substituent analogous to indicated earlier, the 8-keto derivative keto-
morphine, exhibits agonist activity, and the cyclazocine (13)was used in the initial char-
N-phenethyl analog phenazocine shows in- acterization of K receptors by Martin and
creased analgesic potency. Replacing the N- coworkers; the 5-ethyl, 8-keto derivative eth-
methyl with groups such as ally1 or cyclopro- ylketocyclazocine (EKC, 33, Fig. 7.6) has also
pylmethyl (CPM) led to compounds with been used in a variety of studies of K receptors.
antagonist or mixed agonist/antagonist activ- The closely related benzomorphan (-1-brema-
ity (generally agonist activity at K receptors zocine (34, Fig. 7.6) (395) exhibits some pref-
and antagonist activity at other opioid recep- erence for binding to K receptors [Ki (AK)ratio
tors, similar to nalorphine; see Ref. 130). The = 8.2; see Table 7.101 and thus has been used
orientation of the alkyl group at position 9 in- in its tritiated form in radioligand-binding
5 Structure-Activity Relationships of Nonpeptide Opioid Ligands
assays for K receptors (see Section 3.2.3.2); in (see Ref. 130). Although this levo isomer is in-
smooth muscle assays these compounds ex- active at u-PCP receptors, it also produces
hibit mixed agonist/antagonist activity, with dysphoric and psychotomimetic effects in hu-
agonist activity at K receptors and antagonist mans that are antagonized by naloxone, sug-
activity at p and 6 receptors (see Ref. 130). gesting that in this case these adverse effects
A series of analogs with tetrahydrofurfuryl are mediated by K receptors (393). In the case
(396, 397) and furfuryl (398) groups on the of the furyl-substituted analogs the chain
nitrogen have been used as K-receptor ago- length affects the type of activity observed;
nists and antagonists. The tetrahydrofurfuryl thus the N-2-furfuryl derivative exhibits an-
derivative Mr2034 [97, absolute configuration tagonist activity, whereas the longer N-[2-(2-
lR,5R,9R,YS (39711 exhibits high affinity for fury1)ethyll derivative is a potent analgesic
K receptors and has been used as a K agonist, (398). Mr2266 (98),the levo isomer of the 5,9-
but it binds equally well to p opioid receptors diethyl N-2-furfuryl analog, has been used as a
(see Table 7.10); like other benzomorphan K-receptorantagonist in a variety of studies,
mixed agonistlantagonists, it exhibits agonist particularly in early studies when more K-se-
activity at K receptors, but antagonist activity lective antagonists were not available. Similar
at p and 6 receptors in smooth muscle assays to other benzomorphans, its selectivity for K
Narcotic Analgesics
R, R~ (105) Ketobemidone
Figure 7.23. Examples of piper-
idine analgesics with different CH3
substituents on C,. The struc- H
tures of meperidine (7)and fenta- CH2CH=CH2
nyl(8) are shown in Fig. 7.1. H
4-anilidopiperidines (e.g., fentanyl, 8, Fig. ents have been examined (see Ref. 405). Sev-
7.1). Meperidine, a-prodine, and fentanyl are eral phenylalkyl groups [e.g., phenethyl (in
all p opioid agonists (404). All portions of the pheneridine) andp-aminophenethyl (in anile-
piperidine analgesics-the N-substituent, the ridine)] increase potency; N-ally1 or cyclopro-
phenyl ring, the piperidine ring, and the C, pylmethyl groups, however, do not generate
substituent-have been modified. Modifica- antagonists in the meperidine series. Intro-
tions made to the piperidine ring include sub- duction of a 3-methyl group into the piperi- '
stitutions, particularly 3-alkyl substitutions, dine ring yields two isomers, with the 3P-
that affect the preferred conformation of these methyl isomer (with the methyl and phenyl
compounds, introduction of conformational cis) 8-10 times more potent than the a isomer
constraints to fix the conformation of this ring (411); the 3P-methyl group should increase
and the orientation of the 4-aryl ring, and ex- the population of the the axial 4-phenyl con-
pansion or contraction of the piperidine ring. former (283). In conformationally constrained
It is not possible to cover all of the details of tropane derivatives of meperidine (412, 413)
the structure-activity relationships of the pip- the small difference in potency between the a-
eridine analgesics in this chapter, and thus and p-phenyl isomers (the a-phenyl derivative
selected modifications are discussed here. 106 has only 3-4 times the potency of the
More detailed discussions of the SAR are given
in the comprehensive books on opiates (see
Refs. 12, 405) and extensive reviews by Casy
(283, 284, 406); the reviews by Casy discuss
conformational studies in considerable detail.
tionships found in the more rigid opiates, antagonist activity (427).Introduction of a 9P-
particularly with regard to the nitrogen sub- methyl group, which is comparable to the
stituent and its effects on agonist versus an- 3-methyl substituent of the trans-3,4-dimeth-~
tagonist activity, do not hold in the 4-alkyl- ylphenylpiperidines, resulted in compounds
4-arylpiperidine series. In the case of with antagonist activity; the N-phenethyl de-
4-alkylpiperidines, where the axial aryl con- rivative (117) was much more potent than the
formation is preferred; for example, the N-al- N-methyl analog (428). These derivatives stiii
lyl derivative of 4-propyl-4-(m-hydroxyphe- retain rotational freedom around the phenyl-
ny1)piperidine (107, R = allyl), are agonists piperidine bond. Further constraint of the
with only weak antagonist activity (415,416). 5-arylmorphans, with an ether bridge be-
In contrast the 1,3P,4-trimethyl derivative tween the aryl ring and the morphan ring sys-
(109) is an antagonist rather than an agonist tem, yielded compounds with low affinity for
(417).In this series the &methyl group is crit- opioid receptors (see Ref. 2831, but the con-
ical for antagonist activity and an N-ally1 or strained benzofuro[2,3-clpyridin-6-01s(118),
cyclopropylmethyl group did not increase an- in which the phenyl ring is constrained to a
tagonist potency (417). Further variation of dihedral angle of 92" (426), retain p opioid re-
the nitrogen substituent of the trans-3,4-di- ceptor affinity and are potent analgesics (429).
methylphenylpiperidines (108) has resulted In the trans-4a-aryldecahydroisoquinolines
in the synthesis of several potent analogs with (119), in which the aryl ring is constrained to
antagonist activity at both p and K receptors the axial conformation, the N-methyl deriva-
(418-&I), including compounds such as tive has twice the potency of morphine; inter-
LY255582 (110), which has potent anorectant estingly, although the N-methyl derivative is
activity, and the peripherally selective antag- selective for p receptors, the N-CPM deriva-
onist LY246736 (111, now designated AD tive exhibits a slight preference for K receptors
8-2698 or alvimopan) (420), which is under- (430).
going clinical trials for the treatment of gas- Active analogs were also prepared by shift-
trointestinal motility disorders (see Ref. 38 ing the alkyl and aryl substituents to the 3
and Section 2.2.2.1). Potent antagonists that position. All active derivatives of the &methyl-
exhibit some selectivity for K receptors 3-arylpiperines contain the m-hydroxyl on the
[LY227053 (112), LY253886 (113)(4211, and phenyl ring (283). Although the N-methyl de-
(115) (422)], along with p-selective antago- rivatives are weak analgesics, derivatives with
nists (114) (423), have also been identified. an N-arylalkyl substituent are significantly
Conformationally constrained derivatives more potent (12). Some derivatives with N-
of the 4-alkyl-4-arylpiperidines, in which the allyl or N-CPM groups behave as antagonists,
aryl ring is constrained into both axial and similar to the morphinan series. Ring con-
equatorial conformations, have been pre- tracted and expanded analogs have also been
pared. Arylmorphans (116) are constrained prepared, including the mixed agonistlantag-
analogs in which the aryl ring is $quatorial onist meptazinol (120). The pharmacological
(see Ref. 283,406). The N-methyl derivative is effects of this compound are somewhat un-
equipotent with morphine after subcutaneous usual (see Ref. 431) and may involve both opi-
administration to mice (424); the configura- oid and cholinergic mechanisms (432). It has
tion of the more potent dextro enantiomer of
this analgesic [the 1S,5R isomer (42511 is re-
lated stereochemically to the more potent en-
antiomer of 4-arylpiperidines such as a- and
p-prodine (101 and 102, respectively) rather
than to the rigid benzomorphan (-)-metazo-
cine (283). The receptor affinities of both iso-
mers for different receptor types have also
been examined (426). Consistent with the ste- C H ~
reochemical relationships, the N-ally1 and N-
CPM derivatives are agonists with little if any (120) Meptazinol
Narcotic Analgesics
/N
H3C
- -
MA0
0
(121) MPPP (122) MPTP (123) MPP+
been proposed that the opioid actions of ethyl derivative, (CH,),NHC,H, (436)], the
meptazinol are mediated by p1 receptors resulting compound can be more than 1000-fold
(431). more potent than meperidine.
A variety of C, alkylated derivatives of the
5.7.2 4-Arylpiperidines with an Oxygen reversed esters of meperidine were examined
Substituent at C,. Reversal of the ester in me- (see Ref. 405). Incorporation of a methyl group
peridine gives MPPP (N-methyl-4-phenyl-4- at C, yields a- and p-prodine (101 and 102,
propionoxypiperidine, 121) and increases an- Fig. 7.23) (437). The P isomer (102) has five
algesic activity 5- to 10-fold (see Ref. 405). times the potency of the a isomer. X-ray crys-
First described in 1943 (433), this compound tallography and NMR indicate that the pre-
took on new significance in the late 1970s ferred conformation of the prodines is the
when the drug began appearing as a "designer chair form, with the phenyl ring equatorial
drug (a compound with a minor structural
JJ
see Ref. 283); thus substitution on the 4R side propose an alternative mode of interaction to
interferes with drug-receptor interactions, explain how a-allylprodine (103) and other
whereas substituents on the 4 s side are not phenylpiperidines in which the phenyl ring is
involved in interaction with the receptor and equatorial can interact with the same recep-
are well tolerated. A variety of other methyl- tors as morphine and other rigid opiates
and dimethyl-substituted derivatives of the where the phenyl ring is axial. In the initial
reversed esters have been prepared, resulting bimodal binding model proposed by Portogh- '
in compounds with a wide range of potencies ese (82), the m i n e of different opiates was
and with different orientations of the phenyl postulated to interact with a common anionic
ring, depending on the substitution pattern site and the rest of the molecule then would
(see Refs. 283, 405, 440); positions that toler- pivot around the nitrogen to bind in one of two
ate methyl substitution are indicated in struc- possible orientations. The bimodal binding
ture (124) (Fig. 7.26) by an asterisk. Very po- model was subsequently modified to include a
tent analogs have been prepared by bridging second lipophilic site (443), where the equato-
the 3 and 5 positions with a trimethylene rial phenyl ring of phenylpiperidines such as
chain (125, 126) (441). Both the a isomer a-allylprodine (103)was proposed to interact.
A substantial reduction in analgesic po-
tency was also observed for the rn-hydroxyl
derivative of the 2a-methyl reversed ester in
which the preferred conformation of the phe-
nyl ring is axial (444), suggesting that there
can also be differences in receptor interactions
for the axial aryl moiety in this derivative and
in morphine. This in turn may be attributable
to differences in the relative orientation of the
aryl rings in the rigid morphine versus the
phenylpiperidine derivatives in which the aryl
ring is free to rotate.
(131) Carfentanil
strained analogs of fentanyl have been pre- tional conformationally constrained 1,5-ben-
pared [see the previous edition of this chapter zoxazepine derivatives constrained through
(286) for a detailed review]. Generally, confor- cyclization between the ortho position of the
mationally constrained derivatives in which phenyl ring and the 3 position (130) are also
the phenyl is constrained in the P orientation potent analgesics (450).
are inactive (see Ref. 448), but in conforma- A large number of modifications have been
tionally constrained tropane analogs of fenta- made to fentanyl (see Refs. 284,406,445,448).
nyl the p analog (128, Fig. 7.27), in which the Unlike the phenylpiperidines a second sub-
anilido group is equatorial, is more potent stituent at C, is not required, but the addition
than the a isomer (127), in which the propa- of a polar carbon substituent (C0,CH3 or
nanilido moiety is pseudoequatorial (449). CH,0CH3) at this position to give, for exam-
The high potency of the conformationally con- ple, carfentanil (131),enhances potency 27-
strained spirane derivative (129) provides ev- fold over that of fentanyl and results in com-
idence that the phenyl ring may be oriented a pounds 7800 times the potency of morphine
when fentanyl binds to opioid receptors. Addi- (451). In the case of the anilido side chain, the
5 Structure-Activity Relationships of Nonpeptide Opioid Ligands
(134) a-Methylfentanyl
R= (135) Sufentanil
N-N
If \.
\\
R= / N,~H2CH3
(136) Alfentanil
R = C02CH3
R' = C02CH3 (137) Remifentanil
(138) Ohrnfentanyl
tion of the unprotonated amine would be supporting this conclusion. A series of cis/
available to penetrate the blood-brain barrier trans pairs of 3-methylfentanyl analogs was
(406). Other heterocyclic substitutions have reported (460),and in some cases in which the
been made on the piperidino chain (450), re- anilido ring had an ortho substituent, the
sulting in some compounds with less respira- trans isomers were more potent than the cis
tory depression. Incorporation of an ester isomers. This may be due to steric hindrance
functionality into the piperidino substituent between the ortho substituent and the
to give remifentanil (137) (Table 7.1) results 3-methyl group in the cis isomers interfering
in a compound that is 30-fold more potent with the phenyl ring adopting an a orientation
than alfentanil, and that has very rapid onset (see above) (284). As discussed earlier, the
(1.6 min) and offset (5.4 min), which is inde-
conformationally constrained 1,5-benzoxaz-
pendent of the duration of administration
epine derivatives linked through the ortho po-
(455, 456). This is due to the rapid hydrolysis
sition on the phenyl ring and the 3-methyl
by esterases to the acid derivative, which has
very low analgesic activity and is rapidly substituent (130)are active in antinociceptive
excreted. assays (450).
Introduction of a methyl substituent in the Recently, two models of cis-(+)-3-methyl-
3 position of the piperidine ring results in fentanyl (139) and other 4-anilidopiperidines
chiral comvounds. The racemic cis derivative docked to p opioid receptors were described
of fentanyl is more potent than the trans de- (182, 187). The binding mode for cis-3-meth-'
rivative (457). For the cis racemate the dextro ylfentanyl in the two models is different,
isomer has 120 times the potency of the (-)- which has been attributed to different con-
isomer; the absolute stereochemistry of the formations of this compound used for dock-
(+)-cis isomer is 3R,4S (458). The analog of ing to the receptor (187). Comparison of cis-
cis-3-methylfentanyl with a hydroxyl group on 3-methylfentanyl to N-phenethylnormor-
the N-phenethyl chain ohmfentanyl (138) is phine suggested that there was considerable
extremely potent (7000 times morphine) in overlap in the region of the receptor occu-
antinociceptive assays and exhibits remark- pied by the N-phenethyl groups, but that
able selectivity for p receptors [27,000-fold there was no overlap in the region in the
selectivity for p versus 6 receptors (45911. receptor binding pocket occupied by the N-
Comparisons of the 3R,4S isomer of cis-3- phenyl ring of cis-3-methylfentanyl and the
methylfentanyl(139) and the 3S,4S isomer of phenol ring of the morphine analog (see Fig.
p-prodine (102, Fig. 7.28) suggest that al- 7.5 in Ref. 187).
though these compounds are both p agonists, Affinity label derivatives of fentanyl and
they represent different classes of ligands that (+)-cis-3-methylfentanylFIT (198, 461) and
have different modes of interaction with opi- SUPERFIT (4581, which have an isothiocya-
oid receptors (284) (there is an error in the nate group on the phenyl ring of the piperidino
stereochemistry at position 3 of p-prodine in substituent, have been prepared. Unlike the
Ref. 284, p. 504). Fentanyl derivatives con- reversible parent compounds, which are p
taining propyl and ally1 substituents at C3 ex- agonists, these affinity labels irreversibly bind
hibit significant differences in SAR from the to 6 receptors. These compounds are discussed
corresponding prodine analogs (see Ref. 284), further in Section 5.11 on affinity labels.
5 Structure-Activity Relationships of Nonpeptide Opioid Ligands
the (+) isomer. In the case of isomethadone, but activity can be restored by acetylation
which is slightly less potent than methadone, (470); the resulting acetates are more potent
the more active (-) enantiomer has the S con- than the parent ketones. The more active
figuration and has 40 times the potency of the methadol isomers, (-)-a- and (-)+methado1
(+) isomer (see Refs. 284,463). NMR, circular [with absolute configurations 3S,6S and
dichroism (464), and molecular modeling 3S,6R, respectively (47111, both have the same
studies (465) suggest that isomethadone may 3 s configuration, suggesting that the stereo-'
be less flexible than methadone because of the chemistry around the alcohol is more impor-
proximity of the methyl group to the phenyl tant in these derivatives. The more active
rings in isomethadone. 3S,6R isomers of p-methadol and P-acetyl-
These flexible analgesics exhibit their own methadol are derived from the more active
distinct SAR (see Refs. 284, 463, 466). A vari- R-(-) enantiomer of methadone. An interest-
ety of nitrogen substituents have been exam- ing reversal in enantioselectivity occurs in the
ined, and whereas larger acyclic groups mark- a series; although the more potent 3S,6S iso-
edly decrease or abolish activity, compounds mer of a-methadol is derived from the less ac-
containing a cyclic substituent, such as pyrro- tive S-(+) isomer of methadone, acetylation
lidine, piperidine, or morpholine, on the nitro- reverses the enantioselectivity, so that the
gen retain activity. The two-carbon chain more potent isomer is the 3R,6R-(+) isomer
length between the quaternary carbon and the (471). leuo-a-Acetylmethadol (LAAM, 143)
nitrogen is the optimal length, and lengthen- has a longer duration than methadone, requir-
ing the chain abolishes activity. Removal of ing dosing only once every 3 days, and is being
the methyl group from the alkyl chain to give used in the United States for maintenance of
the achiral compound normethadone de- individuals addicted to narcotics. Like metha-
creases potency 6- to 10-fold relative to that of done, the major route of metabolism of this
methadone and isomethadone, respectively. compound is N-demethylation. In the case of
Introduction of a second methyl group at posi- the methadols and acetylmethadols, these sec-
tion 5 yields erythro- and threo-5-methyl- ondary amines-derivatives are active, with po-
methadone (467). The erythro form (141) has tencies similar to those of the parent tertiary
five times the potency of methadone, whereas mines, and probably contribute to the activ-
the threo form is inactive. The more active leuo ity and longer duration of action of LAAM (see
isomer of erythro-5-methylmethadone has the Ref. 463). The ketone of methadone has also
5S,6S configuration (468). Interestingly, one been replaced with a variety of other func-
of the isomers of the inactive threo racemate, tional groups, including esters, ethers, and
with the 5S,6R configuration, combines the amides (see Ref. 463). In the acyloxy series
configurations found in the more active enan- propionyloxyisomethadone shows significant
tiomers of methadone and isomethadone activity (472); further modification of this
(467). NMR analysis suggests that the erythro compound yields propoxyphene (see below).
form exhibits greater conformational flexibil- Most modifications of the phenyls in the
ity than that of the threo form. The authors diphenyl fragment of methadone result in sub-
suggested that the marked difference in anal- stantial loss of analgesic activity (see Ref.
gesic activity may be attributable to different 463). In normethadone analogs replacement
conformational preferences of the threo and of one of the phenyl rings by a benzyl group
erythro forms (467), and that the conforma- abolishes activity (473), but in the isometha-
tion observed for erythro-5-methylmethadone done analog with a propionoxy group in place
in the solid state (469) is the active conforma- of the ketone this modification results in
tion of erythro-5-methylmethadone, as well as propoxyphene (10, Fig. 7.1), which has modest
for (-)-methadone and (-)-isornethadone. analgesic activity (approximately 1/10 the po-
The ketone side chain is also important for tency of methadone 472; see Table 7.1). The
activity (see Ref. 463) and changing the length replacement of one of the phenyl rings by
of this chain decreases activity. Reduction of a benzyl group introduces a second chiral cen-
the ketone to the two possible alcohols, a- and ter into this molecule. The active (+) isomer
P-methadol (142 and 144) decreases activity, has the 2S,3R stereochemistry, which is the
5 Structure-Activity Relationships of Nonpeptide Opioid Ligands 387
Table 7.11 Opioid Receptor Affinities, K Selectivity, and Analgesic Activity of K Opioid
Ligandsa
Receptor Affinity Analgesic Activity MPE,,
(Ki, nM) Ki (mglkg?
Ratio
Compound K" cL" P ~ K i.v. p.0. References(s)
Tifluadom (147)
U50,488 (35)
(- ) isomer
( +) isomer
U69,593 (36)
Spiradoline
(U62,066, 148)
(- ) isomer
(+) isomer
PD 117302 (149)
(- ) isomer
(-1 CI-977 (37)
DuP 747 (150)
S,S isomer
Niravoline
(RU 51599,151)
BRL 5253711 (155)
BRL 52656A (156)
GR 89696 (158)
R-84760 (159)
Apadoline
(RP 60180,160)
ICI 197067 (161)
ICI 199441 (162)
HZ2 (163)
"Data from Ref. 118 except where otherwise indicated.
*Rat paw pressure test, MPE,,, is the dose required to produce 50% of the maximum possible analgesic effect, except
where otherwise indicated.
'Inhibition of [3H]U69,593binding in guinea pig forebrain, except where otherwise noted.
dInhibition of [3HlDAMG0 binding in guinea pig forebrain, except where otherwise noted.
'U50,488 Ki = 6 and 825 nMin K and p assays, respectively (494).
fMouse phenylquinonewrithingassay ED,, S.C.andp.~.,respectively. ED,, for U50,488 = 1.2 mgkg S.C.and 13 mglkgp.0.
(494);0.47 mgkg S.C.and 27 mgkg p.0. (506).
SED,, mouse tail flick assay. U50,488 ED,, = 1.9 mgkg s.c.
'Binding determined in monkey cortical membranes.
'Mouse acetylcholine writhing assays ED,, s.c. ED,, for U50,488 = 0.41 mgkg s.c.
JIC5, values for U50,488 = 7.59 and 571 nM for K and p binding, respectively.
kInhibition of l3H1EKC binding in guinea pig cerebellum.
'Ki (6) = 1.6 nM (l3H1EKC binding in NG 108-15 cells), Ki ratio (61~)= 2.9.
"IC,, values for inhibition of [3Hlbremazocinebinding in guinea pig brain minus cerebellum and [3Hlnaloxonebinding
in rat brain for K and preceptors, respectively. U50,488 IC5, = 95.5 and 14,200 nM, respectively.
"ED,, in mouse acetic acid abdominal constriction assay. U50,488 ED,, = 1.1 mglkg s.c.
"Inhibition of [3HlCI-977 and [3Hlnaloxonebinding in rat brain membranes for K and preceptors, respectively.
its high K selectivity in binding assays (see Ta- finity for K receptors (482) (see Table 7.11).
ble 7.11) and produces analgesia through K re- The absolute stereochemistry of the levo iso-
ceptors in vivo (479,480). mer was determined by X-ray crystallography
U50,488, which was initially characterized of an intermediate to be 1S,2S (481). The pro-
as the racemic mixture, was resolved (481), tective effects of U50,488 against the tempo-
and the levo isomer found to have greater af- rary bilateral carotid occlusion model of cere-
5 Structure-Activity Relationships of Nonpeptide Opioid Ligands
(148) Spiradoline
((+I U62,066)
(151) Niravoline
(RU 51 599)
Figure 7.30. K-Receptor selective agonists. The structures of U50,488 (361, U69,593 (36),and
CI-977 (37) are given in Fig. 7.6.
bra1 ischemia also reside predominantly in the displays preceptor selectivity (see Table 7.11)
levo isomer (483), consistent with the hypoth- (486). Like U50,488 spiradoline exhibits neu-
esis that K receptors may be involved in these roprotective effects and is even more effective
protective effects. than U50,488 in reducing postischemic necro-
Introduction of a spiro ether group on the sis of the vulnerable hippocampal CAI neu-
cyclohexane ring was one of the earliest rons (487). Further examination of C, andlor.
modifications to U50,488 reported, giving (-)- C, methyl ether and spiro tetrahydrofuran
U69,593 (36, Fig. 7.6) (116) and spiradoline substituents indicated that optimal K-receptor
(U62,066, 148, Fig. 7.30) (484). U69,593 ex- selectivity was obtained when the oxygen was
hibits improved K-receptor selectivity over in the equatorial (PI orientation at C, of the
U50,488 (116) (see Table 7.11). The tritiated ring (117).
form of U69,593 was prepared by catalytic tri- From the initial compounds it was clear
tiation of the aromatic chlorines of U62,066 that the spacing between the amide and the
(116), and this highly selective tritiated K li- aromatic ring system in the N-methylamide
gand has been used extensively in radioligand- side chain is critical for K-receptor activity.
binding assays (see Section 3.2.3.2). The X-ray Whereas phenylacetamide derivatives (i.e.,
structure of U69,593 has been reported [see -NCH,COCH&) such as U50,488 and its an-
Ref. 485; however, as noted by Rees (474), alogs exhibit K activity, benzamide derivatives
there is a discrepancy between the X-ray (i.e., -NHCOAr and -NCH,COAr) exhibit mor-
structure as drawn in this paper (5R,7S,8S) phine-like effects (115,488). In both series N-
and that indicated in the title (5S,7S,8S), methyl substitution increases potency over
which has led to some confusion concerning the unsubstituted secondary amides (115,
the absolute stereochemistry of U69,5931. As 489).
is the case with U50,488, the (-) isomer of Numerous variations have been examined
spiradoline is much more potent (>30-fold) in all portions of the U50,488 and spiradoline
than the (+) isomer in analgesic assays (484) structures [see Refs. 287,288,474 and the pre-
and possesses much greater affinity for K re- vious edition of this chapter (286) for detailed
ceptors (see Table 7.11) (486); the (+) isomer reviews]. Figure 7.31 shows the general struc-
Narcotic Analgesics
oral / Block
metabolism
activity
Enhance agonist activity
Figure 7.31. (a)General structure (154) common to most of the analogs of U50,488;(b) summary of
SAR for analgesic activity of U50,488(35) (from Ref. 287).
C")
(158) (R,S) GR 89696
C")
(R) GR 103545
Figure 7.32. K-Receptor selective agonists with alternative ring systems and open chain analogs of
the 1,2-diaminesubstituted cyclohexane ring.
stituted ethyl chain results in compounds with that only those compounds capable of adopt-
weak K-receptor affinity, a substituent a to the ing a conformation similar to that of U50,488
amide functionality yields compounds with were K agonists. The most potent analogs were
high affinity for K receptors (515,516). Exam- the isopropyl (ICI 197067, 161) and phenyl
ination of both isomers indicated that a derivatives (ICI 199441, 162, Fig. 7.32). Fluo-
methyl group at the 1 position with the S con- rescent derivatives of ICI 199441 have been
figuration was active and had reasonable af- prepared by attachment of fluorescein isothio-
finity for K receptors, whereas the isomer with cyanate by a spacer to the meta or para posi-
the R configuration at this position was inac- tion of the central phenyl ring (517). Attach-
tive (516). Conformational analysis suggested ment of an acidic functionality to the meta
5 Structure-Activity Relationships of Nonpeptide Opioid Ligands 393
position of the central phenyl ring of ICI ported to have potent antinociceptive activity
199441 gave ICI 204448, which has limited and, like other K agonists, to be active against
ability to penetrate the CNS (518) (see below). inflammatory pain (522). The quaternary
U50,488 and its analogs are structurally methiodide derivative retains high K-receptor
distinct from the benzomorphan K opiates and affinity, and thus may be useful as a periph-
are considerably more flexible than are the eral K agonist (523).
rigid alkaloids. This raises questions concern- Several of the K-selective ligands have un-
ing their bioactive conformation and how they dergone testing in humans (524-531; see Ref.
bind to opioid receptors compared to the more 475 for areview). Side effects associated with K
rigid alkaloid opiates. Early studies (519-521) receptors include sedation and dysphoric ef- .
attempted to identify possible bioactive con- fects (146). The dysphoric effects are of partic-
formations of U50,488 and its congeners by ular concern and have severely limited the
conformational analysis of the ligands and su- usefulness of the majority of centrally acting K
perimposition of the arylacetamides with ben- agents. Many of the older nonselective com-
zomorphans. With the cloning of opioid recep- pounds possessed high affinity for u and PCP
tors and development of computational sites, raising the possibility that these sites
models of these receptors (see Section 3.2.4.2 might contribute to the dysphoric effects. The
above), several groups (179, 182, 183, 186) benzomorphan Mr2034 (971, which is inactive
have proposed possible binding modes for the at these sites (3931, and the K-selective agonist
arylacetamides docked to the K opioid recep- spiradoline (148) also produce naloxone-
tor. Although all of the models of the arylacet- sensitive dysphoric effects (5321, however, in-
amides docked to K opioid receptors assumed dicating that K receptors mediate psychotomi-
an interaction of the protonated m i n e of the metic effects. Clinical trials of spiradoline for
ligand with Asp13', there are significant differ- the treatment of pain have been discontinued
ences in other proposed receptor-ligand inter- (475). Initial evaluation in humans of enado-
actions in these models (see Ref. 186 for a de- line (CI-977,37, Fig. 7.6) for its diuretic effect
tailed comparison of the models). These found that the dysphoric effects attributable
results illustrate the complexity of modeling to the drug were minimal and not considered
these more flexible ligands and determining clinically significant (5251, although in a sub-
how they interact with their receptors. sequent study of its use in postsurgical pain
HZ2 (163), which is structurally unrelated adverse neuropsychiatric events led to early
to the arylacetamide K-selective agonists, ex- termination of the study (527). Enadoline has,
however, been granted orphan drug status for
the treatment of severe head injuries (475).
Apadoline (RP 60180,160) has been evaluated
in an experimental human pain model and re-
ported to cause fewer side effects than the clin-
ically used agent pentazocine (529). Nirav-
oline (151) was examined for its aquaretic
effect in patients with cirrhosis; the highest
doses examined induced personality disorders,
but moderate doses produced the desired
aquaretic effect and were well tolerated (531).
However, clinical development of this agent
has been discontinued (see Ref. 475). Clinical
investigation of several centrally acting K-se-
hibits reasonable K-receptor affinity (Ki = 15 lective agents (apadoline, DuP 747, enadoline,
nM) with low affinity for p receptors (Ki > and the 4,5a-epoxyrnorphinan TRK-820) has
1000 nM) (521). NMR and molecular modeling continued (288).
studies were performed to compare this novel
bicyclononanone to the arylacetamides and 5.9.2 Peripherally Acting Agonists. Concern
ketocyclazocine (521). The compound is re- over centrally mediated side effects has
Narcotic Analgesics
V- OH
(167) EMD 60400 (168) Asimadoline
(EMD 61753)
prompted attempts to develop peripherally se- amide side chain phenyl ring and found that
lective K opioid agonists. Peripheral opioid re- the 4-trifluoromethyl group resulted in an an-
ceptors can mediate analgesia, particularly in alog with high K-receptor aMinity and less cen-
cases of inflammation (see Refs. 72-74 for re- tral activity compared to that of the parent ICI
views). To limit penetration of the blood-brain 199441 (536); incorporation of additional
barrier (BBB), generally polar andlor charged functionalities into the central phenyl ring
functionalities have been introduced into the (e.g., 166) were examined to further restrict
compounds (see Ref. 534 for a review). Several the compounds to the periphery (537). Re-
modifications to the K-selective agonist ICI searchers at Merck prepared EMD 60400
199441 (162, Fig. 7.32) have been reported to (167) (538) by introducing an amino substitu-
restrict the compounds, access to the CNS. In ent on the phenyl ring of the phenylacetamide
ICI 204448 (164, Fig. 7.33) an acid functional- side chain and a 3-hydroxyl group on the pyr-
ity was introduced on the central phenyl ring rolidine ring; researchers at Glaxo prepared
(518), and Portoghese and coworkers pre- analogs of GR 103545 with reduced penetra-
pared aspartic acid conjugates of ICI 199441 tion of the BBB by the use of a similar ap-
(165) (535) to decrease penetration into the proach (513). In the case of BRL 52974 (169)
CNS. Investigators at the Adolor Corporation (539), an imidazole ring was attached to the
tested various substituents on the phenylacet- piperidine ring to increase hydrophilicity.
5 Structure-Activity Relationships of Nonpeptide Opioid Ligands 395
In asimadoline (EMD 61753,168) (540) an been investigated clinically for the treatment
additional phenyl ring was attached a to the of digestive disorders characterized by abdom-
amide on the phenylacetamide side chain, inal pain, namely, dyspepsia and irritable
which increases lipophilicity. This compound bowel syndrome (see Ref. 548 for a review). In
is also a peripherally selective K agonist (541); Phase 111111 studies for both disease states
studies in knockout mice lacking P-glycopro- (549-551) significant improvement of symp-
tein indicate that asmidaloline is transported toms in the patients treated with fedotozide
by P-glycoprotein, and that transport by this compared to placebo was reported. Fedotozine
protein limits the compound's penetration of also relieves visceral hypersensitivity to gas-
the BBB (542). As noted by Barber et al. (541), tric and colonic distention, which are often ob-
served in dyspepsia and irritable bowel syn-
amphiphilic structures such as asimadoline
drome, respectively (552, 553). Although
generally have greater oral activity than hy-
clinical trials of fedotozine have been discon-
drophilic compounds; consistent with this
tinued (see Ref. 475), peripherally selective K
they found that asimadoline exhibits much agonists remain potentially important thera-
greater potency by the oral route than did ICI peutic agents for treatment of these digestive
204448 (in pressure nociception in inflamma- disorders.
tory hyperalgesia: ID,, for asimadoline = 0.2
mgkg S.C.and 3.1 mg/kg p.0. versus ID,, for
5.1 0 Delta-Selective Agonists
ICI 204448 = 0.8 mgkg s.c. and 30 mgkg p.0.).
Oral absorption of asimadoline was not signif-
icantly altered in the P-glycoprotein knockout 5.10.1 BW373U86, SNC 80, and Ana-
mice, indicating that the intestinal P-glyco- logs. Initially, all of the &selective agonists
protein did not impede absorption after oral were peptide derivatives. The first nonpeptide
administration (542). Unexpectedly, increases agonist selective for 6 receptors, BW373U86
in pain were reported in clinical trials of this (26, Fig. 7.5), was discovered by screening
compound in patients after knee surgery (554). The pharmacology of this compound
(543). Subsequent investigation of this com- has been examined in considerable detail (see
pound in inflammation in the rat found that, Refs. 219, 221 for reviews). This compound
although the analgesic effects of asimadoline has only modest selectivity for 6 receptors in
were K opioid receptor mediated, the adverse binding assays (see Table 7.12). In antindci-
hyperalgesic and proinflammatory effects ob- ceptive assays in mice it appears to function as
served were not mediated by opioid receptors a partial agonist at both 6 and preceptors. The
(543). activity of BW373U86 is highly dependent on
Fedotozine (Jo-1196, 170), which is struc- the route of administration, with effects at the
turally related to the acyclic K agonists, has in spinal level mediated by 6 receptors and su-
vivo antinociceptive effects on duodenal pain praspinal effects involving interactions with p
that appear to be mediated by peripheral K receptors (555). In monkeys BW373U86 did
opioid receptors, but the compound is inactive not produce antinociceptive effects in the
after central administration (544). In binding warm-water tail-withdrawal assay after sub-
assays (in dog myenteric plexus), however, cutaneous administration (556). This com-
this compound exhibits similar affinity (Ki = pound also produces convulsant effects in both
0.3-0.8 f l )for all three types of opioid recep- mice and monkeys (556-558), which appeared
tors (545). Unlike other K agonists, fedotozine to be mediated by 6 receptors.
does not induce diuresis after either S.C. or in- BW373U86 is a racemic mixture and this
tracerebroventricular i c v administration could complicate its pharmacological profile.
(546). Fedotozine also fails to substitute for Therefore Rice and coworkers undertook the
either U50,488 or morphine in animals synthesis and characterization of isomers of
trained to discriminate these drugs (547). The BW373U86 (107).The isomers with the R con-
main effects demonstrated for fedotozine have figuration at the benzylic carbon exhibited
been in the gastrointestinal tract (see Ref. 548 greater affinity for S receptors than did the
for a detailed review of the pharmacology of isomers with the S configuration. One com-
fedotozine), and therefore this compound has pound, SNC 80 (27, Fig. 7 . Q the methyl ether
396 Narcotic Analgesics
Table 7.12 Opioid Receptor Affinities, 6 Selectivity, and Opioid Activity in the MVD
of 6 Opioid Agonistsa
*IC,, or Ki (nM) Ic50 Ic50
Ratio (nM)
Agonist 6 P K CL/6 MVD Reference
BW373U86 (26)
SNC 80 (27)
172
(-) SL-3111(174)
(-) 175
176
177
(?) TAN 67 (28)
(SB205607)
(-1 TAN 67
(SB213698)
(+) TAN 67
(SB213697)
(-) 178
"Data for BW373U86, SNC 80, and TAN 67 fiwm Table 7.8 are included for comparison.
'IC,, in the GPI are 143 and 5500 nMfor BW373U86 and SNC 80, respectively.
'IC,, in the GPI is 26,500 n M
i
1;i 5 Structure-Activity Relationships of Nonpeptide Opioid Ligands 397
i
tified derivatives (e.g., 172) that exhibited ex- diarylaminotropane derivatives (176) were
tremely high selectivity for 6 receptors (see reported by two groups (574, 575), with the
Table 7.12) and were considerably more stable unsubstituted derivative exhibiting high S-re-
to degradation by rat liver microsomes than ceptor affinity and exceptional 6 selectivity
was SNC 80 (568). Interestingly, the analog (see Table 7.12) (575); the N-ally1 and related
DPI 2505 (173) in which the 2-methyl group is derivatives exhibit decreased efficacy and an-
shifted to the 3 position has been reported to tagonist activity in the [35SlGTPyS assay
be an antagonist (228). The basic N4 nitrogen (574). In an alternative approach chosen to
is critical for 6 receptor binding, and opening yield achiral 6 agonists without the compli-
the piperizine ring also results in large de- cated stereochemistry of SNC 80, researchers
creases (>60-fold) in &receptor affinity (563). at R. W. Johnson also prepared a series of
A variety of alkyl substituents on N4 of the piperazinyl benzamidines (576); the highest
piperazine ring are tolerated by the 6 receptor affinity ligand (177) exhibited affinity and se-
(564, 566), but SNC 80 analogs containing a lectivity similar to SNC 80 (see Table 7.12). A
saturated alkyl group exhibit decreased effi- number of other SNC 80 analogs described in
cacy compared to SNC 80 in the [35S]GTPyS the patent literature were reviewed by Dondio
assay; the N-cyclopropylmethyl analog is also (219, 220).
a partial agonist (564). Thus the SAR of the One question is how SNC 80 interacts with
nitrogen substituent of SNC 80 for S-receptor 6 opioid receptors compared to the more clas-
affinity is distinctly different from the SAR of sical morphinan ligands. Dondio et al. (577)
this group in the morphinans for interaction compared SNC 80 to the S antagonist SB
with p receptors. 205588 (78, Section 5.3.3). In this model the
Hruby and coworkers designed a series of basic N4 nitrogen and the oxygenated phenyl
piperazine derivatives as peptidomimetic ana- ring of SNC 80 were superimposed on the cor-
logs of the 6 selective peptide [(2S,3R)- responding groups in SB 205588, with the cen-
TmtllDPDPE (Tmt = 3,2',6'-trimethylty- troid of the second phenyl ring of SNC 80 over-
rosine) (569, 5701, with SL-3111 (174) (see lapped with the pyrrole ring of SB 205588.
Table 7.12) as the lead compound. Although Based on this model, it was hypothesized that
SL-3111 is structurally similar to SNC 80, the the amide group of SNC 80 might function as a
large decrease in S-receptor affinity upon nonaromatic 6 "address" and thus be resp9n-
methylation of the phenol and the high afin- sible for the 6 selectivity of SNC 80. Loew and
ity of SL-3111 for the W284Ll mutated hu- coworkers in their pharmacophoric model for
man 6 receptor is in contrast to the results for a wide range of 6 receptor ligands also overlaid
SNC 80 and more closely parallel those for the the basic N4 nitrogen and oxygenated phenyl
peptide p-C1-DPDPE, suggesting that the ring of SNC 80 with the corresponding groups
binding profile of SL-3111 is more similar to in the epoxymorphinans, with the amide occu-
that of the peptide than to SNC 80 (569). pying the third site in the three-point model
A series of 4-aminopiperidine derivatives for &selective opioid recognition (578). Coop
was designed by researchers at the Research and Jacobson, however, developed a four-
Triangle Institute by transposition of the N4 point pharmacophoric model based on a series
of the piperazine ring and the benzylic carbon of 4,5a-epoxymorphinans with high affinity
(571-573), with the cis (3S,4R) isomer (175) for 6 receptors and found that SNC 80 did not
exhibiting the highest affinity. This transposi- fit the model, suggesting that SNC 80 does not
tion decreased 6-receptor affinity while in- bind to the 6 receptor in the same orientation
creasing preceptor affinity, such that the pi- as oxymorphindole (579).
peridines exhibited somewhat lower 6 Mutational analysis of S opioid receptors
selectivity than the piperazine derivatives (see has found that three residues, Trp284at the
z
Table 7.12). A large series of these 4-aminopi- top of TM6 and Val g6 and in the third
peridine analogs was also prepared by re- extracellular loop, are crucial for the S-recep-
searchers at R. W. Johnson Pharmaceutical tor affinity of SNC 80 as well as other 6-recep-
Institute and subjected to CoMFA analysis tor agonists (166). Computational models of
(188). Recently, a series of constrained 4- BW373U86 (182) and Caminopiperidine ana-
5 Structure-Activity Relationships of Nonpeptide Opioid Ligands 399
logs of SNC 80 (188) docked to the 6 receptor was active in the tail flick assay in diabetic
have been described. In the models the basic mice (58211;antagonism by BNTX but not nal-
nitrogen and oxygenated phenyl ring of these triben suggested that TAN 67 produced its an-
compounds occupy similar regions in the re- tinociceptive effects through 6, receptors
ceptors as the corresponding groups in the ep- (583).
oxymorphinans, although there are some dif- The pharmacological effects of the two en-
ferences in the exact location of the two types antiomers of TAN 67 are distinctly different
of compounds and orientation of their phenyl (see Ref. 584 for a review). (-) TAN 67 (also
rings (see Ref. 182). In both models the benz- named SB 213698 by the Italian group), which
amide ring occupies a region at the TMIextra- has the same absolute configuration as that of
cellular interface, and interacts with one or morphine, shows high affinity and selectivity
more of the residues identified as critical from for 6 receptors (Table 7.121, and is a full ago-
mutagenesis studies. Comparison of the dock- nist in the MVD (5801, whereas the (+) isomer
ing of SIOM with the 4-aminopiperidine com- is inactive in vitro. I n vivo (-1 TAN 67 exhibits
pounds suggested that the benzamide ring oc- antinociceptive activity in the tail flick assay
cupies a similar region to the spiroindane of after both i.c.v. (585) and intrathecal (i.t.1
SIOM, consistent with the pharmacophoric (586) administration, which appears to be me-
models, and thus functions as an "address" to diated by 6, receptors. Whereas (+) TAN 67
target the compounds to the 6 receptor (188). was inactive after i.c.v. administration (585),
after i.t. administration (+) TAN 67 produced
5.1 0.2 Other &Receptor Agonists. Attempts nociceptive behavior (586); interestingly, the
to identify Sselective agonists related to the effects of (+) TAN 67 were blocked by both
epoxymorphinans have concentrated in two naltrindole and ( -) TAN 67 (584). ( -) TAN 67
areas. Modification of the nitrogen substitu- exhibits decreased affinity for the [W284L]
ent of NTI resulted in the identification of mutated 6 receptor compared to the wild-type
SIOM as a 6, agonist (see Section 5.3.3 above). receptor, but the magnitude of the decrease is
Other 6-selective agonists identified have been less than that for SNC 80 (587). In contrast
octahydroisoquinolines. As discussed earlier this mutation increases the intrinsic activity
(Section 5.3.31, octahydroisoquinolines are ei- of SNC 80 (indicated by an increase in the
ther antagonists or agonists, depending on the maximum [35SIGTPyS binding), but it de-
ring size of the spacer. Although compounds creases the intrinsic activity of (-1 TAN 67,
with five-membered ring spacers are brecep- suggesting that these compounds may inter-
tor antagonists, introduction of a six-mem- act with different active receptor conforma-
bered ring spacer, explored by both Japanese tions (587). In a recent report of a pharma-
and Italian researchers, resulted in a new cophoric model for the 6 opioid receptor, low
class of Breceptor agonists (108,358,580; see energy conformations of TAN 67 were identi-
Refs. 109,219 for reviews), with TAN 67 (28, fied in which the phenol, the basic m i n e , and
Fig. 7.5) (108, 109) being the most extensively the second aromatic m i n e could be superim-
studied. The rationale for the design of TAN posed on the corresponding groups in OM1
67 by the Japanese group involved making the and SIOM (588).
phenol ring freely rotatable by removing the Based on their comparison of the antago-
4,5-epoxy and 10-methylene functionalities of nist SB 205588 and SNC 80 and the resulting
the epoxyrnorphinans and using a heteroatom hypothesis that the amide of SNC 80 might
capable of forming a hydrogen bond with the function as a nonaromatic "address", Dondio
receptor as an additional pharmacophoric and coworkers prepared pyrrolooctohydroiso-
group (109). Racemic TAN 67 shows high af- quinolines (178) (Fig. 7.35) lacking the second
finity and selectivity for 6 receptors (Table aromatic ring, which exhibit high 6-receptor
7.12) (log), and is a potent agonist in cloned 6 aMinity and selectivity (see Table 7.12) (577).
human cells (581), and in the MVD (109). I n The analog in which the pyrrole ring was in
vivo racemic TAN 67 exhibits antinociceptive the opposite orientation (179) also retained
activity in the acetic acid writhing assay, but high &receptor affinity (Ki = 3 nM), suggest-
not the tail flick test, in normal mice [TAN 67 ing that this ring functions as a spacer (589).
Narcotic Analgesics
An attempt has also been made to con- exhibited somewhat higher Breceptor affinity
vert 5-(3-hydroxypheny1)-2-methylmorphan, and potency, but the selectivity for 6 receptors
which has negligable &receptor affinity, to was still low [IC,, ratio ( ~ 1 6values
) of 18 and
6-selective agonists by use of the "message- 7, respectively] (591).
address" concept (590). Addition of an indole Recently, a new type of nonpeptidic 6 ago-
ring system to the morphan skeleton, to give nist (182), which lacks a basic nitrogen, was
(180), increased 6-receptor affinity more than identified by high throughput screening (592).
140-fold (IC,, = 7 nM); this structural change Further structural modification of this lead
had little effect on preceptor affinity, how- compound to improve its water solubility led
ever, so that the selectivity of this compound to a series of amide derivatives (e.g., 183) with
for 6 receptors was low [IC,, ratio ( ~ 1 6=) 4.21. modest 8-receptor affinity (IC,, = 37-256 nM)
Based on differences between the SAR of SNC and in vivo potency comparable to TAN 67
80 and the indole phenylmorphan derivatives, (593).
Rice and coworkers postulated that the indole
5.1 1 Nonpeptide Affinity Labels Used to
phenyl ring, not the phenol, of the indole phe-
Study Opioid Receptors
nylmorphans might be structurally analogous
to the methoxyphenyl group on SNC 80. Affinity labels, ligands that interact with re-
Therefore, they prepared a series of methoxy- ceptors in a nonequilibrium manner, are use-
substituted derivatives of (180); the C9' and ful pharmacological tools to study opioid re-
C10' substituted derivatives (181, Fig. 7.35) ceptors and receptor-ligand interactions.
5 Structure-Activity Relationships of Nonpeptide Opioid Ligands
Receptor type A
X
Receptor type B
G'
-
1 " Recognition
N O reaction
Receptor type C
Figure 7.36. Two-step mechanism for covalent binding of an affinity label containing a selective
electrophilic group X with receptor type A. Although receptor types A-C have similar topographical
features that lead to reversible binding (lorecognition), they differ with respect to the reactivity of
the receptor-based nucleophiles (G1and G2)and their locations. Only with receptor type A is the
nucleo~hileG1reactive with resvect to X and within covalent binding distance (2" recognition) (from
Ref. 3i9).
Tritiated affinity labels have been useful in highly reactive intermediates, most often a ni-
rece~torisolation and for determination of the
A
trene or carbene, upon exposure to light of the
molecular weights of solubilized receptors (see appropriate wavelength and these reactive
Refs. 86, 99 for reviews). These compounds species then can react covalently with the re-
can be used to irreversibly block one or more ceptors. Reaction of affinity labels with recep-
receptor type in tissues containing multiple tors involves a two-step mechanism (Fig. 7.36)
receptor types, so that the remaining receptors (319, 594). Initially the ligand binds to the re-
can be studied in isolation. The covalent binding ceptor reversibly, followed in the second step
of a n i t y labels can be used to study receptor- by covalent bond formation between the reac-
l i-m d interactions. Thus Liu-Chen and cowork- tive functionality on the ligand and a group on
ers have characterized the binding of the a n i t y the receptor. Depending on the nature of the
labels p-funaltrexamine (P-FNA)and SUPER- reactive functionality on the affinity label, the
FIT to p and S receptors, respectively, by use second step can enhance receptor selectivity.
of a combination of molecular biology ap- Whereas an electrophilic affinity label may
proaches and protein isolation (see below). bind reversibly to more than one receptor
There are two types of affinity labels based type, covalent bond formation requires the
on the type of reactive functionality. Electro- proper juxtaposition of an appropriate nucleo-
philic affinity labels contain an electrophilic phile on the receptor with the electrophilic
group that can react with nucleophiles on the group on the ligand (see Fig. 7.361, so that
receptor. Photoaffinity labels are converted to covalent binding can occur to only one type of
Narcotic Analgesics
(188) PNTl
0
OH
A variety of affinity labels, mostly nonpep- receptors. [There are conflicting reports on
tide ligands, have been prepared for opioid re- whether the effects of P-FNA on 6 receptors
ceptors. Detailed reviews of the structure-ac- are irreversible (605, 606).] The orientation
tivity relationships for affinity labels have and configuration of the fumaramide func-
been published [see the previous edition of tionality are important for irreversible bind-
this chapter (286) and Refs. 319, 594-5961. ing to p opioid receptors; neither the 6P-male-
Therefore the discussion below focuses on imide with a cis double bond nor a-FNA with'
those ligands that have been most useful in the electrophile in the 6a position is an irre-
the characterization of opioid receptors, and versible p antagonist (605). Examination of
on recent reports of new affinity label deriva- the conformations of a- and P-FNA by X-ray
tives. diffraction (607) offers insight into the differ-
ences in reactivity of these two isomers. The
5.1 1 .I Morphine and Naltrexone Deriva- conformations of the ring system in the two
tives. A variety of morphine and naltrexone compounds are almost identical except for the
derivatives have been prepared that incorpo- C ring, which is in a twist-boat conformation
rate a reactive functionality, often at the 6 po- for the a isomer and a chair conformation for
sition (Fig. 7.37). Incorporation of a nitrogen P-FNA (see 186). The fumarate group is then
mustard at the 6P position of naltrexamine by equatorial in both compounds, which when
Portoghese and coworkers yielded p-chlornal- the morphinan skeletons are superimposed
trexamine (p-CNA, 184) (597, 598), the first places the fumarate double bond in a-FNA
successful opioid antagonist affinity label. more than 2 A away from the double bond in
This compound is a potent affinity label that P-FNA and in the wrong orientation for reac-
because of the reactivity of the nitrogen mus- tion with a nucleophile on the receptor.
tard blocks all opioid receptor types (see Ref. The binding of P-FNA to p opioid receptors
594). It has been a useful tool in studying opi- was examined by Liu-Chen and coworkers by
oid receptors and has been used to single out a use of a combination of molecular biology ap-
specific receptor type in tissues containing proaches and protein isolation. These studies
multiple receptor types. The desired receptor illustrate the utility of using affinity labels to
type for study can be protected by incubation study receptor-ligand interactions. The bind-
with a ligand selective for that receptor and ing of [3H]P-FNA to p / ~receptor chimeras
the tissue subsequently treated with P-CNA to suggested the region of the receptor from the
irreversibly block the remaining opioid recep- middle of the third extracellulular loop (EL31
tors (133, 134, 599). Only one of the chloro- to the C-terminus was necessary for irrevers-
ethyl groups of p-CNA is required for irrevers- ible binding to p receptors (608). Subsequent
ible antagonist activity (600). The nitrogen isolation and partial purification of the labeled
mustard analog of oxymorphone, p-chloroxy- receptor and digestion with CNBr, however,
morphamine (P-COA, 185)(601,6021, has also
-
located the point of attachment of P-FNA to
been prepared. It is a potent irreversible ago- the EL2-TM5 region of the receptor; subse-
nist in the GPI and appears to bind irrevers- quent site-directed mutagenesis of residues in
ibly to opioid receptors in vivo; in vivo, it ini- this region indicated that the point of attach-
tially produces analgesia followed by a long- ment was Lys233at the EL2-TM5 interface,
lasting antagonism of morphine analgesia which is a conserved residue among the opioid
(602,603). receptors (177). Thus the selectivity of P-FNA
To obtain affinity labels selective for a sin- irreversible binding for p opioid receptors ap-
gle receptor type, Portoghese incorporated pears to be due to differences in the tertiary
less reactive electrophiles at the 6P position structure of the receptor, not the primary se-
leading to the preparation of P-FNA (186) quence. This illustrates the subtleties of re-
(604). This compound illustrates how the sec- ceptor-ligand interactions and the importance
ond step in the mechanism of affinity labels of examining receptor-ligand interactions
can enhance selectivity; whereas P-FNA inter- directly.
acts reversibly with K receptors, where it is an Recently, Portoghese and coworkers re-
agonist, it is an irreversible antagonist at p ported the phthalaldehyde derivatives of 6P-
Narcotic Analgesics
naltrexamine (187) and naltrindole (188) as been incorporated into the indole N-benzyl ar-
"reporter affinity labels" (340, 609). Reaction omatic ring of the 6,-selective antagonist N-
of the phthalaldehyde group with an amine benzylnaltrindole (BNTI, 75) to give (190)
and thiol (from Lys and Cys side chains, re- (617). Interestingly, the meta-substituted iso-
spectively, in the receptor) results in a fluores- thiocyanate derivative was an irreversible 6
cent isoindole; detection of fluorescence indi- agonist in the MVD; the ortho- and para-
cates that covalent reaction has occurred. In substituted isothiocyanates and the haloaceti
contrast to naltrindole, the phthalaldehyde amides were 6-receptor antagonists that
derivative PNTI (188) is an agonist in the exhibited time-dependent increases in antag-
mouse vas deferens, leading to the proposal onism consistent with covalent interaction
that the covalent binding of PNTI to 6 opioid with 6 receptors. In uiuo, these compounds
receptors results in a conformational change were less selective for 6, over 6, receptors than
in the receptor and agonist activity (340). BNTI.
Pasternak and coworkers have prepared a A number of 14P-amino substituted deriv-
series of hydrazone derivatives of the 6-ketone atives containing electrophilic groups (Fig.
of naloxone, naltrexone and oxymorphone 7.38) were prepared by Archer and coworkers
(189, Fig. 7.37) (209,210, 610). Prolonged ac- [see the previous edition of this chapter (286)
tions in uiuo and nonequilibrium binding in for a detailed review]. Reactive functionalities
uitro have been reported for several of these that have been attached to the 14P-amino
compounds. The hydrazone naloxazone (48, group include bromoacetamide, thioglycol-
Fig. 7.8) (209) and the corresponding azine amide, and cinnamoyl groups. The naltrexam-
naloxonazine (49) (210) have been used to ine derivative clocinnamox or C-CAM (191)
characterize the postulated pl-receptor sub- (618) has been the most extensively studied
type (see Ref. 208); the azines are 20- to 40- and is a potent long-lasting p antagonist (619-
fold more potent than the corresponding hy- 621). Binding studies indicated that clocin-
drazones (210). Studies with [3H]naloxazone namox selectively decreases the density of p
suggested that a portion of the binding may receptors (621), but not of S or K receptors
involve covalent interaction with pl-receptors (622), without affecting receptor affinity, as
(611); other researchers, however, have not would be expected for an irreversible ligand. A
found evidence for irreversible binding to opi- subsequent examination of [3H]clocinnamox
oid receptors (612, 613). The acylhydrazone binding to mouse brain membranes, however,
naloxone benzoylhydrazone [Nal(Bzo)H, 50, (623) found that the binding was fully revers-
Fig. 7.81 exhibits extremely slow dissociation ible, although half of the binding dissociated
from p receptors ("pseudoirreversible" bind- very slowly (t,,, = 11 h). The p-nitro-substi-
ing), which may be related to interactions with tuted derivative with a 5P-methyl group MET-
a G-protein (614); it also binds reversibly to K CAM0 (194) was the first N-methyl derivative
receptors and the tritiated form has been used reported with long-lasting p-receptor selective
to characterize the proposed K,-receptor sub- antagonist activity with no agonist activity
type (see Section 3.2.4.3) (217,615). (624); it appears to bind irreversibly to p re-
Portoghese and coworkers prepared deriv- ceptors (625,626). Like MET-CAMO, the cor-
atives of the 6-selective antagonist naltrindole responding p-chloro-substituted, 5p-methyl
containing reactive functionalities as affinity derivative MET-C1-CAM0 (195) is also along-
labels for 6 receptors. An isothiocyanate group lasting p-receptor antagonist with no agonist
was incorporated at the 5' position of naltrin- activity, which appears to bind irreversibly to
dole to give NTII (47, Fig. 7.8) (196), which is p receptors (627). The p-methyl derivative of
a potent and selective nonequilibrium S-recep- clocinamox, methocinnamox or M-CAM (193),
tor antagonist. NTII antagonizes the antino- has been reported to be a more p receptor se-
ciceptive activities of [D-Ala2]deltorphin11and lective, long-lasting antagonist in uiuo than
DSLET, but not that of DPDPE, and therefore clocinnamox or P-FNA (628), although in
was proposed to be a selective 6,-receptor an- standard binding assays, like clocinnamox, its
tagonist (616). Electrophilic moieties, either selectivity for p receptors is very low (Kiratio
an isothiocyanate or haloacetamide, have also = 3-8). Recently, the relative importance of
5 Structure-Activity Relationships of Nonpeptide Opioid Ligands
Figure 7.38. Affinity label derivatives of naloxone and morphine containing a reactive functionality
at the 14P position.
the 3-hydroxyl group to the opioid receptor illustrates how much the alkylation step can
affinity of clocinnamox was examined by pre- influence the receptor selectivity of affinity la-
paring both a series of 3-alkyl ether deriva- bels. Both BIT and FIT have been prepared in
tives (629) and the 3-deoxy analog (630). In- tritiated form (631) and r3H]FIT used to
terestingly, in the 3-alkyl ether derivatives the specifically label a 58-kDa protein from
identity of the alkyl group affects efficacy. In NG108-15 cells (632). The enantiomeric pair
vivo, the 0-methyl derivative MC-CAM (192) of the cis-&methyl derivatives of FIT were
is a p-partial agonist, the propargyl ether is a synthesized and one of these isomers, the (+)-
potent agonist, and the cyclopropylmethyl an- 3R,4S enantiomer, SUPERFIT (198) was
alog is a long-lasting antagonist with little ag- found to be a very potent (5-10 times the po-
onist activity; both the methyl and propargyl tency of FIT) and selective irreversible ligand
ethers exhibit delayed long-lasting antagonist for 6 receptors (458). SUPERFIT was used to
activity. The 3-deoxy analog exhibits high p purify 6 receptors from NG108-15 cells to ap-
opioid receptor affinity comparable to that of parent homogeneity (633). Liu-Chen and co-
clocinnamox, indicating that the C,-hydroxyl workers used similar approaches to those de-
does not play a significant role in the binding scribed above for P-FNA to study the binding
of these 14P-cinnamoyl epoxymorphinans to of SUPERFIT to 6 opioid receptors. The re-
opioid receptors; the deoxy derivative exhibits sults for wash-resistant inhibition of binding
greater selectivity for p over 6 receptors than to A6 chimeric receptors suggested that the
clocinnamox. segment from the start of the first intracellu-
lar loop to the middle of TM3 of 6 receptors is
5.1 1.2 Other Nonpeptide Affinity Labels. important for the selective irreversible bind-
Rice and coworkers prepared a variety of affin- ing of SUPERFIT (634). The enantiomeric
ity labels on the basis of the structures of eto- pair of the trans-3-methylfentanyl isothiocya-
nitazine, fentanyl, and oripavine (Fig. 7.39) nates have also been prepared (199). The (+)-
(198, 461). The etonitazene derivative BIT 3S,4S isomer was a Bselective acylating agent
(196) selectively inactivates p receptors, in vitro, with potency similar to that of SU-
whereas the fentanyl derivative FIT (fentanyl PERFIT, and has been used to selectively de-
isothiocyanate,197) and the oripavine deriva- plete S, binding sites (see Section 3.2.4.3
tive FA0 (fumaramido oripavine, 200) selec- above) (200).
tively inactivate 6 receptors. The selective al- Several derivatives of K-selective com-
kylation of 6 receptors by FIT, which is a pounds containing an isothiocyanate have
derivative of the p-selective ligand fentanyl, been prepared as potential irreversible ligands
Narcotic Analgesics
S=C=N
iN(cH2cH
Q- dNLC
abQ I
0CH2CH3
N
R1 0 .>
for K receptors (Fig. 7.39). de Costa et al. pre- pig brain. This led de Costa and coworkers to
pared analogs of U50,488 (201) and (202) prepare UPHIT (202), the chlorine-contain-
(635-637). In the series of enantiomerically ing analog of (201), to improve affinity and
pure analogs of (201) the (-1-1S,2S isomers selectivity (636). In contrast to (201), this
generally exhibited wash-resistant inhibition compound inhibited binding to K receptors af-
of binding of [3H]U69,593 to guinea pig brain ter i.c.v. administration (636). In vivo, in mice
membranes, but none of the compounds irre- UPHIT antagonizes antinociception produced
versibly inhibited the binding of [3H]bremazo- by U69,593, but not by bremazocine, provid-
cine to either guinea pig or rat brain mem- ing additional supporting evidence for K-re-
branes (635, 637), supporting the conclusion ceptor subtypes (638).
of heterogeneity of K receptors. The (-)-o-iso- Isothiocyanate derivatives of the K-selec-
thiocyanate isomer of (201) exhibited selec- tive agonist ICI 199441 have also been de-
tive wash-resistant inhibition of [3HlU69,593 scribed. Chang et al. prepared the m-isothio-
binding, and was the most potent in vitro, but cyanate derivative DIPPA (203) (639, 6401,
it failed to produce any irreversible inhibition which exhibits wash-resistant inhibition of
of K receptors after i.c.v. injection into guinea L3H1U69,593 binding and long-lasting K re-
6 Opioid Peptide Analogs 407
ceptor antagonism in vivo. Liu-Chen and co- tive. This clinically used analgesic is a mixed
workers examined the binding of the corre- agonistlantagonist (645) (see Table 7.3) with
sponding p-isothiocyanate derivatve to p / ~ partial p agonist activity; it may also have ac-
chimeric receptors and determined that the tivity at K receptors (see Ref. 307). The cyclo-
region from TM3 to the C-terminus of the K hexane derivative tramadol (Ultram, 206) is
receptor is important for the binding of this an atypical analgesic (see Refs. 307, 646, 647
compound (641). Nelson and coworkers in- for reviews), which appears to produce analge-
corporated the isothiocyanate in the phenyl- sia through both opioid and nonopioid mecha-
acetamide phenyl ring of ICI 199441 (642). nisms (648). Tramadol antinociception ap-
These isothiocyanate analogs all exhibited pears to be mediated through both activation
wash-resistant inhibition of binding, whereas of p opioid receptors, where it exhibits low af-
the parent compound with an unsubstituted finity (Ki = 2 @ I)
and by ,
inhibition of mono-
phenylacetamidephenyl ring was completely re- amine uptake. Examination of the isomers of
moved by washing; the lead compound ICI tramadol (649) found that although the (+)
199441, with chlorines on this ring but without enantiomer had higher affinity for p receptors
the isothiocyanate group, however, was not (Ki = 1.3 piW, the activities of the isomers
completely removed by the washing procedure. were complementary and synergistic. Other
A number of photoaffinity label derivatives recently reported compounds with opioid
of opiates have also been prepared [see the receptor affinity and activity include pyrroli-
previous edition of this chapter (286) and Ref. dinylnaphthalenes, which are structurally
594 for more detailed reviews]. Azide deriva- related to heterosteroids (650). Highly con-
tives of a number of different opiates, includ- strained tricyclic piperazine derivatives (207)
ing etonitazene, carfentanil, and 6a- and 6P- and (208) were prepared, which are structur-
substituted naltrexamine derivatives, have ally related to the 4-anilidopiperidines and
been prepared (see Ref. 286). A significant which exhibit reasonable affinity (Ki values of
problem with using opioid photoaffinity labels 10 and 7 nM,respectively) and selectivity [Ki
is the sensitivity of opioid receptors to destruc- ratio (p/6/~)= 1/230/300 and 1/71/110] for p
tion by short-wavelength UV irradiation receptors; in vivo, (208) is a sixfold more po-
(643). Incorporation of a nitro group into the tent analgesic than morphine (651). 3-Amino-
aromatic ring bearing the azide functionality 3-phenylpropionamide derivatives were pre.-
shifts the absorption maximum so that photo- pared as small molecule mimics of the peptide
lysis can be conducted at longer wavelengths, antagonists CTOP and CTAP and analogs
where little if any photodestruction of opioid identified with high affinity for p and K opioid
receptors occurs. receptors (e.g., 209) (652).
5.1 2 Miscellaneous Nonpeptide Opiates
6 OPlOlD PEPTIDE ANALOGS
A variety of compounds in other chemical
classes have also been identified that have an-
6.1 Introduction
algesic activity (see Ref. 466). In addition to
the K-receptor selective arylacetamides, such The identification of the opioid peptides in the
as U50,488 and related compounds, the ben- mid-1970s opened up a whole new area for the
zodiazepine tifluadom (147) is a K agonist (see development of opioid receptor ligands. The
Section 5.9). The benzimidazole etonitazene endogenous opioid peptides, both mammalian
(204, Fig. 7.40) is a potent analgesic, having and amphibian (see below), have served as
approximately 1500 times the potency of mor- lead compounds that have been extensively
phine in mice (6441, and is a preceptor ago- modified to enhance potency, receptor type se-
nist. The aminotetralin dezocine (205) bears lectivity, stability, and/or decrease conforma-
some resemblance to the benzomorphans and tional flexibility. Peptide ligands selective for
contains a phenol and basic amino group; in both p and S opioid receptors are found in
contrast to other opiates, however, the m i n e more than one type of peptide sequence. Thus
group is a primary amine. Dezocine is the levo some enkephalin analogs, as well as the re-
isomer of the p epimer; the (+) isomer is inac- cently discovered endomorphins, analogs of
Narcotic Analgesics
Etonitazene
-
(205) Dezocine (206) Tramadol
the peptide P-casomorphin (derived from ca- tagonists, have been prepared, and novel pep-
sein) and the amphibian peptide dermorphin, tides with opioid receptor affmity have been
preferentially interact with p opioid receptors identified through the use of combinatorial ap-
(see Sections 6.2.1, 6.4.1, and 6.5.1 below). proaches (see Section 6.6). Affinity label deriv-
Other enkephalin analogs, as well as analogs atives of opioid peptides that bind irreversibly
of the amphibian peptides the deltorphins, to opioid receptors have also been identified
preferentially interact with 6 opioid receptors (see Section 6.7). In addition to preparing an-
(see Sections 6.2.2 and 6.5.2 below). For K re- alogs of opioid peptides, inhibitors of opioid
ceptors the endogenous opioid peptides identi- peptide metabolism have been developed as an
fied to date have been limited to one class of indirect approach to using opioid peptides as
peptides, the dynorphins (see Section 6.3 be- potential therapeutic agents (see Section 6.8).
low). Peptides with aMinity for opioid recep- Much of the early SAR of the enkephalins
tors, which have sequences completely differ- has been discussed in a classic review by Mor-
ent from those of the endogenous opioid ley (653) and in The Peptides, Volume 6 (654).
peptides, have also been identified. Analogs of Subsequent reviews of opioid peptides include
somatostatin, which are p opioid receptor an- those by Hruby (655, 656) and Schiller (657,
6 Opioid Peptide Analogs
[D-~la~ldeltorphin
I (deltorphin C, 214) Tyr-D-Ala-Phe-Asp-Val-Val-GlyNH2
[D-~la~ldeltorphin
I I (deltorphin B, 25) Tyr-D-Ala-Phe-GIu-Val-Val-GlyNH,
658),which contain extensive tabular data. An 6.1 .I Opioid Peptides from Amphibian Skin.
issue of Biopolymers (Peptide Science) (Vol.
Based on their finding amphibian skin pep-
51, Number 6, 1999) is devoted solely to re-
tides, which were counterparts to other mam-
views of peptide and peptidomimetic ligandsmalian bioactive peptides, Erspamer and co-
for opioid receptors. The reader is referred to
workers examined amphibian skin for opioid
these reviews for additional references to the
peptides (see Ref. 663 for a review). This led
literature. first to the isolation and characterization of
In addition to the four classes of opioid pep-
dermorphin (212, Fig. 7.41), which is a p-se-
tides discussed earlier (Section 3.4), other pep-
lective peptide (see Table 7.13), from the skin
tides of mammalian origin with opioid activity
of South American Phyllemedusinae hylid
have also been identified. p-Casomorphin frogs in the early 1980s (664). Inspection of
(210),obtained by enzymatic digestion of the
the sequence of one of the cloned cDNAs for
milk protein casein (659, 6601, exhibits some
the precursor of dermorphin suggested the ex-
selectivity for p receptors. Human p-casomor-
istence of another heptapeptide with a similar
phin (211) differs from the bovine sequence in
N-terminal sequence (665). This then led to
two positions; the human p-casomorphin pen-the isolation of deltorphin (also called dermen-
tapeptide and tetrapeptide fragments are less
kephalin or deltorphin A, 213, Fig. 7.41), the
potent than the corresponding bovine pep- first &selective amphibian opioid peptide,
tides (661). Other peptides with affinity for
from these frogs (666, 667). Synthesis con-
opioid receptors include peptides derived from
firmed that the amino acid in position 2 of
hemoglobin (see Ref. 662). deltorphin was D-methionine rather than
L-methionine (666, 668, 669). Two additional
Bovine p-casomorphin (2 10) peptides [D-Ala2]deltorphinI (also referred
to as deltorphin C, 214, Fig. 7.41) and
Tyr-Pro-Phe-Pro-Gly-Pro-Ile [D-Ala2]deltorphin I1 (also referred to as del-
Human p-casomorphin (211) torphin B, 25, Fig. 7.5) were subsequently dis-
covered (106) which exhibited even greater
Tyr-Pro-Phe-Val-Glu-Pro-Ile &receptor affinity and exceptional selectivity
Table 7.13 Opioid Receptor Affinities and Opioid Activity in the GPI and lVND of Peptides
from Amphibian Skina
Ki (nMIb K,Ratio I(& (nM)
Peptide P 6 p/8/K GPI MVD
Dermorphin (212) 0.70 62 1/89/> 14,000 1.4 2.4
Deltorphin (213) 1,630 2.4 680/1/>4,000 5,000 1.4
[D-Ala2]Deltorphin1 (214) 3,150 0.15 21,000/1/>66,000 >1,500 0.21
[D-Ala2]Deltorphin11(25) 2,450 0.71 3,400/1/> 14,000 >3,000 0.32
"Data from Ref. 106.
bK,> 10,000 nM for K receptors for all of the amphibian skin peptides.
Narcotic Analgesics
(see Table 7.131, making them the most selec- decreases in potency when this residue was
tive of the naturally occurring opioid peptides. substantially modified. A D-amino acid in posi-
[D-Ala2]deltorphinI1 has been used to study tion 2 was one of the early modifications exam-
the proposed 8,-receptor subtype (see Section ined to decrease the cleavage of the Tyr-Gly
3.2.4.3 above). The pharmacology of these am- bond by aminopeptidases (671). Incorporation
phibian opioid peptides has been discussed in of a D-amino acid at this position increases po-
a recent review (670). tency at both p and 6 receptors and is found &
The unique feature of these amphibian the vast majority of enkephalin derivatives; an
skin opioid peptides is the sequence between L-amino acid at position 2 significantly de-
the important aromatic residues. In contrast creases potency at both receptors. In the eri-
to the enkephalins and other mammalian opi- kephalins the 3 position is very intolerant of
oid peptides that contain the Gly-Gly dipep- substitution, and therefore enkephalin deriv-
tide sequence between Tyr and Phe, the am- atives generally retain a glycine at this posi-
phibian opioid peptides contain a single tion. There are significant differences between
D-amino acid (see Fig. 7.41), which apparently p and 6 receptors, however, in the structural
arises from a post-translational conversion of requirements for residues in positions 4 and 5,
the L-amino acid to its D isomer (665). The and frequently modifications in these posi-
identification of these unusual opioid peptides tions have been used to impart selectivity for
expanded our understanding of the structural one of these opioid receptor types (see below).
requirements for interaction with opioid re-
ceptors and provided new lead compounds for 6.2.1 Mu-Selective Enkephalin Analogs
further modification (see Sections 6.5.1 and 6.2.1.1 Linear Analogs. Enkephalin ana-
6.5.2 below). logs with substantial structural changes from
the endogenous peptides, particularly in the
6.2 Enkephalin Analogs
C-terminus, retain affinity for p opioid recep-
The enkephalins have been the most exten- tors and can exhibit improved preceptor se-
sively modified of the opioid peptides, and lectivity. Thus the aromatic moiety of Phe4 is
thousands of analogs of these pentapeptides not an absolute requirement for interaction
have been prepared (see Refs. 653-658 for re- with p receptors, and a cyclohexane ring (672)
views). The naturally occurring enkephalins or leucine side chain (673, 674) is tolerated.in
exhibit some selectivity for 8 receptors (see this position. Significant variation is also tol-
Table 7.9), but these peptides are rapidly de- erated in residue 5 and the C-terminus by p
graded by a variety of peptidases (see Section receptors, and modifications in this region of
6.8 below). Therefore one major goal of struc- the peptides have been very useful in differen-
tural modification of these small peptides has tiating p versus 8 receptors. Thus, the C-ter-
been to increase metabolic stability. Depend- minus can be amidated, reduced, or elimi-
ing on the nature of the modifications made, nated completely with retention of p-receptor
both p- and &selectiveenkephalin derivatives affinity and often with a substantial increase
have been prepared (enkephalin derivatives in preceptor selectivity.
generally have very low affinity for K opioid One of the most commonly used p-selective
receptors). These derivatives have included ligands DAMGO (15,Fig. 7.4) (675) contains a
both linear peptides and conformationally reduced C-terminus and is available in triti-
constrained derivatives. Conformational con- ated form. Substitution of the glyol function-
straints have included cyclizations between ality of DAMGO with Met(0)ol (to give FK
residues in the peptide chain and local con- 33824) increases 6-receptor affinity 10-fold,
straints by incorporation of an amino acid decreasing p-receptor selectivity (130). Other
whose side-chain conformation is restricted. related tetrapeptide analogs with a modified
Early SAR studies (see Ref. 653 for a re- C-terminus syndyphalin-25 (Tyr-D-Met-Gly-
view) identified important structural features NMePheol) (676) and LY 164929 (NMeTyr-
of the enkephalins and which positions could D-Ala-Gly-NEtPhe[9(CH2NMe2)],2 15, Fig.
be readily modified. The importance of Tyrl 7.42) with a reduced C-terminal amide (677)
for opioid activity was apparent from the large exhibit significantly higher selectivity for p re-
6 Opioid Peptide Analogs
"I, 0
Figure 7.42. p-Receptor selective enkephalin analogs. The structure of DAMGO (15) is shown in
Fig. 7.4.
ceptors than DAMGO (Table 7.14). Shorter Fig. 7.43) in position 1 of [Leu51enkephalin
tripeptide amide analogs with a branched al- results in large increases in both I*. and 6 affin-
kyl chain in place of the second phenyl ring ity and an analog with some selectivity for F
also exhibit preceptor selectivity, although receptors (682), whereas incorporation of
their potency in the GPI is significantly lower 2',6'-dimethylphenylalanine(Dmp, Fig. 7.43)
(IC,, = 240-320 nM) (678). in position 4 results in increased preceptor
6.2.1.2 Conformationally Constrained Ana- selectivity as a result of decreased breceptor
logs. Local restriction of conformational free- affinity (683). Interestingly, combining the
dom can be accomplished by incorporating two modifications results in a weak antagonist
conformationally constrained amino acids (see (PA, = 6.90) with some selectivity (20-fold)for
Refs. 679,680 for reviews). In the case of the p receptors (683).
enkephalins, incorporation of 2-amino-6-hy- Enkephalin derivatives selective for p re-
droxy-2-tetralincarboxylic acid (Hat, Fig. ceptors have also been prepared by cyclization
7.43) in place of Tyrl in [Leu5]enkephalin between a D-amino acid and the C-terminus.
methyl ester results in a p-selective compound Tyr-cyclo[NY-D-Dab-Gly-Phe-Leu1 (216, Fig.
[ICE, ratio (MVDIGPI) = 5.11, whereas the ana- 7.42; Dab = a,y-diaminobutyric acid) (684) ex-
log containing 2-hydroxy-5-hydroxy-2-indane- hibits both high potency and preceptor selec-
carboxylic acid (Hai) is virtually inactive (681). tivity (Table 7.14), whereas the corresponding
Incorporation of 2',6'-dimethyltyrosine (Dmt, linear peptide [D-Dab2,Leu5]enkephalinamide
412 Narcotic Analgesics
Table 7.14 Opioid Receptor Affinities and Opioid Activity in the GPI and MVD of p-Selective
Enkephalin Analogsa
Ki
Ki (nM) Ratio Ic50(nM)
Peptide P 6 alp GPI MVD Reference
"Data for DAMGO from Table 7.8 are included for comparison.
bIC,, values.
is not preceptor selective (685). This was the accommodate an aliphatic residue in position
first demonstration that receptor selectivity 4 of enkephalins, S receptors generally require
could be imparted by conformational restric- an aromatic moiety in this position. At the C-
tion. Related analogs with D-Omor D-Lysin terminus 6 receptors prefer a free carboxylic
position 2 also exhibit high potency for p re- acid, and lengthening of the peptide with a
ceptors but decreased selectivity in radioli- residue such as Thr can result in increased 6
gand-binding assays (see Table 7.14) (686). selectivity.
The conformations of these peptides have Several linear enkephalin analogs have en-
been examined by both computational meth- hanced selectivity for 6 opioid receptors (Ta-
ods (687-690) and NMR spectroscopy (689, bles 7.8 and 7.15). DADLE (21; see Fig. 7.5 and
691). As expected, the ring structure reduces Table 7.8) (695) was an early analog prepared
conformational flexibility, but some flexibility that shows a slight preference for 6 opioid re-
in the ring remains, particularly for the larger ceptors. Roques and coworkers prepared sev-
ring sizes, and different intramolecular hydro- eral [Leu5,Thr61enkephalin analogs contain-
gen bond patterns have been proposed for the ing D-Ser, D-Thr, or a derivative in position 2,
cyclic structures. Modifications to the peptide which have greater selectivity for these recep-
bonds in these cyclic peptides, e.g., thioamide tors. Thus incorporation of D-Ser in position 2,
replacement of the Gly3-Phe4 peptide bond which yields DSLET (22) (6961, and substitu-
(692) or partial retro-inverso analogs of (216) tion of D-Thr in this position to give DTLET
e.g., Tyr-cyclo[~-Glu-Gly-gPhe-D-Leu1 (218, (23) (697) enhances breceptor selectivity. X-
Fig. 7.42) (6931, can further enhance p-recep- ray structures for both DADLE (698) and
tor selectivity (see Table 7.14). DTLET (699) have been reported; in both
cases a single bend conformation is found in
6.2.2 Delta-Selective Enkephalin Analogs the crystals (see Ref. 700 for a review). The
6.2.2.1 Linear Analogs. Differences in the steric bulk of the residues in positions 2 andor
SAR of enkephalin analogs for interaction 6 was increased by incorporating the t-butyl
with 6 versus p opioid receptors have been ex- ether derivatives of D-Ser andor Thr. The re-
ploited to develop Bselective derivatives. Thus sulting derivatives DSTBULET ([D-Ser
more hydrophilic D-amino acids, such as D-Ser (OtBu)', Leu5,Thr61enkephalin) and BUBU
or D-Thr, can be incorporated into position 2 to ([D-Ser(OtB~)~,Leu~,Thr(OtBu)~]enkephalin)
impart breceptor selectivity, whereas p opioid (696) exhibited decreased affinity for p recep-
receptors prefer a more hydrophobic residue tors and thus enhanced selectivity for 6 recep-
in this position (674).Whereas receptors can tors. Replacement of the ether in the side
6 Opioid Peptide Analogs
chain at position 2 by a sulfur to give BUBUC temic administration is relatively weak (702).
([D-Cy~(StBu)~,Leu~,Thr(OtBu)~]enkephalin) Therefore modifications have been made to
(701) further decreases affinity for p recep- enkephalin analogs in an attempt to enhance
tors, resulting in an analog with a 1000-fold BBB penetration. Attachment of D-glucose
selectivity for 8 over p receptors (Table 7.15). through an 0-linkage to Ser6 in the DTLET
The penetration of the blood-brain barrier analog Tyr-D-Thr-Gly-Phe-Leu-SerNH, re-
(BBB) by enkephalin analogs is relatively low sulted in a peptide that retains similar opioid
and therefore their analgesic activity after sys- receptor affinity and potency in smooth mus-
41 4 Narcotic Analgesics
Table 7.15 Opioid Receptor Affinities and Opioid Activity in the GPI and lVIVD of &Selective
Enkephalin Analogsa
Ki
Ki (nM) Ratio IC50 (nM)
Peptide ti P 1 MVD GPI Reference(s)
m
Agonists
DSLET (22) 1.8
DTLET (23) 2.7
DSTBULET 2.8
BUBU 1.7
BUBUC 2.9
[D-Thr2,Leu5]enkephalin-Ser(OR)6
R=H 2.4
R = D-glucose 3.4
DPDPE (24) 2.7
DPLPE 2.8
[Phe@-C1)41DPDPE 1.6
cyclo[~-Pen2,Cys5]enkephalin-Phe61.4'
cyclo[~-Pen~,Phe@-X)~,Pen~]-
enkephalin-Phe6
X=F 0.43'
X = Br 0.20'
[2'-MeTyrl]DPDPE 0.8gb
[(2S,3R)-TmtllDPDPE 5.0
[(2S,3S)-TmtllDPDPE 210
[Hatl]DPDPE 2.36'
[(2S,3S)-p-MePhe41DPDPE 10
Antagonists
ICI 174,864 (29) 190
NJV-Dibenzyl Leu-enkephalin 78
[(2S)-Mdpl,~-Ala2,Leu5]enkephalin
amide 12
"Data for DSLET, DTLET, DPDPE, and ICI 174,864 from Table 7.8 are included for comparison. Data from Ref. 130
except where otherwise indicated.
bICSOvalues.
=1c,, = 5 f l .
dAntagonist, K, values (nM).
"Not active.
cle assays to the parent peptide (see Table its 150-260 times higher affinity for 6 and p
7.15) (702). Glycosylation led to significant in- receptors than the E isomer (705). For the
creases in both enzymatic stability and BBB peptides containing cyclopropylphenylalanine
permeability, resulting in significantly im- (VPhe), one of the Z isomers exhibits high af-
proved analgesia after i.v. administration finity for [3H]naloxone binding sites in rat
(703). brain (706), whereas the 2R,3S isomer of
6.2.2.2 Conformationally Constrained Ana- VEPhe exhibits high affinity (Ki = 13 nM) and
logs. Local restriction of conformation in the selectivity (Ki ratio = 250) for 8 opioid recep-
linear enkephalins has included incorporation tors (707); the latter compound, however, was
of dehydroamino acids [e.g., dehydrophen- essentially inactive in smooth muscle assays
ylalanine (APhe, Fig. 7.43)] and cyclopropyl- (IC5, = 2000-4000 nM).
methylphenylalanine (VPhe)into the peptides Delta-selective enkephalin analogs were
(see Ref. 704 for a review). In the case of the prepared by cyclization through disulfide bond
dehydrophenylalanine (APhe4) derivatives of formation. Cyclic enkephalin analogs contain-
[~-Ala~,Leu~]enke~halin, the Z isomer exhib- ing D- or L-Cys residues in positions 2 and 5
[
f
P
6 Opioid Peptide Analogs
E.
E
were first synthesized by Sarantakis (708) and Pen5]enkephalin (DPLPE) had only a modest
Schiller (709). These initial cyclic enkephalin effect on &receptor affinity or potency in the .
analogs exhibit only a slight preference for 6 MVD, but halogenationof the resulting DPLPE-
over p receptors, but introduction of methyl Phe resulted in increased Sreceptor affinity and
groups on the P carbons of the cysteine resi- exceptionally high Sreceptor selectivity and po-
dues by incorporation of penicillamine (Pen) tency (see Table 7.15) (721); the p-chloro and
in positions 2 and/or 5 markedly enhances p-bromo derivatives also exhibited enhanced
&receptor selectivity (710-713). The most penetration of the BBB (717).
&selective compounds in the series were the Glycopeptide derivatives of cyclo[n-
bis-penicillamine derivatives DPDPE (cyclo[n- Cys2,Cys5]enkephalin and DPDPE have
Pen2,n-Pen5]enkephalin, 24, Fig. 7.5) and been prepared by attachment of Ser(p-D-
DPLPE (cyclo[n-Pen2,~-Pen51enkephalin) (713). glucose)-GlyNH, to the C-terminus (722).
The individual contributions of the P-methyl After peripheral administration, the cyclo[n-
groups in residue 2 to &receptor affinity and Cys2,Cys5]enkephalin derivative exhibits
selectivity were examined by preparing significant analgesic activity that appears to
cyclo[(3S)Me-D-Cys2,n-Pen5]- and cyclo[(3R)- be centrally mediated, indicating that the
Me-D-Cys2,n-Pen5]enkephalin(714). The glycopeptide penetrates the blood-brain bar-
P-methyl groups in the 2 position had only rier. The penetration of the BBB was ini-
a minor affect on 6 opioid receptor affinity, tially postulated to be through interaction
and the similar affinity for p receptors of with the glucose transporter GLUT-1, but
cyclo[(3S)Me-D-Cys2,n-Pen5]- and cyclo[n- subsequent studies have proved this to be
Cys2,n-Pen5]enkephalin suggested that ad- incorrect (see Ref. 723 and references cited
verse steric interactions with t h e pro-R therein). I n a series of cyclo[n-Cys2,n-
methyl group are responsible for the low p-re- Cys5,Ser6,Gly7]enkephalinamide analogs in
ceptor affinity, and therefore &receptor selec- which the sugar attached to Ser6 was varied,
tivity, of DPDPE. the nature of the sugar affected analgesic
Additional modifications to DPDPE can fur- potency after i.v. administration; the a-glu-
ther enhance Sreceptor affinity andlor selectiv- cose derivative showed the highest analgesic
ity (see Ref. 656 for a review, including tables of potency and 6-receptor affinity of the glyco-
analogs). Halogen substitution on thepara posi- sylated analogs examined (723). Recently, a '
tion of the phenylalanine ring in position 4 en- prodrug derivative of DPDPE, in which PEG
hances both &receptor potency and selectivity (polyethylene glycol) is attached to the N -
(7151, and [Phe(p-C1)41DPDPEhas been used in terminus, has been reported to also exhibit
tritiated form in radioligand binding assays. Ha- enhanced analgesia compared to that of
logenation can also increase blood-brain barrier DPDPE after i.v. administration; this pro-
permeability (716, 717). [L-A~~~IDPDPE was drug has very weak affinity for 6 opioid re-
prepared and exhibits an unusual spectrum of ceptors, but is converted to DPDPE in vivo
activity (718). In the mouse vas deferens, (724).
[L-A~~~IDPDPE is a potent (IC,, = 12 nM) Sse- The conformation of DPDPE has been ex-
ledive agonist, whereas at central 6 receptors it amined by NMR and computational methods
is a moderately potent 6,-receptor antagonist (see Ref. 725 and references cited therein) and
and weak agonist with no apparent affects on an X-ray structure has been obtained (725).
central 6,-receptors. The conformation of There is generally good agreement between
[L-Ala31DPDPE has been compared to that of these studies on the conformation of the 14-
DPDPE (719),and the differences in conforma- membered ring, but there is still considerable
tion around residues 2 and 3 were proposed to conformational flexibility around Tyrl in
explain the differences in efficacy observed for DPDPE. This is evident in the crystal where
the two analogs. A C-terminal extension of cy- three distinct structures, with essentially
clo[n-Pen2,~-Cys5]enkephalin (DPLCE) with identical conformations for the 14-membered
phenylalanine resulted in an analog with ex- ring but with different conformations for
tremely high potency in the MVD (IC,, = 0.016 Tyrl, were found (725). The activity of the
nM)(720).A similar extension of cyclo[~-Pen2,~- Hat1, P-MeTyrl, and Tmt derivatives (see be-
Narcotic Analgesics
low) suggested that the preferred conforma- P-MePhe4 increases 6-receptor affinity and
tion of the side chain of residue l in DPDPE is potency 6- to 10-fold, so that [(2S,3S)-p-
trans (726-728). Based on the activity of the MePhe(p-N02)4]DPDPEexhibits potency at 6
P-MePhe4 derivatives (see below), the pro- receptors similar to that of DPDPE. Further
posed side-chain conformation for Phe4 is constraint of Phe4 by incorporation of 2',6',P-
gauche (-) (xl = -60") (729). trimethylphenylalanine (Tmp, Fig. 7.43),
Therefore, other modifications to DPDPE however, results in decreased 6 receptor bind-
have been made to incorporate additional con- ing and selectivity; in the case of the 2S,3S
formational constraint into the aromatic resi- isomer affinity for the p receptor increases,
dues in the peptide. Constrained Tyr analogs, resulting in a peptide with 10-fold selectivity
including 2'-methyltyrosine, Dmt, P-methyl- for p receptors (733).
tyrosine, 2',6',/3-trimethyltyrosine(Trnt, Fig. 6.2.2.3 Enkephalin Analogs with Antagonist
7.43), and Hat (726-728, 730), have been in- Activity at 6 Receptors. The first antagonists
corporated at position 1. Substitution of Dmt for 6 opioid receptors were prepared by N,N-
enhanced affinity at both 6 and p receptors dialkylation of the N-terminus of enkephalins.
and increased both in vitro and in vivo potency The first 6-selective antagonists reported were
(730). Peptides containing 2'-MeTyr and Hat N,N-diallyl Leu-enkephalin methyl ester and
were also potent analogs with high 6-receptor the derivative ICI 154,129 containing a
selectivity (726) (see Table 7.15). Of the four thioether in place of the peptide bond between
peptides containing P-MeTyr, the (2S,3R)-p- residues 3 and 4 (734),although the potency of
MeTyrl derivative has the highest affinity for these peptides was weak (K, against Leu-en-
6 receptors and the greatest potency in the kephalin in the MVD = 254 nM for ICI
MVD (726). In the Tmt derivatives, which 154,129). Substitution of Aib (a-aminoisobu-
combine methyl groups on the phenyl ring tyric acid) in positions 2 and 3 of NJV-diallyl
with the P-methyl substitution, only the pep- Leu-enkephalin to give ICI 174,864 (29, Fig.
tide containing the 2S,3R isomer of Tmt re- 7.5, Table 7.15) enhanced potency approxi-
tains high 6 receptor potency and selectivity mately 10-fold and 6-receptor selectivity at
(727,728), whereas the peptide containing the least fivefold compared to that of ICI 154,129
diastereomer 2S,3S exhibits much lower S-re- (110), and so ICI 174,864 has been frequently
ceptor affinity and selectivity (Table 7.15). In- used as a 6-selective antagonist in a number of
terestingly, in vivo [(2S,3S)-Tmtl]DPDPE is pharmacological studies. The structure-activ-
the more potent analog in the tail flick test ity relationships for the N,N-diallyl deriva-
after i.c.v. administration (731). The antinoci- tives as 6 antagonists are distinctly different
ceptive activity of both peptides is antagonized from those of the unsubstituted agonist series
by P-FNA as well as by DALCE (731),suggest- (735). Thus the N,N-diallyl derivatives of a va-
ing that in contrast to DPDPE the antinoci- riety of potent enkephalin agonists such as
ceptive activity of both these Tmt analogs is DADLE are weak nonselective antagonists.
partially mediated by p receptors as well as 6, Moreover, although ICI 154,129 is a selective 6
receptors; these results are surprising for antagonist, the corresponding [Gly3V(CH2S)-
[(2S,3R)-Tmtl]DPDPE, given its high selec- Phe4,Leu5]enkephalin is virtually inactive as
tivity for 6 receptors in binding assays. an agonist. Other modifications have been
Further conformational constraint in DPDPE made to the N,N-diallyl enkephalins, includ-
has also been examined by incorporation of ing replacement of Gly2-Gly3 with a rigid
constrained residues in position 4. All four 4-aminobenzoic acid spacer (736), whibh
isomers of P-methylphenylalanine have been yields a S antagonist with potency similar to
incorporated into DPDPE (732). [(2S,3S)- that of ICI 154,129. Substitution of benzyl
P-MePhe4]DPDPE (see Table 7.15) exhibits groups for the ally1 groups in N,N-diallyl Leu-
the highest 6-receptor affinity of the four pep- enkephalin enhances 6-antagonist potency,
tides, although it is 6- to 10-fold less potent whereas N,N-dialkylation with other alkyl
than DPDPE; it also exhibits the highest groups (e.g., phenethyl) result in derivatives
selectivity for 6 over EL receptors. Incorpo- with agonist activity (737). Interestingly, al-
ration of a p-nitro group into (2S,3S)- though ICI 174,864 is more potent than N,N-
6 Opioid Peptide Analogs
dibenzyl Leu-enkephalin in the MVD (737), In the tetrapeptide series biphalin [(Tyr-D-
the reverse was true in binding studies that Ala-Gly-Phe-NH),, 2201, with only a hydra-
used cloned 8 receptors (738), suggesting pos- zine spacer, is not selective for p or 8 receptors
sible differences in 8 receptors in the two prep- (Table 7.16) (742). This peptide exhibits po-
arations. NJV-DialkylLeu-enkephalin analogs tent analgesic activity in vivo (742, 743), even
bearing a reactive functionality recently have though its potency in smooth muscle assays is
been described (738) (see Section 6.7 below). modest (Table 7.16). Biphalin is equipotent
Recently, the Leu-enkephalin analog con- with morphine, but produces little if any phys-
taining a novel derivative of Dmt lacking a ical dependence after systemic 6.p.) adminis-
basic amine [(2S)-2-methyl-3-(2,6-dimethyl-4-tration. The truncated dimer Tyr-D-Ala-Gly-
hydroxypheny1)propanoicacid, (2s)-Mdp,Fig. Phe-NHNH(Phe)retains high binding affinity
7.431 was reported; this peptide is an antago- and potency in smooth muscle assays, indicat-
nist in both the MVD and GPI smooth muscle ing that the entire sequence of biphalin is not
preparations, with fivefold higher potency in required for activity (744). Modification of the
the MVD (739). residue in the 4 position can alter opioid recep-
tor affinities and impart some selectivity for
6.2.3 Dimeric Enkephalin Analogs. A num- either p or 8 receptors. Thus introduction of
ber of dimeric enkephalin analogs have been L-0-MePheor naphthylalanine in this position
prepared (Fig. 7.44). Their receptor selectivity results in analogs that preferentially interact .
depends on the peptide sequences and the with p receptors (see Table 7.16) (7451,
length of the spacer between the two peptides. whereas p-halogenation or nitration of the
The tripeptide dimer (DTRE), [(Tyr-D-Ala- phenyl rings enhances 8-receptor affinity
Gly-NH),(CH,),, 219, Table 7.161 is p selec- more than preceptor affinity (746);para chlo-
tive, whereas the corresponding monovalent rination also increases penetration across the
tripeptide T y r - D - A ~ ~ - G ~ ~(TRE)
N H , is much BBB (747,748).In situ brain perfusion studies
less potent and only moderately p selective of [1251-Tyr]biphalinsuggest that part of the
(740). Replacement of the Tyr residue in one transport of this compound may use the large
half of DTRE, with Phe or D-Tyr significantly neutral amino acid carrier (749).
decreased potency, which suggests that p opi- Other dimeric enkephalin derivatives have
oid receptors contain two similar sites that are been prepared that exhibit 8-receptor selectiv-
in close proximity to one another (741). ity. The dimer (DTE),, (221) with a long
Table 7.16 Opioid Receptor Affinities and Opioid Activity in the GPI and MVD of Dimeric
Enkephalin Analogs
spacer (n = 12) between the two tetrapeptides posal. This helical structure of dynorphin A
is a &selective ligand (the monomer Tyr-D- has been docked to a computational model of K
Ala-Gly-PheNH, is p selective) (750). In the opioid receptors (181).
pentapeptide series (DPE), the dimer with n Studies of dynorphin A have been compli-
= 2 (222) exhibits the greatest S selectivity in cated by its metabolic lability (758). In addi-
this series (751),but the selectivity of this pep- tion to inactivation by peptidase cleavage jn
tide is low [IC,, ratio (d6) = 6.51. Dimeric the N-terminus, cleavage in the C-terminus
analogs with antagonist activity were also yields shorter active peptides, which may have
examined. The dimeric derivative of the an- different receptor selectivity profiles from that
tagonist N,N-diallyl Leu-enkephalin (223) of the parent peptide. Dynorphin A-(1-8),
is approximately ninefold more potent than which is the predominant form of dynorphin A
the monomeric N,N-diallyl-Tyr-Gly-Gly-Phe- present in rat brain (759,7601,is less selective
LeuNHEt (7521, but the antagonist activity of
for K receptors than the longer peptides (see
the truncated dimer N,N-diallyl-Tyr-Gly-Gly-
Ref. 761). C-Terminal amidation enhances the
Phe-Leu-NHCH,CH,NH(AcPhe-Leu) sug-
gests that the enhanced activity of the full metabolic stability of dynorphin A-(1-13)
dimer is not due to bridging two 6-receptor (758), and therefore this modification is typi-
binding sites. cally incorporated into dynorphin A analogs.
The peptide bonds in dynorphin A-(I-11)NH2
have been replaced by reduced amide bonds to
6.3 Dynorphin Analogs increase metabolic stability (762, 763); this
Dynorphin has not been nearly as well studied modification was well tolerated by K receptors
as other smaller opioid peptides. Although in the C-terminal "address" sequence, but led
several peptides with high affinity for K recep- to decreased opioid receptor affinity when
tors are obtained from prodynorphin (see Sec- incorporated in the 16message" sequence.
tion 3.4 above), SAR studies have focused on An analog of dynorphin A - 1 8 E2078
derivatives of dynorphin A (see Refs. 656, 753 ([NMeTyr1,NMeArg7,~-Leu8]dynorphin A-(1-
for reviews). Dynorphin A is a heptadecapep- 8)NHEt) (764, 765), containing modifications
tide, but dynorphin A-(1-13) accounts for es- to stabilize the peptide to metabolism has
sentially all of the activity of the larger peptide been studied extensively in vivo. It exhibits a
(754). Further truncation of dynorphin A-(1- slight preference for K receptors in binding as-
13) from the C-terminus identified the basic says (see Table 7.17) and produces analgesia
7 after both i.v. and S.C.administration (764-
residues Arg and Lysl1 as important for K
receptor potency and selectivity (263). Thus, 766), apparently by spinal K receptors (767).
typically, dynorphin A-(1-13) or A-(1-11) has
been used as the parent peptide for further
modification.
The possible conformations of dynorphin A
have been studied by a variety of spectral tech-
niques (see Ref. 753 for a review). Like other
linear peptides, a variety of conformations 6.3.1 Linear Analogs. Early structural modi-
have been observed for dynorphin A, which fications were made in the C-terminal "ad-
depend on the experimental conditions. dress" sequence of dynorphin A (263) and fo-
Schwyzer (755) proposed a "membrane-assist- cused on the nonbasic residues (see Refs. 656,
ed" model for dynorphin's interaction with K 753). D-Pro was incorporated in place of Prolo
receptors, in which the N-terminal "message" in both dynorphin A-(1-11) (768) and A-(1-13)
sequence adopts an a-helical structure from (769) and was reported to enhance K-receptor
residues 1-9 when it binds to the receptor. selectivity (see Table 7.17). Ala and D-Ala were
NMR studies of dynorphin A bound to dode- substituted for Iles with retention of K-recep-
cylphosphocholine micelles (756, 757) ob- tor affinity and selectivity (769, 770). Replace-
served a helical structure in the N-terminal ment of the basic residues individually by
portion of the peptide, supporting this pro- N'-acetylated lysine (771) indicated that
6 Opioid Peptide Analogs 419
Table 7.17 Opioid Receptor Affinities and Opioid Activity in the GPI and MVD of Analogs of
Dynorphin A (Dyn A)
Ki (dfl KiRatio IC5, (nM)
Peptide K I*. 6 rdd6 GPI MVD Reference(s)
-
Agonist analogs
Dyn A-(1-13) 0.15 1.3 4.1 118.6127 1.7 78
Dyn Ia (225) 0.25 6.7 71 11261280 0.5 236
[~-Pro'~]Dyn A-(1-11) 0.032 2.0 7.5 1/621230 - -
E2078 (224) 1.9 4.5 27.2 112.4114 0.3 7.4
[N-Allyl,~-Pro'~]]Dyn
A-(1-11) 0.049 11 450 1/220/9,160 18 -
[N-CPM,D-P~O'O]D~~ A-(1-11) 0.020 9.6 560 1/480/28,000 2.2 -
[N-Benzy1,~-Prol0]Dyn
A-(1-11) 0.029 31 175 11107016,080 990 -
[Ala3]DynA-(1-11)NH2 1.1 210 730 1/190/660 1.7 -
[~-Ala~lDyn A-(1-11)NH2 0.76 260 1,000 1/350/1,300 8.1 -
[(R)-Atc4,~-Ala81Dyn
A-(1-ll)NH2 0.89 33 >10,000 1/37/>6,000 - -
[(s)-Atc4,D-Ala8]Dyn
A-(1-ll)NH2 9.5 88 >10,000 1/9/>1,000 - -
~~clo[C~s~,C~s'~]Dyn
A-(1-ll)NH2 0.28 0.27 1.6 llll6 1080 420
~~clo[Cys~,~-Pen'~]D~n
A-(1-ll)NHz 1.1 31 240 11281220 690 -
cyclo[~-Asp~,Da~~]Dyn
A-(1-13)NH2 0.22 0.49 10 112146 0.16 -
cyclo[~-hp~,Dap~]Dyn
A-(1-13)NH2 8.0 75 3,300 1191400 >5000 -
cyclo[~-hp6,DapglDyn
A-(1-13)NH2 2.6 4.4 48 1/2/19 46 -
cyclo[~-hp~,Lys~]Dyn
A-(1-ll)NH2 4.9 310 130 1/64/27 600 -
Antagonist analogs
[N&-Didlyl,~-ProlO]Dyn
A-(1-11) 21 135 350 1/6.5/17 190" -
[N&-D~CPM,D-P~O'~ID~~
A-(1-11) 0.19 3.9 166 1/21/880 -b -
Dynantin 0.82 213 163 112601200 3.9, -
0.63'
[Pro3]Dyn A-(1-11)NH2 2.7 5,700 8,800 112,10013,260 244, -
494'
Arodyn 10 1,700 5,800 1/170/580 -b -
JVA-901 (venorphin) 20 250 5,300 1/131270 -b -
made to reduce the motor effects observed 11)NH2(782). Interestingly, the peptide con-
with dynorphin Ia (774). Labeled derivatives taining (??)-Atc, which corresponds to a con-
of dynorphin A were prepared by attaching formationally constrained D-Phe analog,
functionalities to the C-terminus [DAKLI, possesses higher affinity for K and preceptors
[Arg1',13]dynorphin A-(1-13)- Gly-NH than the peptide containing the S isomer (Ta-
(CH2),NH-R, where R is fluorescein, lZ5I- ble 7.171, even though the ~ - P h eanalog ~
labeled Bolton Hunter reagent, or biotin exhibits relatively low affinity for these redp-
(775)l or to the side chain of Lys13 (biotin) in tors (Ki values of 8.9 and 146 nM, respec-
dynorphin A-(1-13) amide (776). tively). Both peptides exhibit negligible &n-
ity for 6 receptors. A novel conformational con-
Dynorphin Ia (225) straint, 4-aminocyclohexanecarboxylic acid,
has been incorporated into dynorphin A-(1-
13) amide in place of Gly2-Gly3 (783). Al-
Pro-Lys-Leu-Lys-Tyr-Leu-Phe-Am-Gly-Pro though the affinities of the two peptides for K
receptors is significantly reduced compared to
Several studies have focused on modifica- the parent peptide, it is interesting to note
tions in the N-terminal sequence of dynorphin that the peptides containing the cis and trans
A. Interestingly, the most K-selective deriva- isomers of 4-aminocyclohexanecarboxylicacid
tives of dynorphin A have been prepared by exhibit similar affinity for K receptors.
modifications in this 66message" sequence Cyclic derivatives of dynorphin A have
rather than in the C-terminal "address" se- been prepared by formation of both disulfide
quence. The N-terminal monoalkylation of and lactam linkages (see Ref. 656 for a review
[D-Prol0]dynorphinA-(1-11) with an allyl, cy- including extensive tables). The first cyclic
clopropylmethyl, or benzyl group results in dynorphin analog reported was cyclo[~-
marked enhancement in K-receptor affinity by Cys2,Cys5]dynorphin A-(1-13) (784), which is
a more potent agonist in smooth muscle assays
decreasing preceptor &nity (Table 7.17)
(777); NJ-dialkylation results in analogs than dynorphin A-(1-13). Subsequently, a va-
with antagonist activity (see below). An ala- riety of cyclic dynorphin analogs containing a
nine scan (770)verified the importance of Tyrl disulfide linkage in the C-terminus were de-
and Phe4 in dynorphin A-(1-13) for potency in scribed (785-787; see Ref. 656 for a tabular
smooth muscle assays and receptor affinity. summary of these analogs). Except for the an-
Although incorporation of D-amino acids in po- alogs cyclized through a D-amino acid in posi-
sition 2 can enhance metabolic stability and tion 5, these peptides with a constraint in the
yields analogs with high affinity for K recep- C-terminus retain high affinity for K receptors
tors, this modification significantly increases in radioligand binding assays. Three analogs
preceptor affinity and thus yields p-selective cyclized between positions 5 and 11 (cyclo-
analogs (263,778). In contrast, substitution of [Cys5,0-Pen1']-, cyclo[Pen5.11I-, and cyclo-
either Ala or D-Ala in position 3 of dynorphin [Pen5,~-Penl']dynorphin A-(1-11)NH2) show
A-(1-11)NH2 markedly enhances K-receptor increased K-receptor selectivity in the radioli-
selectivity (Table 7.17) (779); incorporation of gand-binding assays compared to that of the
other amino acids in this position, however, is linear parent peptide (Table 7.17). Interest-
ingly, a number of these cyclic disulfide-con-
generally less well tolerated (780). Recently,
[Pro3] dynorphin A-(1-11)NH2 was reported taining peptides, particularly those in which
to be a highly selective K-receptor antagonist position 5 is involved in the disulfide linkage,
show much lower potency in the GPI than ex-
(see below) (781).
pected from the binding assays; these discrep-
6.3.2 Conformationally Constrained Ana- ancies between the two assays were postulated
logs. Local constraints have been incorpo- to be due to different receptor subtypes
rated into dynorphin A. The conformationally present in the brain versus peripheral tis-
constrained phenylalanine derivative Atc (2- sues (785-787). A conformational search of
aminotetralin-2-carboxylicacid, Fig. 7.43) has s ~ ~cyclo[Cys5,~-Alas,Cys"l-
c y c l o [ C y ~ ~ , C ~and ]-
been incorporated in [D-Ala8]dynorphinA-(1- dynorphin A-(1-11)NH2 found a low energy
6 Opioid Peptide Analogs
Dynantin (2S)-Mdp-Gly-Gly-Phe-Leu-Arg-Arg-lle-Arg-Pro-LysNH2
TIPP-Dyn A Tyr-Tic-Phe-Phe-Leu-Arg-Arg-lle-Arg-Pro-LysNH2
Extacet AcArg-Phe-Met-Trp-Met-Arg-Arg-D-Ala-Arg-Pro-Ly~NH~
Arodyn AcPhe-Phe-Phe-Arg-Leu-Arg-Arg-D-Ala-Arg-Pro-Ly~NH~
JVA-901 1 AcTyr-Lys-Trp-Trp-Leu-Arg-Arg-D-Ala-Arg-Pro-Ly~NH~
venorphin +
a-helical conformation for the disulfide- agonist in the GPI, while retaining reasonable
bridged ring in these peptides, suggesting that affinity for K receptors in the radioligand-
this conformation may be important for the binding assays (792, 793). For the i to i + 4
potency of these peptides at central K recep- derivatives (794) cyclo[o-Asp3,Lys7]dynorphin
tors (788). When ~yclo[C~s~,Cys~~ldynorphin A-(1-11)NH2 exhibited the highest K-receptor
A-(1-11)NH2 bound to micelles was examined affinity and selectivity (Table 7.17); the cyclic
by NMR and molecular dynamics, conforma- peptide cyclo[Lys5,~-Asp9]dynorphinA-(1-
tions resulting from cis-trans isomerism 11)NH2also exhibited nanomolar affinity for
around the Arg9-Prolo amide bond were ob- both K and p receptors. The synthesis of novel
served (789),with the trans isoform exhibiting N-terminal to side-chain cyclic dynorphin A
5 8
a p turn from residues Cys to Ile , whereas analogs was recently reported (795,796).
the cis isoform contained a type I11 P turn
from residues Arg7 to Pro1'. 6.3.3 Dynorphin A Analogs with Antagonist
Conformational constraint through lactam Activity. Early attempts to prepare K-selective
formation has been examined in both the N- antagonists by modification of dynorphin A
terminus and C-terminus of dynorphin A met with limited success, and the analogs re-
(790-794). The initially reported derivatives ported generally exhibited weak antagonist
exhibited low Ke values for antagonism by nal- activity, residual agonist activity, and/or low
oxone (K, = 1.5-4.5 nM) in the GPI, suggest- selectivity for K receptors (797,798). In recent
ing that these peptides preferentially inter- years antagonist analogs with improved p h d -
acted with p receptors (affinities for K macological profiles have been identified.
receptors were not reported) (790). More re- Modifications of Tyrl have resulted in dynor-
cently, two series of dynorphin A-(1-13) amide phin A analogs with antagonist activity.
analogs constrained through a lactam linkage Incorporation of Phe in place of Tyrl in
between residues in either the i and i + 3 po- [D-Ala8]dynorphinA-(1-11)NH, resulted in a
sitions (791-793) or i and i + 4 positions (794) peptide that antagonized dynorphin A (Ke =
have been reported (Table 7.17). These con- 30-65 nM) in the GPI, but still exhibited weak
straints were chosen to be compatible with the agonist activity (772). The NJV-terminal dial-
helical conformation proposed by Schwyzer as kylation of dynorphin A fragments with either
the bioactive conformation for the N-terminal ally1 or cyclopropylmethyl (CPM) groups re-
sequence of dynorphin A (755). For deriva- sulted in analogs with antagonist activity
tives constrained between a D-Aspin position i (142, 799-801); the NJV-di(cyclopropy1-
and Dap (a$-diaminopropionic acid) in posi- methyl) derivative exhibits higher K-receptor
tion i + 3, the constraint is well tolerated by K selectivity than that of the NJV-diallyl peptide
receptors between positions 2 and 5 (791) and (142, 801). Recently, novel analogs of 2',6'-
also between positions 6 and 9 (792, 793), dimethyltyrosine, which lack a basic m i n e
whereas moving the constraint to residues 3 (Dhp and Mdp, Fig. 7.43), have been incorpo-
and 6 markedly decreases affinity for opioid rated in place of Tyrl in dynorphin A-(1-
receptors (792, 793). Consistent with the re- 11)NH2, resulting in [(2S)-Mdpl]dynorphin
sults for the cyclic disulfide-containing ana- A-(1-11)NH2 (dynantin, Fig. 7.45), which is a
logs cyclized through position 5, c y c l o [ ~ - highly K-selective peptide that is a potent an-
Asp5,Daps]dynorphin A-(1-13)NH2 is a weak tagonist at K opioid receptors (see Table 7.17)
Narcotic Analgesics
(802). [Pro3]dynorphin A-(I-11)NH2 was also which exhibits antagonist activity and greatly
reported to be a highly selective, but relatively enhanced affinity for cloned K receptors (808).
weak, K-receptor antagonist (see Table 7.17) In spite of the structural similarities to dynor-
(781). phin A, the SAR of venorphin is completely
Chimeric dynorphin A analogs, in which different from that of dynorphin A, suggesting
the N-terminal "message" sequence of dynor- that these two peptides interact with K recep,
phin A is replaced by small peptides with opi- tors in different ways (809,810). In venorphin
oid antagonist activity (Fig. 7.45), have also only Trp3, and not Tyr' or a basic functional-
been prepared as potential antagonists. Addi- ity, is required in the N-terminal "message"
tion of the C-terminal "address" sequence in sequence for high affinity for K receptors
these peptides significantly enhances K-recep- (8091,and Arg9, which is not important for the
tor affinity, but the receptor preference of the K-receptor affinity of dynorphin A, is the most
N-terminal sequence still often predominates. critical residue in the C-terminus (810).
Thus incorporation of the &receptor antago-
nist TIPP (Tyr-Tic-Phe-Phe) in place of the 6.4 Opioid Peptides with the Tyr-Pro-Phe
N-terminal sequence of [ ~ - P r o ' ~ ] d ~ n o r ~ h iSequence
n
A-(1-11)NH2 or the corresponding acid re-
sulted in peptides with affinity and antagonist
activity at K receptors, but which still prefer- 6.4.1 P-Casomorphin Analogs and the En-
entially bind to 6 receptors (803, 804). Incor- domorphins. p-Casomorphin was identified
poration of only Tic2 in position 2 produced over 20 years ago, so a number of analogs of
similar results for the dynorphin A-(I-11)NH2 this peptide have been reported. The hep-
derivative (805) or a slight (twofold) prefer- tapeptide p-casomorphin (2101, although ex-
ence for 6 over K receptors for the hibiting some selectivity for p receptors, is a
[D-Prol0]dynorphinA-(1-11) analog (804); in- weak opioid agonist [IC,, in the GPI = 57 pit4
corporation of N-MePhe in position 2 of (811)l. Shortening the peptide to the pen-
[D-Prol0]dynorphin A-(1-11) resulted in a tapeptide and tetrapeptide increases potency
greater (eightfold) preference for K over 6 re- (811,812),so generally analogs have been pre-
ceptors but little selectivity for K over p recep- pared of one of these smaller fragments. Con-
tors (804). version of the tetrapeptide to the C-terminal
Attachment of the C-terminal "address" se- amide to give morphiceptin (226; see Table
quence from [D-Ala81dynorphinA-(1-11)NH, to 7.18) (812) substantially increases both po-
the acetylated hexapeptide [Arg6]acetalin, tency and p-receptor selectivity. Further mod-
which is a preceptor antagonist, to give the ification of morphiceptin by incorporation of
peptide extacet increases K-receptor affinity D-Pro at position 4 and N-methylation at posi-
65-fold, resulting in nanomolar affinity for K tion 3 yields PL017 (227) (813), which is sig-
receptors (Ki = 6.6 nM), but it still preferen- nificantly more potent than morphiceptin and
tially binds to preceptors (Ki = 1.1 nM)(804). more p selective (see Table 7.18). D-Pro or D-
Examination of analogs of this lead peptide by Pip (pipecolic acid) in position 4 of p-casomor-
use of a combinatorial library led to the iden- phin-(1-5) also enhances potency in the GPI
tification of arodyn, a novel dynorphin A ana- (814).
log with higher affinity, much higher selectiv-
ity, and antagonist activity at cloned K Morphiceptin (226) Tyr-Pro-Phe-ProNH,
receptors (see Table 7.17) (806).
A Boc-protected tetrapeptide derived from
a sequence found in Philippine cobra venom Cisltrans isomerization occurs around
has been reported to have weak antagonist ac- amide bonds involving the nitrogen of proline
tivity at K receptors (807). Attachment of the and other N-alkyl amino acid residues. NMR
C-terminal "address" sequence of [ ~ - & i ~ ] - studies of both morphiceptin and PL017 found
dynorphin A-(1-11)NH, to this sequence re- that, whereas the major isomers were the all-
sulted in a novel acetylated dynorphin A ana- trans conformers, the second most common
log, JVA-901 (now referred to as venorphin), isomer (25%) had a cis arnide bond between
1r 6 Opioid Peptide Analogs
I
C
Table 7.18 Opioid Receptor Affinities and Opioid Activity in the GPI and MVD of p-Selective
[ Opioid Peptide Analogs
kI K, (nM) K, Ratio IC,, (nM)
1
k
Peptide P 6 SIP GPI MVD Reference
1 P-Casomorphin analogs <
if Morphiceptin (226)
PL017 (227)
63
11
30,000
7,250
475
660
318
34
4800
240
813
130
i m-cyclo[~-Om-Phe-D-Pro-Gly]
I (232) 0.88 13.2 15 2.1 4.9 825
1
L T~~-c~c~o[D-O~-~-N~~-D-P~O-
1I Glyl (233) 5.9 17.2
Tyr-CyClo[ D - O ~ ~ - ~ - N ~ ~ - D - P ~ O - D -
2.9 384 K, = 200-270" 825
i
Alal 0.76 72 95 600 K, = 5.4-6.0" 828
I
I Dmt-cyclo[~-Om-2-Nd-~-Pro-
I Glyl (234) 0.46 0.46 1 7.9 K, = 2.1-3.3" 830
1 CHO-Dmt-cyclo[~-Om-2-Nd-~-
1 Pro-Glyl 218 33 0.15 K,=216" K,= 16" 829
Dermorphin analogs
TAPP (Tyr-D-Ala-Phe-PheNH,) 1.5 625 409 255 780 831
DALDA (242) 1.7 19,200 11,400 3.23 800 831
[DmtllDALDA (243)b 0.14 2,100 14,700 1.4 23 832
Tyr-cyclo[~-Om-Phe-AspINH,
(244) 10.4 2,220 213 36.2 3880 833
Tyr-cyclo[~-Om-Phe-GluINH, 0.98 3.21 3.3 1.2 1.1 834
Tyr-cyclo[~-Om-Aic-GluINH, 4.21 209 50 7.21 36.5 834
JOM-6 (246) 0.29 24.8 86 - - 835
JH-54 ([PhellJOM-6, 247) 1.36 1,020 750 9.1 - 835
[D-HatllJOM-6(248) 0.39 58 150 -C -C 836
"Antagonist.
*K,( K ) = 22 nM.
"EC,, = 1.4 and 1500 nMin GTPyS assays using cloned p and 6 receptors, respectively
Tyrl and Pro2 (815). Therefore, Goodman and ration, were inactive. This led Goodman and
coworkers incorporated 2-aminocyclopentane coworkers to propose that the cis conforma-
carboxylicacid (2-Ac5c, 228, Fig. 7.46) into po- tion around the Tyr-Pro amide bond is re-
sition 2 of morphiceptin analogs to eliminate quired for the opioid activity of morphiceptin
possible cisltrans isomerization (816, 817). Of and its analogs (817). A more detailed model
the four possible stereoisomers only morphi- for the bioactive conformation of morphicep-
ceptin analogs containing cis-(1S,2R)-2-Ac5c, tin was developed based on the comparison of
which exhibits a structure similar to the cis a series of active and inactive analogs (818).
conformation of the Tyr-Pro amide bond, are Like morphiceptin and PL017, cisltrans
potent opioids, whereas the analogs with cis- isomerization occurs around the Tyr-Pro
(1R,2S)-2-Ac5c,which is similar to morphicep- amide bond in endomorphin-1, with similar
tin with the Tyr-Pro bond in a trans configu-
populations (75%trans, 25% cis) found for the
two conformations (819). Based on structural
comparison of the cis and trans conformations
of endomorphin-1 to other p- and &selective
opioid peptides, Podlogar et al. proposed that
the trans conformation was the bioactive form
(819). Recently, Schiller and coworkers re-
ported endomorphin-2 and morphiceptin ana-
Figure 7.46. Proline analogs. logs containing a pseudoproline derivative in
424 Narcotic Analgesics
Table 7.19 Opioid Receptor Affinities and Antagonist Activity in the GPI and MVD of TIPP
and Selected Analogsa
Ki (nM) Ki Ratio K, (nM)
Peptide 6 P 4 6 MVD GPI Reference(8)
TIPP (30) 3.0-5.9 -
TIPPNH, 14-18 IC,, = 1,700b
Tyr-D-Tic-Phe-PheNH, IC,, = 454b IC,, = 37'
TIPP[$I (31) 2.1-2.9 -
DIPP (236) 0.15 IC,, = 770'
DIPPNH2 (237) 0.20 IC,, = 18b
DIPP[$]-NH, (239) 0.54 IC,, = 7.7'
TICP (Tyr-Tic-Cha-Phe) 0.44
TICP[$I 0.22
Dmt-TicOH 5.7
- - - - - -
"Data for TIPP and TIPP[+] from Table 7.8 are included for comparison. Data for a number of other analogs are given in
Ref. 837.
'Agonist.
"Not active.
dIC,,.
'30% inhibition at 30 a.
fK, ((K)= 68 nM, EC5, = 0.18 nM for GTPyS binding at cloned preceptors.
SK, (K) = 1.3 nM;EC,, = 0.65 and 6.9 for GTPyS binding at cloned 6 and K receptors, respectively. Inactive at p receptors. ,
of the 6 antagonist TIP and the p agonist Tyr- ity compared to the parent peptides and excep-
D-Tic-PheNH, by molecular mechanics found tional 6-receptor selectivity (see Table 7.19).
compact structures for both peptides, but with This modification also enhances the metabolic
different patterns of aromatic ring stacking stability of the peptides. Whereas the dike-
(838). Superimposition of low energy confor- topiperazine of Tyr-Tic is inactive, the Dmt
mations of these two peptides found that the analogcyclo[Dmt-Tic] (235, Fig. 7.48) exhibits
Phe3 residues were on opposite sides of the 6-receptor affinity (Ki = 9.6 nM) and weak h-
plane defined by the Tic residue, providing a tagonist activity (K, = 3.98 pI4) in the MVD
possible explanation for the differences in ac- (845).
tivity observed for the two peptides. Compar- A variety of structural modifications have
ison of TIP to the nonpeptide 6 antagonist nal- been made to all positions of TIPP (see Refs.
trindole found good spatial overlap of the 837, 846 for recent reviews), and these can
N-terminal m i n e and aromatic rings in the have a profound effect on the activity profile of
peptide with the corresponding groups in the these peptides. Numerous substitutions have
alkaloid. In this model the Tyr-Tic peptide been made in position 3, and the results indi-
bond is trans. In an alternative model based on cate that an aromatic residue is not required
the weak dipeptide 6 antagonist Tyr-TicNH, in this position for 6 antagonist activity. Thus
this bond is cis (839). the cyclohexylalanine (Cha) analogs of both
Further examination of the possible confor- TIPP and TIPP[qI (TICP and TICP[Vl, re-
mations of a series of TIPP analogs, including spectively) are approximately 10-fold more po-
derivatives of TIPP[qI (311, by molecular me- tent as 6-receptor antagonists than the parent
chanics found low energy conformations consis- peptides (847) (see Table 7.19). Interestingly,
tent with the model containing a trans Tyr-Tic incorporation of D-amino acids containing a
bond, but the model with a cis amide bond still P-methyl group can have a profound effect on
remained plausible (840). Other modeling stud- the efficacy at 6 receptors (848,849). Thus, the
ies involving TIPP found conformations consis- (2R,3R)-P-MePhe3derivative of TIPPNH, is a
tent with both the trans (841, 842) and the cis 6 antagonist, whereas the (2R,3S)-p-MePhe3
conformations (842). Therefore Schiller and isomer is a 6 agonist, and incorporation of
coworkers prepared TyrlIr[CH,NH]Tic-Phe- p-Me-~-Tic' derivatives in Tyr-D-Tic-Phe-
PheOH and Tyrq[CH,NH]MeTic-Phe-PheOH PheNH, converts the peptide from an agonist
(MeTic = 3-methyl-1,2,3,4-tetrahydroisoquino- to an antagonist. Iodination of Tyrl in TIPP
line-3-carboxylic acid, Fig. 7.43) in which a cis converts the peptide from an antagonist to a
amide bond between the first two residues is 6-selective agonist (850); the Tyr(3'-1)' ana-
sterically forbidden (843). The Sreceptor affin- logs of TIPP[qI, TICP, and TIP, however, are
ity and antagonist activity of these peptides, al- 6 antagonists, thus illustrating the subtleties
though 20- to 40-fold lower than TIPP, is consis- of the effects of modifications on efficacy at 6
tent with a trans conformation as the bioadive receptors (837,846).
conformation for TIPP. Interestingly, these Substitution of Dmt in position 1 of TIPP
modifications substantially increased p-recep- and analogs yielded DIPP (Dmt-Tic-Phe-Phe,
tor affinity, so that Tyrq[CH,NH]MeTic-Phe- 236, Fig. 7.48) and related peptides. DIPP is
PheOH was a nonselective antagonist at both p an extremely potent 6 antagonist (Table 7.19)
and 6 receptors (< a two-fold difference in K, and like TIPPNH, is also a full agonist in the
values in the GPI and MVD). GPI (851, 852). As noted earlier the nonpep-
During examination of TIPP by NMR in tide naltrindole can prevent the development
deuterated DMSO spontaneous degradation of morphine tolerance and dependence in mice
through diketopiperazine formation occurred (2251, and therefore compounds with mixed p
(844). This led Schiller and coworkers to pre- agonist/S antagonist activity could have ther-
pare TIPP[q] (31, Fig. 7.5) and the tripeptide apeutic potential. Thus, Schiller and cowork-
TIP[qI (112) containing a reduced peptide ers also incorporated Dmt into position 1 of
bond between Tic2 and Phe3. These analogs TIPPNH, and TIPP[YINH2 to enhance p ag-
exhibit increased 6-receptor antagonist po- onist activity; the resulting DIPP[q]-NH,
tency in the MVD and higher S-receptor affin- (239) was the first compound with balanced p
6 Opioid Peptide Analogs
HO.
agonistI8 antagonist properties (229, 853). consisted of only the Tyr-Tic dipeptide rather
DIPP[V]-NH, is a potent analgesic after i.c.v. than the tripeptide Tyr-Tic-Phe, Temussi and
administration (three times more potent than coworkers synthesized Tyr-IJD-Tic-NH, and
morphine) and produces less acute tolerance Tyr-LID-Tic-AlaNH,(839). Although they ex-
than morphine and no physical dependence hibited much lower potency than TIPP and
with chronic administration (229).The tripep- TIP, the shorter peptides containing Tyr-L-Tic
tide analogs Dmt-Tic-Ala-X (where X = OH or were &selectiveantagonists; peptides contain-
NH,) also exhibit high affinity for 8 receptors ing the Tyr-D-Tic sequence were nonselective
and are potent antagonists in the MVD; the agonists. Lazarus and coworkers explored the
arnide derivative retains appreciable p-recep- structure-activity relationships of Tyr-Tic in
tor affinity and is a weak agonist in the GPI considerable detail (see Refs. 837,855,856 for
(854). Replacement of Dmtl with N,2',6'-tri- reviews, including extensive tables of ana-
methyltyrosine (NMeDmt) in DIPPNH, de- logs). The dipeptide Dmt-TicOH was initially
creases efficacy at p receptors, so that reported to have exceptionally high 8-receptor
NMeDmt-Tic-Phe-PheNH, is a partial agonist affinity and selectivity (854), although other
and NMeDmt-TicV[CH,NH]Phe-Phe-NH, is researchers subsequently reported lower val-
an antagonist in the GPI (229). ues (857, 858) (see Table 7.19). Modifications
Based on the hypothesis that the "mes- examined have included N-terminal alkyla-
sage" domain in these 8 antagonist peptides tion, which enhances 8-receptor antagonism
Narcotic Analgesics
(Ki = 2.0 pM), but this C-terminal extension morphins (264). TAPP analogs in which either
does not enhance potency in the GPI. Dimeric Phe3 or Phe4 was nitrated were prepared to ex-
derivatives of dermorphin fragments have amine whether the Phe3 aromatic ring of der-
been prepared by bridging two monomers morphin interacts with a different subsite on
with hydrazine or diamines of various lengths opioid receptors than the Phe4 aromatic ring of
(873). Di-dermorphin, [(dermorphin-NH-),I, the enkephalins. Examination of Phe(p-NO,)-
in which two dermorphin molecules are linked containing analogs of dermorphin, morphicep-'
by hydrazine, exhibits fivefold higher affinity tin, and various enkephalin analogs found that
for preceptors and similar p-receptor selectiv- p-nitro substitution in Phe3 of dermorphin and
ity as dermorphin. Di-tetra-dermorphin, morphiceptin causes large decreases in potency
[(Tyr-D-Ala-Phe-Gly-NH-CH,),] and other in the GPI, whereas this substitution in Phe4 of
dimeric derivatives exhibit greater affinity for p- and &selectiveenkephalin analogs enhances
6 receptors, and hence decreased p-receptor potency in smooth muscle assays (880).Incorpo-
selectivity compared to that of the monomer. ration of Phe(p-NO,) in position 3 of TAPP de-
Glycosylated derivatives of dermorphin creases receptor affinity, whereas incorporation
were prepared by attaching p-D-glucoseto the in position 4 increases affinity, consistent with
hydroxyl of a Ser or Thr, or galactose through the concept of two distinct receptor subsites for
a C-alinkage to Ala in position 7 (874-876). these aromatic residues on opioid receptors
Although glycosylation decreased p-receptor (881). Bulky aromatic amino acids such as tryp-
affinity twofold, the penetration of the blood- tophan or naphthylalanine are well tolerated in
brain barrier was significantly higher for the positions 3 and 4 of TAPP and yield even more
glycosylated derivatives and they exhibited lipophilic peptides (881).
twice the antinociceptive activity of dermor- Incorporation of D-Arg in position 2 of der-
phin (875, 876). The enhanced BBB penetra- morphin and tetrapeptide analogs yields pep-
tion by the C-a-galactoside analog suggested tides that are potent opioids in antinociceptive
that the glucose transporter was not involved assays in mice (879,882). The tetrapeptide de-
in the transport (875). rivative Tyr-D-Arg-Phe-Sar(TAPS) is a potent
Amino acid substitutions have been exam- opioid in antinociceptive assays and causes re-
ined in every position of dermorphin (see Ref. spiratory stimulation, rather than respiratory
663 for a review). An alanine scan of the pep- depression, that is antagonized by naloxona-
tide indicated that substitutions in positions zine (883);TAPS also antagonizes the respira-
4, 6, and 7 are well tolerated, whereas substi- tory depression caused by dermorphin. On the
tution particularly in positions 1 or 2, but also basis of these results TAPS has been postu-
in positions 3 or 5, results in large decreases in lated to be a p, agonist and a p2antagonist in
potency in the GPI (877). A D-amino acid in vivo (883). In contrast, incorporation of L-Tic
position 2 is important for activity, and the in position 2 of dermorphin converts the pep-
~-Ala'peptide is virtually inactive (<0.1% the tide to a &receptor antagonist (884).
potency of dermorphin). Tetrapeptide analogs Schiller and coworkers postulated that pos-
containing D-methioninesulfoxide in position itively charged ligands should display p-recep-
2 are also potent p-selective agonists (878). tor selectivity (831) on the basis of Schwyzer's
Substitutions for Gly4 are well tolerated, proposal that p receptors are located in an an-
particularly in the tetrapeptide derivatives. ionic membrane compartment (885). Consis-
Sarcosine (NMeGly, Sar) at position 4 in tet- tent with this concept they found that incor-
rapeptide derivatives enhances opioid activity poration of a positively charged residue in
in antinociceptive assays (879). Substitution position 4 of tetrapeptide dermorphin deriva-
of Phe in position 4 of the tetrapeptide amide tives enhances p-receptor selectivity by de-
yields the dermorphidenkephalin hybrid TAPP creasing affinity for 6 receptors (831). The
(Tyr-D-Ala-Phe-PheNH,)(831), which is a po- combination of a D-Argin position 2 with a
tent p-selective agonist (see Table 7.18). This second basic residue in position 4 yielded
peptide can also be considered an analog of en- the polar peptide Tyr-D-Arg-Phe-LysNH,
domorphin-2, although TAPP was synthesized (DALDA, 242), which carries a net charge of
several years before the discovery of the endo- +3 (831) and exhibits exceptional p-receptor
Narcotic Analgesics
selectivity in binding assays (see Table 7.18). 6.5.1.2 Conformationally Constrained Ana-
Quaternization of the side chain amine of Lys4 logs. Dermorphin analogs containing a local
in DALDA and related analogs is well toler- constraint were prepared by incorporation of a
ated, and the resulting analogs retain potent dipeptide mimetic in place of Phe3-Gly4. The
in vivo antinociceptive activity in the mouse Aba3-Gly4 (see Fig. 7.43) analog of dermor-
writhing assay after S.C.administration (886). phin retains high affinity at p receptors and
The antinoceptive effects of DALDA and the potency in the GPI, whereas this structural
quaternary derivatives are substantially re- modification increases S-receptor affinity and
duced by the quaternized antagonist N-meth- potency in the MVD 17- to 25-fold (892). A
yllevallorphan, suggesting that these peptide number of heterocycles are also tolerated as
analogs have a high degree of peripheral an- bond replacements for the Phe3-Gly4 peptide
bond (893).
tinociceptive activity in this assay. The distri-
Cyclic tetrapeptide analogs of dermorphin
bution of DALDA to the CNS is limited (887),
with the structure Tyr-cyclo[~-X-Phe-ylNH2
and the antinociceptive activity of DALDA in were prepared by Schiller and coworkers. The
the hot plate test after S.C. administration is 13-membered ring cyclic peptide Tyr-cyclo[~-
low (888).The in vivo distribution of DALDA Om-Phe-AspINH, (244, Fig. 7.49) exhibits
has been examined in pregnant sheep (889), high selectivity for p receptors (see Table
and DALDA was not detected in any of the 7.181, whereas the more flexible peptide Tyr-
fetal plasma samples. This is in contrast to cyclo[~-Lys-Phe-GluINH, with a 15-mem-
meperidine and morphine, which undergo bered ring is nonselective (833). Peptides
rapid transfer across the placenta to the fetus, Tyr-cyclo[~-Asp-Phe-OrnINH, (894) and Tyr-
and suggested that DALDA could be a promis- cyclo[~-Asp-Phe-DablNH,(Dab = a, y-diami-
ing opioid for obstetrical use. nobutyric acid) (895) in which the lactam link-
Replacement of Tyrl by Dmt results in a age is reversed also exhibit high affinity and
peptide [DmtllDALDA (243) (832) with 10- p selectivity. The antiparallel cyclic dimers
fold higher affinities for both p and S receptors (Tyr-D-Om-Phe-AspNH,), and (Tyr-D-Asp-
and 200-fold higher affinity for K receptors Phe-OrnNH,),, obtained as by-products dur-
(890). [DmtllDALDA is 220 and 3,000 times ing the synthesis of the cyclic monomers, have
more potent than DALDA and morphine in similar affinities for both p and 8 receptors
the rat tail flick test after i.t. administration. (894).
[DmtllDALDA also inhibits norepinephrine Various modifications to Phe3 in these cy-
uptake in spinal cord synaptosomes, and this clic peptides have also been examined. Many
dual action may contribute to its antinoci- of the modifications (e.g., p-nitro or N-methyl
ceptive potency. Both [Dmtl]DALDA and substitution, or shortening the side chain) de-
DALDA exhibit longer durations of antinoci- crease preceptor affinity by 25-fold or more
ceptive action than morphine after i.t. admin- (895). Although the cyclic structure restricts
istration to rats (7 and 13 h, respectively, ver- the conformation of the peptide backbone,
sus 3 h) (890). In sheep these peptides have there is still considerable conformational flex-
longer elimination half-lives (1.5 and 1.8 h, ibility around the Tyrl and Phe3 side chains
respectively) than that of either morphine (896). Conformational constraint of Phe3 by
(20-30 min) or the much more hydrophobic incorporation of 2-aminoindan-2-carboxylic
p-selective peptide DAMGO (15 min) (891). In acid (Aic, Fig. 7.43) into the relatively nonse-
contrast to morphine and DALDA, [Dmt1]- lective peptide [Ki ratio (Sip) = 31 Tyr-cyclo[~-
DALDA did not cause respiratory depression Om-Phe-GluINH, markedly enhances p-re-
at the doses examined (832),suggesting that it ceptor selectivity (see Table 7.18) (834). This
could be a drug candidate for intrathecal anal- is a direct consequence of conformational re-
gesia. striction because incorporation of the acyclic
derivatives a-methylphenylalanine or 2'-
X-D-Arg-Phe-LysNH, methylphenylalanine in position 3 does not
DALDA (242) X=Tyr significantly change preceptor selectivity. In-
[DmtllDALDA (243) X = Dmt terestingly, the peptides containing L- and D-2-
6 Opioid Peptide Analogs
aminotetralin-2-carboxylic acid (Atc, Fig. = 4.4 and 12 for the L-Leu and D-Leu analogs,
7.43) in position 3 had similar preceptor af- respectively]. The analog Tyr-cyclo[~-Dab-
finity and high p-receptor selectivity (834). Phe-gPhe-S-mLeu] containing a reversed
The restricted conformation of the side chain amide bond between Phe4 and Leu5 is also a
of residue 3 in the K c analogs was apparent p-receptor selective agonist. Interestingly, re-
from comparisons of molecular dynamics sim- versal of a second amide bond in the side chain
ulations for the Phe3 versus Kc3-containing lactam linkage to give Tyr-cyclo[~-Glu-Phe-
peptides (897). Further conformational con- gPhe-rLeu1 results in a &elective derivative
straint of Tyr-cyclo[~-Om-Kc-GluINH,by re- [IC,, ratio (GPIIMVD) = 111,which exhibits a
placement of Tyrl with L- or D-Hat yields pep- considerably different conformation from that
tides with only two freely rotatable bonds exhibited by the other retro-inverso analogs
(245, Fig. 7.49) (897); interestingly, both di- that preferentially interact with p receptors
astereomers exhibit reasonable preceptor af- (899).
finity and selectivity [Ki ( p ) = 20-30 nM, Ki In the case of peptides cyclized through a
ratio (Sip) = 12-191. disulfide or dithioether linkage, the receptor
Cyclic pentapeptide derivatives of Tyr-cy- selectivity depends on the linkage. The cyclic
C~O[D-Dab-Phe-Phe-(D- or L-)Leu] containing disulfide analog Tyr-cyclo[~-Cys-Phe-Cysl-
retro-inverso modifications were prepared by NH, exhibits 10-fold greater affinity for S re-
Goodman and coworkers (898). The parent ceptors than the corresponding lactam Tyr-
peptides 'lj~-cyclo[~-Dab-Phe-Phe-D/L-Leu] are cyclo[~-Asp-Phe-Om]-NH,and is therefore
potent agonists in the GPI with relatively low less p-receptor selective [IC,, ratio ( 6 1 ~=) 341
p-receptor selectivity [IC,, ratio (MVDIGPI) (895). The cyclic tetrapeptide JOM-13 Tyr-cy-
Narcotic Analgesics
"Data for [D-Alaz]deltorphinI and I1 from Table 7.13 are included for comparison.
bPartial agonist.
exhibited high 8-receptor affmity and selectiv- acid causes a large decrease in 8-receptor af-
ity, the analog with L-Met had very low affinity finity (909). As with most opioid peptides,
and potency in the MVD, indicating the impor- other modifications of Tyrlare also generally
tance of a D-amino acid in this position. As was not tolerated, except for substitutions by con-
found for deltorphin, the D-Ala in position 2 of formationally restricted derivatives of Tyr
[D-Ala2]deltorphin1is also important for S-re- (see Section 6.5.2.2 below). A variety of struc-
ceptor affinity and selectivity, and the ~-Ala' tural modifications have been examined at po-
analog has extremely weak activity in the sition 3 of [D-Ala2]deltorphin I, including in-
MVD (IC,, > 1 a) (106). The exception to corporation of aromatic, heterocyclic, and
the requirement for a D-amino acid in position nonaromatic amino acids (see Ref. 904) and
2 is [Tic2]deltorphin I, which retains high af- conformationally constrained derivatives (see
finity and exhibits increased selectivity for 8 Section 6.5.2.2 below). The effects of substitu-
receptors (Table 7.20) (907); this substitution tion on the phenyl ring of Phe3 vary with the
decreases efficacy, however, and in the MVD substituent. para-Substitution with a halo-
this analog is a partial agonist. In deltorphin gen, but not an m i n e , nitro, hydroxyl, or
substitution of D-Ala, as is found in methyl group, in [ ~ A a ~ l d e l t o r p hIi nis well
[D-Ala2]deltorphinI and 11, for o-Met is well tolerated by 6 receptors, and p-bromo substi-
tolerated at 8 receptors, although it increases tution enhances 8-receptor selectivity in both
preceptor affmity and potency in the GPI binding and smooth muscle assays (910-912).
(908, 909). In contrast, substitution of D-Met The heterocyclic phenylalanine analog 342-
in position 2 of [D-Ala2]deltorphinI or 11 thieny1)alanine is well tolerated in position 3
causes a large decrease in 8-receptor aMinity by 8 receptors, but other heterocyclic aromatic
and selectivity (908, 909). amino acids, such as the pyridylalanine deriv-
Modifications to the aromatic residues in atives or His, significantly decrease 8-receptor
the N-terminal "message" sequence have been affmity (912). An aromatic residue is not re-
examined, mostly in [D-Ala2]deltorphinI or 11. quired in position 3 of [D-Ala2]deltorphinI,
Replacing either Tyrl or Phe3 by a D-amino and the analogs with Cha and even Leu or
Narcotic Analgesics
norleucine (Nle) exhibit &receptor affinity due in position 4. Replacement of Asp and Glu
only six- to sevenfold lower than that of the in this position with Asn and Gln, respectively,
parent peptide (Table 7.20). QSAR analysis of results in compounds that maintain brecep-
the effect of substitution in position 3 sug- tor affinity, but have enhanced p-receptor af-
gested that the binding site around this side finity and thus significantly decreased 6-recep-
chain is very similar for 6 and p receptors tor selectivity (908, 914, 915). A variety of
(913). Substitution of either the a-carbon or other amino acid substitutions in this position
3
nitrogen of Phe with a methyl group, which are well tolerated by 6 receptors but signifi-
can alter the conformation of the peptide cantly enhance p-receptor affinity (908, 919).
backbone, is not tolerated (911). Thus the negative charge at position 4 is not
The C-terminal truncation of the deltorphins necessary for &receptor interaction, but con-
can affect both &receptor affinity and selectiv- tributes significantly to 6-receptor selectivity
ity. Deamidation of both [D-Ala21deltorphinI by interfering with interaction with p recep-
and I1 causes significant decreases in &receptor tors. Consistent with this, substitution of Asp/
affinity (25- to 50-fold) and 6 selectivity (908, Glu by His, the residue found in this position
914, 915). Although there is one report of a of deltorphin, does not affect &receptor affin-
large (90-fold) decrease in 6-receptor affinity ity but increases preceptor affinity (908,919).
upon conversion of deltorphin to the C-termi- Replacement of Asp/Glu by hydrophobic resi-
nal acid (914),there are other reports that de- dues [e.g., Phe (see Table 7.20), a-aminobu-
amidation of this heptapeptide causes only a tyric (Abu), or a-aminoisobutyric acid (Aib),
small decrease (916), or even a slight increase and the conformationally restricted l-amino-
(9151, in &receptor affinity. Shortening of ei- cycloalkane derivatives1 substantially in-
ther [D-Ala21deltorphinI or deltorphin from creases p-receptor affinity, resulting in ana-
the C-terminus causes progressive decreases logs with nanomolar affinity for both 6 and p
in &receptor affinity and selectivity and po- receptors, which has been referred to as "opi-
tency in the MVD (908, 915, 916). The oid infidelity" (903). QSAR analysis of substi-
hexapeptide and pentapeptide derivatives of tutions in this position has also been per-
[D-Ala2]deltorphinI retain 6-receptor selectiv- formed (913), and suggests that the receptor
ity (908); the corresponding deltorphin frag- binding site for this residue is quite different
ments, although retaining high affinity for 6 for 6 versus preceptors. Analysis of binding to
receptors, are nonselective (916). The N-ter- 6 receptors suggested at least a partial positive
mind tetrapeptide amide derivative of deltor- charge in the binding pocket, with both elec-
phin is selective for preceptors (917,918) [the trostatic and van der Wads forces, but not hy-
tetrapeptide acid exhibits low affinity for both drogen bonding, contributing to receptor
p and 6 receptors (91811, indicating that the binding.
C-terminal "address" sequence is capable of In positions 5 and 6 of [D-Ala2]deltorphin11
changing receptor type selectivity. Indeed, replacement of Val5 and Val6 individually by
swapping the C-terminal tripeptide se- Ala suggested that Val5 is more important
quence of dermorphin and deltorphin re- than Val6 for &receptor selectivity (920). Re-
verses the receptor selectivity profile [i.e., placement of valine in both positions by nor-
5 6 7
p
[Leu ,Met ,As ]dermorphin is S selective leucine, Ile, or y-methylleucine enhances
(91611. The N-terminal tetrapeptide amide both 6-receptor affinity and selectivity four- to
fragment of [D-Ala2]deltorphin1 (Tyr-~-Ala- eightfold (920). Replacing Gly7 in [~-Ala']-
Phe-AspNH,) shows a slight preference for p deltorphin I with Asp, which is found in this
receptors (908,915), but its affinity (Ki = 100- position in deltorphin, decreases affinity for
195 nM) and selectivity for p receptors is both 6 and p receptors approximately 10-fold
much lower compared to that of the N-termi- (919).
nal tetrapeptide amide fragment of deltorphin Amino acid substitutions in the C-terminus
[Tyr-D-Met-Phe-HisNH,;Ki (p) = 8.0 nM]. of deltorphin have also focused mainly on the
In the C-terminal "address" sequence of charged residues His4 and Asp7. As was found
[D-Ala2]deltorphinI and I1 considerable atten- for Asp4/G1u4 in [D-Ala2]deltorphin I and 11,
tion has focused on the role of the acidic resi- His4 appears to play an important role in the
F
6
Ej
P
&receptor selectivity, but not affinity, of del- riety of other substitutions for Met6 in deltor-
torphin. Thus a variety of amino acid substi- phin are well tolerated (see Ref. 9041,
tutions for His4, including Gly and aromatic suggesting that residue 5 is more important
amino acids, are well tolerated by 6 receptors, for receptor interaction. However, incorpora-
but they enhance preceptor affinity and thus tion of an additional acidic residue in position
decrease 6-receptor selectivity (915). Interest- 6 decreases breceptor affinity (914,923).
ingly, substitution by Lys markedly decreases 6.5.2.2 Conformationally Constrained And-?
&receptor affinity (915), and substitution by logs. The conformationally restricted tyrosine
Asp, as is found in [D-Ala2]deltorphinI, de- analogs Dmt and Tmt (Fig. 7.43) have been in-
creases both 6- and preceptor affinity (908, corporated into position1of [D-Ala2]deltorphin 1-
914, 915). Replacement of His4 by D-H~s only or 11. Substitution of Dmt for Tyr in
decreases 6-receptor affinity and selectivity [D-Ala2]deltorphin I1 markedly enhances p-re-
4-6-fold, whereas des-His4 analogs have ceptor affinity, resulting in a compound with
markedly lower &receptor affinities and selec- nanomolar affinity for both receptors (Table
tivities (909),suggesting that His4 may play a 7.20) (924). In contrast [D-Ala2]deltorphinI
role in maintaining the necessary spatial ori- analogs containing either of the 2S isomers of
entation of the C-terminal sequence of deltor- Tmt, which has the additional P-methyl
phin relative to the N-terminus. Further mod- group, exhibited high Breceptor affinity and
ifications of His4 have been examined (921). selectivity; the 2S,3S isomer exhibited greater
N-Methylation on either nitrogen of the 6-receptor selectivity than that of the parent
imidazole enhances breceptor selectivity, peptide (IC,, ratio = 5,740 versus 3,500)
whereas N"-methylation of His4 decreases (728).
both &receptoraffinity and selectivity. Substi- Linear peptides containing conformationally
tution of the conformationally constrained constrained Phe analogs in position 3 have also
residue Tic for His4 is well tolerated by 6 re- been prepared. The conformationally restricted
ceptors, but decreases 6-receptor selectivity phenylalanine analogs Aic and both isomers of
(921).The charge on Asp7 also does not appear Atc (Fig. 7.43) are well tolerated at this position
to be critical for interaction with 6 receptors, by 6 receptors, although the Aic3 analog is less
but contributes to 6-receptor selectivity by ad- selective for 6 receptors than are the Atc3deriv-
versely affecting preceptor affinity. Thus re- atives of [D-Ala2]deltorphin I (907, 911). The
placement of Asp7 by a nonacidic amino acid similar potencies of both diastereomers of
decreases 6-receptor affinity by less than two- [D-A12,Atc3]deltorphinI are in sharp contrast
to threefold, but enhances preceptor affinity to the large (13- to 50-fold) decrease in potency
so that 6-receptor selectivity decreases (914, of the ~ - P h eanalog
~ compared to that of
2
915,919). [D-Ala ]deltorphin I, suggesting that the recep-
A number of residues have been incorpo- tor binding mode of the ~ - A t analog
c~ may be
rated in position 5 of deltorphin, and generally different from that of the ~ - P h derivative
e~
the 6 receptor affinities of these analogs corre- (907). The configuration of Atc in the diastereo-
late with the hydrophobicity of the residue in meric peptides was not determined in these ini-
this position (see Ref. 904). Branched hydro- tial studies, although it was in subsequent deriv-
phobic amino acids Val, Ile, and y-methyl- atives of [~-Ala~,Ile~,~]deltorphin 1and 11(929).
leucine have been incorporated into this posi- The four isomers of p-MePhe were incorpo-
tion of deltorphin to enhance metabolic rated into both [D-Ala2]deltorphinI and del-
stability; these analogs have Breceptor affini- torphin (930). The peptides containing the 2 s
ties and selectivities similar to those of the isomers exhibit higher Breceptor affinity
parent peptide (902). N-Alkyl amino acids and agonist activity in the MVD than the
were also introduced at position 6 to decrease analogs with the 2R isomers for both
metabolism of the Leu5-Met6 bond; these an- [~-Ala~]deltor~hin I and deltorphin, consis-
alogs possess the desired stability to degrada- tent with the preference for L- over D-Phein
tion by both rat brain and plasma, and the this position. The stereochemistry of the
N-nBuGly6 analog exhibits exceptional selec- methyl group at the P position, however, had
tivity for 6 receptors (Table 7.20) (922). A va- different effects on the 6 affinity and agonist
Narcotic Analgesics
activity of the two peptides, with the 3 s iso- reduced susceptibility to conformational per-
mer preferred in [D-Ala2]deltorphinI and the turbation, of the tetrapeptide. Other substitu-
3R isomer preferred in deltorphin. Substitu- tions that affect only the side chain of residue
3 4
tion with the dipeptide mimetic Aba -Gly is 3, particularly homophenylalanine (Hfe), had
also reasonably well tolerated in [ ~ - A l a ~ l - significantly different effects on &receptor af-
deltorphin I1 (IC,, = 5.0 nM) (892).Other con- finity in the two series. Hfe substitution does
strained residues in position 3 e . Tic or not adversely affect breceptor affinity or po-
NMePhe) result in drastic decreases in S-re- tency in the MVD when it is incorporated into
ceptor affinity (907,911). JOM-13 (although it does increase preceptor
As in the case of cyclic enkephalin analogs, affinity 20-fold so that &receptor selectivity
conformationally constrained &selective pep- decreases), but it decreases breceptor affinity
tides with the sequence Tyr-D-X-Phe were also
and potency in the MVD 100- and 25-fold
prepared by cyclization through a disulfide
when incorporated into DPDPE (932). para-
bond. Cyclization between D-Cys in position 2 3
and D-Pen in position 4 yielded the tetrapep- Substitution of Phe with fluorine, chlorine,
tide derivative Tyr-cyclo[~-Cys-Phe-D-Pen]-or a nitro group in JOM-13 is well tolerated by
OH (JOM-13, 249), which contains a fairly 6 receptors, similar to the results found for
DPDPE analogs (933). An aromatic residue at
position 3 is not essential for &receptor inter-
action, and incorporation of Cha at position 3
of JOM-13 causes less than a threefold de-
crease in breceptor affinity (934); not surpris-
ingly, this modification also enhances p-recep-
tor affinity so that [Cha3]JOM-13 exhibits
only a fourfold lower Kivalue for 6 receptors
than for p receptors.
The conformation of residue 3 has been re-
stricted by incorporation of p-MePhe (935)
and dehydrophenylalanine (APhe) (927) in
this position. Both the 3S and 3R diaste-
rigid 11-membered cyclic structure and which reomers of L-p-MePhe were compatible with
exhibits high affinity and selectivity for 6 re- interaction with S receptors, with the peptide
ceptors (Table 7.20) (900). Conformational containing the 2S,3S isomer exhibiting ap-
analysis by NMR, X-ray crystallography, and proximately eight-fold higher affmity than
computational analysis (931) indicated a sin- that of the peptide containing the 2S,3R iso-
gle preferred conformation for the cyclic tri- mer. Surprisingly, the peptide containing the
peptide portion of JOM-13, but the key phar- 2R,3R isomer of D-p-MePhealso exhibits high
macophoric elements, the exocyclic Tyrl and affinity and selectivity for 8 receptors. Exami-
3
the Phe side chains, still exhibit conforma- nation of this latter peptide by molecular mod-
tional flexibility. eling suggested a side chain conformation of
A variety of substitutions have been made Phe3 in JOM-13 with x1 about -60" when the
for Phe3 in JOM-13 (932-935). Comparison of peptide interacts with S receptors (935). On
3
Phe substitutions in JOM-13 to the same sub- the basis of these results, it was predicted that
4
stitutions for Phe in DPDPE found that mod- the JOM-13 analog containing AZPhe (Fig.
ifications that would be expected to affect the 7.43), but not AEPhe, could be superimposed
conformation of the peptide backbone (e.g., in- on the resulting proposed bioactive conforma-
corporation of NMePhe or D-Phe) had delete- tion of JOM-13; as predicted, [AZPhe3lJOM-
rious effects on breceptor affinity and potency 13 exhibited nanomolar affinity and higher
in the MVD in both series (932); the effects of &receptor selectivity than JOM-13, whereas
these substitutions were generally greater in incorporation of AEPhe in position 4 resulted
the pentapeptide series, however, which may in a 60-fold decrease in 8-receptor affinity
be due to the greater rigidity, and therefore (927).
6 Opioid Peptide Analogs
Table 7.21 Opioid Receptor Affinities and Antagonist Activity in the GPI and MVD
of Peptide Antagonistsa
Ki (nM) Ki Ratio PA2 -
Peptide EL 6 alp GPI ($1
CTP 3.7 1,150 310 7.1
CTOP (19) 4.3 5,600 1,300 6.4"
CTAP (20) 2.1 5,310 2,530 7.1
TCTP 1.2 9,320 7,770 8.1
TCTOP 1.4 20,400 11,400 7.4 .
TCTAP 1.2 1,270 1,000 8.7
"Data from Ref. 944.
CTAP (20), respectively (105, 941), which ex- sequence of P-casomorphin,and the unrelated
hibit greatly reduced affinity for somatostatin peptides Trp-Trp-Pro-Lys-His-X-NH, (949)
receptors and enhanced affinity and selectiv- with affinity for p receptors. Hexapeptide li-
ity for p receptors. Analysis of CTP by NMR braries have also been examined for affinity
suggested that the central Tyr-D-Trp-Lys-Thr for 6 receptors, resulting in the identification
sequence adopts a type 11' P turn (942). The by positional scanning of several peptides that
conformation of these peptides was further re- share some similarity to the enkephalins [Tyr-
stricted and preceptor selectivity further en- X-Y-Z-Leu-ValNH,, where X-Y-Z = Gly-Met-
hanced by incorporation of the constrained His, His-Gly-Trp (9501, and Gly-Phe-His
amino acid D-Tic in position 1; the resulting (94611.
peptides TCTP, TCTOP, and TCTAP are po- A variety of novel acetylated peptides with
tent p antagonists with exceptional selectivity sequences unrelated to known opioid peptides
for p receptors (Table 7.21) (943,944). Incor- have been identified for all three opioid recep-
poration of either isomer of P-Me-~-phe in po- tors from combinatorial libraries (see Ref.
sition l decreases p-receptor affinity and se- 946). Initially, Houghten and coworkers used
lectivity 10- and 40-fold, respectively (945). p-receptor binding as the screening assay to
Other p-receptor antagonists have been identify peptides from an acetylated hexapep-
identified from combinatorial libraries (see be- tide amide library with affinity for p receptors.
low). These peptides, termed acetalins (250), are po-
tent p-receptor antagonists in the GPI (951).
6.6.2 Peptides and Peptidomimetics from Further exploration of the acetylated hexapep-
Combinatorial Libraries. Mixture-based com- tide library resulted in the additional identiha-
binatorial peptide libraries have been exten- tion of AcPhe-Arg-Trp-Trp-Tyr-X- NH, and
sively explored by Houghten and coworkers AcArg-Trp-Ile-Gly-Trp-X-NH,; AcPhe-Arg-
and have led to the identification of a variety of Trp-Trp-Tyr-MetNH, is an agonist, whereas
peptides with affinity for opioid receptors (see AcArg-Trp-Ile-Gly-Trp-Arg-NH, is an antago-
Ref. 946 for a review). Early hexapeptide li- nist at I*, receptors (949). Screening of an
braries, deconvoluted by binding to p opioid acetylated hexapeptide library of all D-amino
receptors and either iterative deconvolution acids led to the identification of Ac-arg-phe-
(947) or positional scanning (948; see Ref. 946 trp-ile-asn-lys-NH, (D-amino acids indicated
for a description of these deconvolution tech- by use of all lowercase letters for the amino
niques), identified sequences related to the en- acid) as a ligand for opioid receptors; inter-
kephalins. Other nonacetylated peptide li- estingly, this peptide is a potent agonist that
braries have identified more varied peptides, produces analgesia after peripheral adminis-
some that resemble opioid peptides and some tration (952). A nonacylated, all D-amino acid
that do not. Iterative deconvolution of a hexapeptide library also yielded peptides (ile-
hexapeptide library resulted in identification phe-trp-tyr-argNH, and ile-met-ser-trp-trp-
of both Tyr-Pro-Phe-Gly-Phe-XNH, (X = one glyNH,) with affinity for p opioid receptors
of 20 natural amino acids), reminiscent of the (946). An acetylated decapeptide library was
6 Opioid Peptide Analogs
6.7 Peptide Affinity Label Derivatives binds with high affinity to 6 receptors and has
been used to characterize 6-receptor subtypes
Peptide-based affinity labels have been princi-
(see Section 3.2.4.3). 3-Nitro-2-pyridinesulfe-
pally photoaffinity labels, including the azide nyl (Npys)-containing derivatives of cysteine
derivatives of several enkephalin analogs and were incorporated into opioid peptides to yield
CTP (see Refs. 286,594 for detailed reviews). potential affinity label derivatives (969-972).
As noted earlier (Section 5.11.21, the use of S-Activated enkephalin analogs containing
photoaffinity labels, however, has been lim- the Npys group attached to the C-terminus
ited because opioid receptors are susceptible label p opioid receptors in a dose-dependent
to inactivation by UV irradiation (643). manner (969),whereas the Npys-protected de-
The preparation of opioid peptide deriva- rivative of DALCE reacts with 6 receptors
tives containing electrophilic affinity labels (971). Incorporation of Cys(Npys) into posi-
has been limited to a few compounds. The tion 8 of dynorphin A-(1-9) yields a peptide
chloromethyl ketone DALECK (Tyr-D-Ala- that decreases the B,, value, but does not
Gly-Phe-LeuCH,Cl) (957, 958) is one of the affect the K, value, for binding to K receptors
best studied electrophilic peptide derivatives. (970); recovery of binding after treatment
It selectively alkylates p opioid receptors (959, with dithiothreotol suggests that the dynorphin
960),and [3HlDALECKhas been used to label analog binds to K receptors through a disulfide
and characterize these receptors (959, 961, bond. [D-Ala2,Cys(Npys)8]-dynorphinA-(1-9)-
962). DAMK (Tyr-D-Ala-Gly-MePheCHfl), NH, is reported to label all three opioid recep-
the chloromethyl ketone derivative of tors, whereas [D-Al$,Cy~(Npys)~~ldynorphin
DAMGO, was also prepared and binds selec- A-(1-13)NH, apparently labels p and 6, but not
tively to p opioid receptors (963). The chlo- K , receptors (972).
romethyl ketone of dynorphin A-(1-10) (964)
6.8 Peptidase Inhibitors
inhibits binding to frog brain membranes in a
wash-resistant manner, although the affinity All of the ligands discussed so far in this chap-
of this peptide for K receptors is relatively ter interact directly with opioid receptors. An
weak (apparent IC,, for irreversible blockade alternative approach to producing analgesia is
-10 m. The C-terminal maleamide deriva- to inhibit the metabolism of the endogenous
tive of DSLET was recently reported to exhibit opioid peptides, thus increasing their concen-
wash-resistant inhibition of binding to 6 re- tration and occupancy of opioid receptors (see
ceptors at micromolar concentrations (965). Refs. 973-976 for reviews). The two major en-
Enkephalin analogs containing melphalan zymes involved in metabolism of the opioid
(Mel), the nitrogen mustard derivative of p- peptides are the aminopeptidases, especially
aminophenylalanine, were also prepared (see the bestatin-sensitive aminopeptidase-N
Refs. 286, 966). Recently, the first isothiocya- [APN (EC 3.4.11.2)], and the neutral endo-
nate derivatives of opioid peptides were de- peptidase-24.11 [NEP or neprilysin (EC
scribed (738, 967); the Phe(p-N=C=iSI4 de- 3.4.24.11) (974)1,which cleaves at the Gly-Phe
rivative of N,N-dibenzyl enkephalin and both bond. NEP has been cloned, and the crystal
the Phe(p-N=C=S)3 and p h e ( ~ - N = = C + 3 ) ~ structure of the extracellular domain of the
derivatives of TIPP exhibited wash-resistant enzyme complexed with the inhibitor phos-
inhibition of binding to cloned 6 opioid recep- phoramidon has recently been reported (977).
tors. The Phe(p-br~moacetamide)~ derivative Both of these enzymes have a broad substrate
of TIPP is even more potent than the p-iso- specificity and cleave other peptides in addi-
thiocyanate derivative at inhibiting binding to tion to the opioid peptides. NEP particularly
cloned 6 opioid receptors in a wash-resistant exhibits much lower activity toward longer
manner (968). opioid peptides, and thus the opioid effects re-
Thiol-containing derivatives of opioid sulting from inhibition of this enzyme are
peptides have been prepared that potentially probably mainly due to interfering with the
can form disulfide linkages with cysteine resi- metabolism of the enkephalins and possibly
dues on opioid receptors. DALCE ([D- the heptapeptide Met-enkephalin-Arg6-Phe7
Ala2,Leu5,Cys6]enkephalin, 45, Fig. 7.8) (194) (973). Other minor metabolic routes include
6 Opioid Peptide Analogs
II
-C- HNCHC-HNCHC02- Enkephalin (enzyme substrate)
I
0
0
II
X = CNH (253) Thiorphan
0
II
X = NHC (254) Retrothiorphan
(255) Acetorphan
(256) Kelatorphan .
(257) RB38A
(258) RBI 20
(259) RBI 01
and does not generalize to morphine, suggest- kinetics for the hydrolysis of the two lipophilic
ing that this inhibitor may have low abuse po- groups were different, with the carboxylate es-
tential (991). These major differences in the ter undergoing much more rapid hydrolysis in
side effect profile of these enzyme inhibitors plasma than in rat brain membranes; the
compared to morphine has been attributed to deprotection of the phosphinate group was
the low tonic release of the endogenous en- slower, resulting in approximately 55 and 80%
kephalins in brain regions relevant to these of the completely deprotected compound after
side effects of morphine (see Refs. 975, 976 a 1-h incubation with plasma and rat brain
and references cited therein). membranes, respectively (995).
Recently, a-aminophosphinic derivatives
(260) have been identified as both selective
7 RECENT DEVELOPMENTS
and domain-swapped dimer modes of dimer- There is also high sequence homology with
ization (1004). The growing evidence for opioid receptors in the intracellular loops, con-
dimerization of opioid receptors prompted sistent with ORLl receptors coupling to the
Portoghese to revisit some of his earlier work same G-proteins as opioid receptors. Splice
on bivalent ligands and their possible interac- variants of the ORLl gene have been reported
tions with dimeric receptors (321). Computa- (see Refs. 87,90 for reviews), and the results of
tional models of possible dimeric opioid recep- some pharmacological studies have suggested
tors are only beginning to appear in the receptor heterogeneity (see Ref. 871, but the
literature (321). existence of subtypes of the ORLl receptor
has not been firmly established.
7.2 Opioid-Receptor-Like 1 (ORL1) Receptor
The ORLl receptor exhibits high selectiv-
and Its Endogenous Ligand Orphanin
ity for its endogenous ligand, and has very low
FQ/Nociceptin (OFQ/N)
affinity for most opioid ligands. Site-directed
During attempts to clone different opioid re- mutagenesis and chimeric receptor studies
ceptor types, several laboratories isolated a have examined possible structural reasons for
cDNA for a receptor with high homology to this selectivity of the ORLl receptor. Point
opioid receptors (see Refs. 87,89,90 for recent mutations of only four residues in TM6
reviews). Because this receptor, referred to by CVQV276-278IHIl and TM7 LT302II of the
Mollereau et al. as opioid-receptor-like 1 ORLl receptor were sufficient to impart bind-
(ORL1) receptor (911, did not display affinity ing affinity for Dyn A fragments without af-
for classical opioid ligands, it was classified as fecting the affinity or potency of OFQ/N (176).
an "orphan receptor." Subsequently, two The additional mutation of a residue in TM5
groups isolated a 17-residue peptide as the en- [A213K] enhanced affinity for several opioid
dogenous ligand for this receptor (see Fig. 7.9) alkaloids, particularly antagonists (1007), but
(92,93).This peptide was referred to as orpha- this mutant no longer bound OFQ/N. Alanine
nin FQ by one group because it was the ligand mutation of several TM residues that are con-
for the orphan receptor (F and Q are the N- served with opioid receptors yielded mutant
and C-terminal residues, respectively, of the receptors with reduced affinity for OFQ/N
peptide) (92) and named nociceptin by the (1008), suggesting that the binding pocket in
other group, since in the initial studies this the ORLl receptor may be similar to that
peptide was reported to produce hyperalgesia found in opioid receptors. Alanine replace-
(93). The OFQ/N precursor protein preprono- ment of GlnZa6at the C-terminus of TM6 in
ciceptin (93) contains additional biologically hORLl results in a mutant to which OFQIN
active peptides related to OFQ/N. Nocistatin still binds with high affinity, but which cannot
(Fig. 7.9) (271) blocks a number of the effects mediate inhibition of forskolin-stimulated
of OFQ/N (see Ref. 1005 for a review), but no- CAMPformation (1008),implicating this resi-
cistatin does not interact with ORLl recep- due in ORLl receptor signal transduction.
tors. A second 17-residue peptide, referred to OFQ/N and the ORLl receptor exhibit the
as orphanidnociceptin 11,is also found in pre- greatest similarity to dynorphin A and K opi-
pronociceptin (2701, along with a longer pep- oid receptors, respectively, but OFQ/N exhib-
tide OFQN160-187 (1006); these peptides, its very low affinity for K receptors and, con-
however, have not been as well characterized versely, Dyn A exhibits low affinity for ORLl
as OFQ/N (see Ref. 87 for a recent review). receptors. To study the structural reasons for
Like opioid receptors, the ORLl receptor is this selectivity, chimeric receptors have been
a G-protein-coupled receptor, consisting of constructed between ORLl and K receptors
seven transmembrane (TM) regions plus ex- (1009, 1010). Replacement of the N-terminal
tracellular and intracellular domains. The region of the K receptor up through the top of
ORLl receptor exhibits high sequence homol- TM3 with the corresponding sequence of the
ogy to opioid receptors, with 60-62% identity ORLl receptor imparted affinity for OFQ/N,
for the whole transmembrane domain (resi- but low potency in an adenylyl cyclase assay,
dues 52-342 in the human ORLl receptor) without affecting the binding or potency of
(90) and higher homology in TM2, 3, and 7. Dyn A. Further incorporation of extracellular
7 Recent Developments
loop 2 (EL21from the ORLl receptor restored fects, however, were subsequently classified as
efficacy for OFQ/N, again without affecting antianalgesic effects (89, 1011) based on re-
the ability of Dyn A to bind and activate the evaluation of the controls and the effects of
receptor. Thus, EL2 appears to be required for stress-induced analgesia accompanying the.
activation of ORLl receptors, but not K opioid experimental procedures. Effects reported for
receptors. OFQ/N administered i.c.v. have ranged f r o q
Based on the experimental data, a compu- hyperalgesia, analgesia, or anti-analgesic ac-
tational model of OFQ/N bound to ORLl re- tivity to a combination of these effects (87,89,
ceptors has been proposed (189).In this model 1011). A similar range of activities have been.
the N-terminal sequence containing the two reported after spinal (intrathecal) administra-
Phe residues binds in a highly conserved
tion (87,89,1012).A number of factors appear
pocket formed by TM3, 5, 6, and 7, which is
to influence these often contradictory find-
similar to that proposed for opioid receptors.
ings, including the noxious stimuli studied,
Residues 5-7 (Thr-Gly-Ala)of OFQIN are then
positioned at the TM-EL2 interface in a the species and strain of animal used, the dose
largely nonconserved region; unfavorable of OFQ/N, stress, and the physiological state
side-chain interactions in this region of the of the animal (see Ref. 87 for a detailed discus-
receptor are then used to explain the selectiv- sion). The most robust and consistently ob-
ity of the ORLl receptor for OFQ/N over Dyn served effects of OFQ/Nare the anti-analgesic
A. The positively charged C-terminus of the effects after supraspinal administration.
peptide is proposed to make multiple contacts OFQ/N acts as a functional antagonist of opi-
with the highly acidic EL2. oid receptors and blocks analgesia produced
by a wide variety of opioids (see Refs. 87, 89).
7.2.1 Physiological and Pharmacological Ef- At the spinal level the predominant effect of
fects. Since their discovery, interest in this re- OFQ/N appears to be inhibitory, resulting in
ceptor and its endogenous ligand has in- analgesia and/or anti-hyperalgesidanti-allo-
creased exponentially. There have been a dynia (see Refs. 87, 89, 1012). Several studies
number of excellent reviews covering the com- have reported anti-hyperalgesic or anti-allo-
plex pharmacology of this system (see Refs. dynic activity for OFQ/N in rat models of in-
87-89 for recent reviews), including a special flammation and nerve injury (89, 1012). Be-
issue of the journal Peptides (Vol. 21, Number cause morphine appears to have reduced
7, 2000) devoted solely to the ORLl receptor effectiveness in treating neuropathic pain af-
and OFQ/N. ter nerve injury, the activity of OFQ/N in this
Consistent with the sequence similarities type of pain could have important therapeutic
between the ORLl and opioid receptors, acti- implications. OFQ/N has also been implicated
vation of the ORLl receptor triggers the same in the development of tolerance to morphine
signal transduction mechanisms as used by (see Refs. 87, 89).
the opioid receptors. Thus activation of ORLl OFQ/N and the ORLl receptor are also in-
receptors inhibits both forskolin-stimulated volved in a number of other physiological ef-
adenylyl cyclase and Cat+ currents and acti- fects (see Refs. 87,891. One of the most signif-
vates several other effectors, including inward icant effects is the anxiolytic activity of
rectifying K+ channels, protein kinase C, mi- OFQ/N (1013), which has been postulated to
togen-activated protein kinase (MAP kinase) be one of OFQ/N's most fundamental actions,
and phospholipase C (see Ref. 89 for a review). and may help explain the effects of OFQ/N on
There has been considerable controversy other phenomena [e.g., locomotion, reward,
over the roles of ORLl receptors and OFQ/N and feeding (8711. A small molecule ORLl ag-
in response to painful stimuli (see Refs. 87,89, onist has also demonstrated anxiolytic activity
1011 for recent reviews). When administered (10141,demonstrating an important potential
by intracerebroventricular (i.c.v.) injection therapeutic application of these compounds.
OFQ/N was initially reported to produce hy- Like opioids, OFQ/N inhibits electrically in-
peralgesia (92,93),hence the name nociceptin duced contractions in the GPI and MVD
for the endogenous peptide ligand. These ef- smooth muscle preparations; these effects are
Narcotic Analgesics
not naloxone reversible, indicating that they oid receptors, particularly K and p receptors
are not mediated by opioid receptors (see Ref. (see Refs. 1015, 1016). Unlike in the opioid
89 for a review). peptides, an aromatic amino acid is not re--
quired in position 1, and Phel can be replaced
7.2.2 Structure-Activity Relationships of by the aliphatic residues cyclohexylalanine
OFQ/N and Other Peptidic Ligands for the and leucine (1022). In contrast, an aromatic
OR11 Receptor. Shortly after the identifica- residue is required in position 4, and replace:
tion of the endogenous ligand, several re- ment of Phe4 with an aliphatic residue results
search groups began exploring the SAR of this in loss of activity (1022). An Arg in position 8
peptide. Several recent reviews (88, 1015, appears to be critical, and replacement with
1016) have described the details of the SAR of Lys results in large decreases (>loo-fold) in
OFQ/N, so the discussion here focuses on affmity and potency (1016). Incorporation of
some of the key findings. Tyr in place of Leu14 was used to obtain an
Truncation studies have been performed to analog that could be radioiodinated or triti-
identify the minimum sequence required for ated for use in radioligand binding assays (92);
ORLl affinity and activation. These studies both labeled derivatives are commercially
revealed that, like opioid peptides, the N-ter- available.
minal aromatic residue was important for bi- A series of chimeric peptides between
ological activity (1017, 1018), although one OFQ/N and Dyn A were prepared to explore
study (1017) reported that the further N-ter- the structural reasons for the selectivity of
minal truncated fragments OFQ/N-(6-17) OFQ/N for ORLl over K opioid receptors
and OFQ/N-(12-17) retain affinity and activ- (1023). The results from this study suggested
ity for ORLl receptors. In contrast to dynor- that residues Thr 5 and Gly6, in addition to
phin A, where shorter fragments retain opioid Phel, are responsible for the activity and se-
activity (263), 13 of the 17 residues of OFQ/N lectivity of OFQIN for ORLl receptors; the
appear to be required for ORL1-receptoraffin- chimera Dyn A-(1-5)/OFQ/N-(6-17) (261)
ity and activation (1017, 1018). The amide de- was able to bind and activate both ORLl and K
rivative of OFQ-(1-13) is a considerably more opioid receptor.
potent agonist in the mouse vas deferens assay
than the acid derivative, apparently because of
decreased metabolism (1016); therefore this
fragment is typically used as the parent struc-
ture in further SAR studies (see below). OFQ/
N-(1-ll), however, has been reported to be Ser-Ala-Arg-Lys-Leu-Ala-Am-Gln
active both in vitro (1019) and in vivo (10201,
despite its low affinity for cloned ORLl recep- Early studies of the pharmacology of the
tors in binding assays, resulting in the pro- ORLl system were hindered by the lack of an-
posal of receptor heterogeneity for OFQIN tagonists for this receptor. Therefore there
(1019). Results from binding studies have also was considerable excitement in the field when
suggested receptor heterogeneity (see Ref. the first report of an antagonist appeared in
87). the literature (1024). The reduced amide de-
A number of analogs of OFQ/N have been rivative of OFQ/N [ P ~ ~ ~ I ) ( C H , N H ) G ~ ~ ~ ] O F Q /
examined for their pharmacological activity N-(1-13)NH2 (262, Fig. 7.51; referred to as
(see Refs. 88, 1015, 1016 for recent reviews). [F/G]NC(l-13)NH, by Calo and coworkers),
Shortly after identification of the endogenous which was synthesized to protect the peptide
ligand the results of an alanine scan of OFQ/N from metabolism by aminopeptidases, was ini-
were reported, identifying Phel, Phe4, and tially reported to be an antagonist of OFQ/N-
Ar$ as critical residues in the sequence (1017, (1-13)NH2 in smooth muscle preparations
1021). Phel can be replaced by tyrosine (1018, (1024). Subsequent examination of this com-
1021), resulting in an analog that retains af- pound in a variety of assays, however, indi-
finity and potency at ORLl receptors, but also cated that the activity observed depended on
exhibits increased affinity and activity at opi- the assay and that although (262) was an an-
tagonist in some assays, it was a partial or full literature, with leads identified from screen-
agonist in a number of other assays, including ing assays (see Refs. 1015, 1016, 1029 for re-
forskolin-stimulated CAMP accumulation in cent reviews). Starting from a lead (267, Fig.
Chinese hamster ovary cells expressing cloned 7.52), identified by high throughput screening
ORLl receptors (see Refs. 88,1015 for detailed as a nonselective ORLl ligand, a group from
reviews). Subsequently, the N-substituted Hoffmann-La Roche explored a number ofr
glycine analog [NphellOFQ/N-(1-13)NH2
(263) was reported to be an ORL1-receptor to identify more selective and potent ORLl
antagonist (1025).Although the potency of the agonists (1030-1033). The pharmacology of- -
compound is weak (PA, < 6 in most assays), it one of these compounds, Ro 64-6198 (268),
is an antagonist in all of the assays examined
has been examined in considerable detail
to date (see Refs. 88, 1015 for reviews).
(1014,1033-1036), including determination of
The use of combinatorial libraries has re-
sulted in the identification of peptidic and pep- the affinities of the different stereoisomers for
tidomimetic ligands for the ORLl receptor opioid receptors as well as ORLl receptors
that are not structurally related to OFQ/N. (1033).The stereochemistry at positions 1 and
Houghten and coworkers identified acetylated 3a has significant effects on ORL1-receptoraf-
hexapeptides with high affinity for the ORLl finity, whereas the affinities for the opioid re-
receptor, with Ac-RYYRXK-NH, (264; X = W ceptors are comparable for the different iso-
or I) having the highest affinity (1026); in mers; the 1S,3aS isomer exhibits the highest
most assays these peptides exhibit partial ag- affinity for ORLl receptors (pKi = 9.41) and
onist activity (see Ref. 1015). A combinatorial therefore the highest selectivity for ORLl over
library of conformationally constrained pep- opioid receptors (1033). This compound is a
tides resulted in the identification of 111-BTD full agonist at ORLl receptors and produces
(265) that exhibits moderate affinity (K, = 24 dose-dependent anxiolytic effects in several
nM) but only modest selectivity [Ki ratio rat models of anxiety, with an efficacy and po-
(oRLl/p/~/a)= 1/4.6/6.1/221 for ORLl recep- tency after systemic administration compara-
tors; this compound acts as an antagonist at ble to those of benzodiazepine anxiolytics such
ORLl receptors, whereas it exhibits partial as diazepam (1014).
agonist activity at opioid receptors (1027). The Researchers from the Banyu Tsukuba Re:
related conformationally constrained peptide search Institute reported the first nonpeptide
111-Haic (266) was nonselective, exhibiting antagonist for ORLl receptors (1037). Start-
modest affinity for ORLl and the three opioid ing from a lead (269) from their chemical li-
receptors (K, = 50-125 nM) (1027). brary, which again exhibited reasonable affin-
ity but poor selectivity for ORLl receptors,
7.2.3 Nonpeptide Ligands for the OR11 Re- these researchers identified 5-113397 (270),
ceptor. Because of the potential therapeutic which exhibits high affinity (Ki = 1.8 nM) and
applications of ORLl receptor ligands, there high selectivity for ORLl receptors over opioid
has been considerable interest in identifying receptors (1037-1039). This compound is an
nonpeptidic compounds that interact with this antagonist of OFQ/N in uitro and in viuo after
receptor. Several opiates have been reported subcutaneous administration (1038, 1039). A
to exhibit some affinity for ORLl receptors series of 4-aminoquinolines (271) has also
(see Refs. 1015,1016) with some opiates, most been examined for antagonist activity, based
notably the fentanyl analog lofentanyl(1018), on the weak affinity of the lead compound
naloxone benzoylhydrazone [Nal(BzO)H, 501 (271)in a random screen (1040). JTC-801
(see Refs. 1015,1016),the naltrexamine deriv- (272) was selected because it showed the best
ative TRK-820 (65, Fig. 7.16) (311), and bu- bioavailability. After oral administration this
prenorphine (1028), exhibiting reasonable af- compound was shown to antagonize the ef-
finity andlor potency. Reports by groups from fects of OFQ/N and to produce an analgesic
the pharmaceutical industry of both nonpep- effect that was not antagonized by naloxone;
tidic selective agonists and antagonists have this compound is reported to be undergoing
begun to appear in the scientific and patent clinical trials (1040).
Narcotic Analgesics
Ac-Arg-Tyr-Tyr-Arg-X-LysNH2
pounds that do not cross the blood-brain bar- This approach is still in its infancy, however,
rier, but that are still orally bioavailable. The so its therapeutic application in clinical trials
peripherally selective antagonists methylnal- still remains to be demonstrated.
trexone and alvimopan are currently undergo- There has also been considerable advance-
ing clinical trials for treatment of opioid-in- ment in the identification of agents that pro-
duced constipation and related GI side effects. duce analgesic effects through different mech-
The ability of 6-receptor antagonists to de- anisms than interaction with opioid receptors
crease the development of tolerance and de- (see Refs. 1041-1044 for reviews). Thus
pendence to morphine (see Section 3.3.1) has NMDA (N-methyl-D-aspartate) antagonists,
considerable therapeutic potential and has GABA (y-aminobutyric acid) agonists, nico-
prompted the search for compounds with both tinic acetylcholine receptor agonists (e.g., epi-
p agonist and 6 antagonist activity. Although batadine), antagonists of substance P at NK-1
peptide derivatives with both activities have receptors, and a number of other compounds
been studied in some detail (see Section 6.4.2)... targeting different receptors exhibit antinoci-
nonpeptide opioids with this activity profile ceptive activity in animal models. These com-
have been reported only recently. This ap- pounds do not cause the side effects associated
proach of using S antagonism together with p with the clinically used opiates, but they have
agonism, either combined in a single drug or their own distinct side effect profiles, which in
by coadministering two agents, to minimize some cases (e.g., NMDA antagonists, which
the side effects of p agonists is very exciting. cause psychotomimetic effects) have resulted
Narcotic Analgesics
in termination of clinical studies (see Ref. structures exhibiting affinity for opioid recep-
1042). Also, the promising antinociceptive ac- tors, there is still ample opportunity for the
tivity observed in animal models has not al- development of new therapeutic agents that,
ways translated into clinical efficacy in hu- hopefully, will bring us closer to the goal of
mans. Thus a number of NK-1-receptor identifying optimal analgesics.
antagonists are highly effective in animal
models of pain and exhibit excellent pharma- 9 WEB ADDRESSES AND RECOMMENDED
cokinetic profiles in humans, but have failed in READING FOR FURTHER INFORMATION
phase I1 clinical trials for treatment of pain
and migraine (see Ref. 1042). Thus, whether 9.1 Clinically Used Agents
any of these novel targets will ultimately re-
sult in clinically used agents for pain remains Drug Facts and Comparisons, Wolters Klu-
to be determined. wer, St. Louis, 2001. May be accessedat www.
Clearly, one very exciting target for the de- factsandcomparisons.com
velopment of potential therapeutic agents is C. Stein, Ed., Opioids in Pain Control: Basic
the ORLl receptor (Section 7.2). Because of and Clincial Aspects, Cambridge University
the complexity of ORLl receptor involvement Press, Cambridge, 1999. This book contains
in pain perception, however, it is not clear at several chapters discussing the clinical use
this time whether ligands for this receptor will of opioids in different types of pain and clin-
prove to be clinically useful analgesics. The ical settings in addition to chapters discuss-
anxiolytic activity of ORLl agonists, however, ing the pharmacology of opioids.
represents a promising approach for the devel-
opment of novel clinically useful agents for 9.2 Pharmacology
this indication.
A major unmet therapeutic need is effective 0 H. B. Gutstein and H. Akil in J. G. Hard-
treatments for substance abuse, including man, L. E. Limbird, and A. G. Gilman, Eds.,
abuse of opioids, cocaine, and amphetamines. Goodman and Gilman's The Pharmacolog-
Results for ligands interacting with either 6 or ical Basis of Therapeutics, 10th ed.,
K receptors hold some promise in this area (see McGraw-Hill Medical Publishing Division,
Section 3.31, but whether such agents will be New York, 2001, pp. 569-619.
clinically useful remains to be demonstrated. A. Hem, H. Akil, and E. J. Simon, Eds., Opi-
Although the results of some initial studies in oids I and Opioids 11, Handbook of Experi-
humans have been promising [e.g., an im- mental Pharmacology, Vols. 104/I and 1041
proved positive response of opioid-dependent 11, Springer-Verlag, Berlin, 1993. Two
individuals to a "functional" K antagonist (bu- volumes containing comprehensive reviews
prenorphine in the presence of naltrexone to of opioid pharmacology.
block p-agonist activity) as compared to nal-
trexone alone (24911, other studies have been 9.2.1 Opioid Receptor Structure and Molec-
disappointing [although the K-selective ago- ular Biology
nist enadoline reduced the positive subjective
effects of cocaine, neither enadoline nor the 0 University of Minnesota Center for Opioid
mixed agonist butorphanol modified self-ad- Research and Design (CORD), http://www.
ministration of cocaine in humans (255)l. opioid.umn.edu. This site includes computa-
The goal of identifying potent analgesics tional models of the opioid receptors, a com-
free of the side effects of morphine and other plete list of chimeric receptors and point
narcotics has remained elusive. As more infor- mutations in opioid receptors with pharma-
mation has become available about opioid re- cological data and literature references, and
ceptor structure, opioid pharmacology, and re- links to other relevant sites.
lated systems (i.e., the ORLl receptor), this 0 P. Y. Law, Y. H. Wong, and H. H. Loh, Mu-
has provided new challenges to medicinal tational Analysis of the Structure and Func-
chemists to prepare compounds with unique tion of Opioid Receptors, Biopolymers, 51,
pharmacological profiles. With the diversity of 440-455 (1999).
9 Web Addresses and Recommended Reading for Furtht?r Information 45 1
9.2.2 Dimerization of Opioid and Other C- (Parts I and 111,Expert Opin. Ther. Patents,
Protein-Coupled Receptors 7,1075-1098 (1997); 9,353-374 (1999).
a A. F. Casy and R. T. Parfitt, Opioid Analge- P. W. Schiller, Ed., Peptide and Peptidomi-
sics: Chemistry and Receptors, Plenum metic Ligands of Opioid Receptors, Biopoly-
Press, New York, 1986. mers, 51, 377-458 (1999). This issue of
0 G. R. Lenz, S. M. Evans, D. E. Walters, and Biopolymers contains reviews covering opi-
A. J. Hopfinger, Opiates, Academic Press, oid receptor structure as well as peptide and
Orlando, FL, 1986. peptidomimetic ligands for these receptors:
9.6 OR11 Receptor and Orphanin
The above two comprehensive books con-
FQ/Nociceptin
tain detailed reviews of the early literature.
0 M. Nassi, C. Polidori, G. Cal6, and D. Regoli,
0 A. F. Casy and G. H. Dewar, The Steric Fac-
NociceptinIOrphanin FQ and Its Receptor,
tor in Medicinal Chemistry: Dissymmetric
Peptides, 21,891-1154 (2000). This issue of
Probes of Pharmacological Receptors, Ple-
Peptides contains reviews of all aspects of
num Press, New York, pp. 429-501 and
ORLl receptor and orphanin FQInociceptin
503-548.
pharmacology.
0 G. Calb, R. Guerrini, A. Rizzi, S. Salvadori,
9.4 Delta and Kappa Opioid Receptors and and D. Regoli, Pharmacology of Nociceptin
Selective Ligands and Its Receptor: A Novel Therapeutic Tar-
get, Br. J. Pharmacol., 129, 1261-1283
9.4.1 Delta Opioid Receptor Pharmacology (2000).
and 6-Receptor-Selective Ligands 0 L. M. Harrison and D. K. Grandy, Opiate
Modulating Properties of NociceptinlOr-
0 A. Coop and K. C. Rice, Role of 6-Opioid Re- phanin FQ, Peptides, 21, 151-172 (2000).
ceptors in Biological Processes, Drug News 0 J. S. Mogd and G. W. Pasternak, The Molecu-
Perspect., 13,481-487 (2000). lar and Behavioral Pharmacology of the Or-
0 G. Dondio, S. Ronzani, and P. Petrillo, Non- phanin FQJNociceptin Peptide and Receptor
peptide 6 Opioid Agonists and Antagonists Family, P h a m o l . Rev., 53,381-415 (2001).
Narcotic Analgesics
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CHAPTER EIGHT
Antidepressants
University of Oxford
F Department of Pharmacology
Oxford, United Kingdom
i
g RICHARD A. GLENNON
Virginia Commonwealth University
School of Pharmacy
;, Richmond, Virginia
Contents
1 Introduction, 484
2 Clinical Use, 484
2.1 Available Agents and Classification, 484
2.2 Clinical Efficacy of Antidepressants, 486
2.3 Adverse Side Effects, 490
2.3.1 Introduction, 490
2.3.2 Monoamine Oxidase (MA01 Inhibitors,
491
2.3.3 Tricyclic Antidepressants, 492
2.3.4 Serotonin-Selective Reuptake
Inhibitors, 493
2.3.5 Other Agents, 494
2.4 Pharmacokinetics, 496
2.4.1 Tricyclic Antidepressants, 496
2.4.2 SSRI, 496
2.4.3 MA0 Inhibitors, 497
2.4.4 Other Antidepressants, 498
3 Physiology and Pharmacology, 498
3.1 Monoamine Transporters, 498
3.1.1 Discovery, 498
3.1.2 Monoamine Transporters, 498
3.1.3 Drugs as Inhibitors of Monoamine
Transporters, 499
3.2 Serotonergic Agents, 502
3.2.1 Receptor Populations, 502
3.2.2 5-HT, Receptors, 503
3.2.3 5-HT,, Receptors, 503
3.2.4 Mixed-Function Ligands, 505
3.2.5 Other 5-HT Receptors,
- 506
3.3 Alpha Adrenergic Receptors, 506
Burger's Medicinal Chemistry and Drug Discovery 3.4 Monoamine Oxidase, 507
Sixth Edition, Volume 6: Nervous System Agents
-
3.5 Other Proposed Mechanisms of Action, 507
Edited by Donald J. Abraham 4 History, 508
ISBN 0-471-27401-1 O 2003 John Wiley & Sons, Inc. 4.1 Discovery of the First Antidepressants, 508
Antidepressants
4.2 Case History: Fluoxetine (Prozac), 509 6.1 Monoaminergic Drugs, 516
5 Structure-Activity Relationship and Metabolism, 6.2 NMDA Receptor Antagonists, 516
510 6.3 Drugs Acting at Neuropeptide Receptors,
5.1 Reuptake Inhibitors, 510 517
5.2 Monoamine Oxidase Inhibitors, 512 6.3.1 Substance P-NK1 Receptor
5.3 Serotonergic Agents, 513 Antagonists, 517
5.4 Other Agents, 515 6.3.2 CRF Antagonists, 517
6 Recent Developments and Things to Come, 516
apy on an SSRI, for example, are more time of existing agents, are required to
likely to complete a course of adequate dose overcome this problem.
and duration of antidepressant therapy 4. Finally, there is a certain population of pa-
than patients who initiate therapy with a tients who are resistant to current therapies.
TCA (4). In a review of more than 100
cases, patients receiving SSRIs tended to
Most antidepressants in clinical use today
discontinue medication less often than
act by enhancing the neurotransmission of se-
those on TCAs or heterocyclic antidepres-
sants. The difference was statistically sig- rotonin [5-hydroxytryptamine (5-HT)], nor-
nificant when SSRI use was compared with epinephrine [NE; noradrenaline (NA)], or
use of TCAs, but the differences were not as both. They do so either by blocking the re-
significant when the SSRIs were compared uptake (transport) of neurotransmitter,
to the heterocyclic antidepressants. The blocking the metabolism of neurotransmitter
findings were attributed more to the side [i.e., monoamine oxidase (MAO) inhibitors],
effects associated with the TCAs than with or by direct action on a neurotransmitter re-
the therapeutic efficacy of the agents (5). ceptor. Hence, the antidepressants can be
3. Most antidepressants have a delayed time classified on the basis of their putative mech-
of onset; currently available antidepres- anisms of action (Table 8.2 and Figs. 8.1-8.4).
sants require administration for at least Agents that block neurotransmitter reuptake
2-4 weeks before effects are evident. can be further divided into those that are non-
Newer agents with shorter onset times, or selective (e.g., tricyclic antidepressants with
newer strategies for enhancing the onset mixed action), serotonin-selective reuptake
486 Antidepressants
a0
\ I
CH2CH2CH2N(CH3)2
Dothiepin (7)
\ I
CH2CH2CH2N(CH3)2 lmjpramjne R
Doxepin (8)
YR I /CH3
CH3
= -H (11)
Trimipramine R = -CH3 (25)
'I
I
NH-CH3
Desipramine (6)
9
a
0 NH-CH3
Maprotiline (13)
1 I
\ CHCH2CH2NH-CH3
I
Nortriptyline (17)
QQJ
1 I N \
I
CH2CH2CH2NH-CH3 CH3 0
Protriptyline (20) Lofepramine (12)
Figure 8.1. Structures of tricyclic antidepressants.
Clinical trials invariably employ self-report- drug treatment is usually defined as a de-
ing of symptoms, using standardized ques- crease of at least 50% in the baseline HAM-D
tionnaires, the tool most often used being score.
the 17- or 21-item Hamilton Rating Scale for There are several puzzling features of anti-
Depression (HAM-D). A positive response to depressant drug action. The first is that re-
488 Antidepressants
CI H
Paroxetine (18) Sertraline (22) Reboxetine (21)
Venlafaxine (26)
Figure 8.2. Structures of the SSRIs (citalopram, fluoxetine, fluvoxamine, paroxetine, sertraline),
the NSRI reboxetine, and the SNRI venlafaxine.
Figure 8.4. Structures of some of the newer atypical antidepressants: bupropion, mirtazepine,
nefazodone, and trazodone.
sant drug (9). The placebo effect is variable; it tween the drug-treated and placebo groups.
is larger, for example, in patients with milder The various classes of antidepressant drugs
depression than in those with more severe exhibit few differences in their clinical effi-
forms of the illness. It is not uncommon for cacy; the advantages of the newer compounds
clinical trials of antidepressant drugs to fail to are related to their improved side-effect
show a statistically significant difference be- profiles rather than to a more powerful anti-
depressant action (8, 10). The results of a SSRIs in particular have come to be used in-
meta-analysis of antidepressant drug trials il- creasingly in a variety of conditions other than
lustrate this point (Table 8.3). major depression. Table 8.4 summarizes the
A meta-analysis of 186 randomized con- uses for which SSRIs have been approved,
trolled trials with amitriptyline (1)indicated based on the finding of significant beneficial
that although this drug is less well tolerated effects in controlled clinical trials. In addition,
than other tricyclic or SSRI antidepressants agents in this class in controlled trials have
there was a small but significant 2.5% higher shown usefulness in premenstrual dysphoria,
proportion of responders compared to that of the borderline personality disorder, obesity,
other drugs (12). A systematic search of 108 smoking cessation, and alcoholism (17).
other meta-analyses suggested that combined The remarkable success of the SSRIs has
serotoninlnorepinephrine reuptake inhibitors prompted the question of whether genetic de-
have slightly superior efficacy to that of the fects in the serotonin (SERT) transporter
SSRIs (13). Recent studies of the norepineph- gene or in the regulation of its expression
rine-selective reuptake inhibitor reboxetine (21) might explain the etiology of mood disorders.
showed it to have comparable efficacy to that of The gene coding for human SERT is localized
other antidepressants, but it improved social on chromosome 17q11.2. It spans over 35 kilo-
functioning more than did the SSRIs (14,15). bases and is organized in 14 introns. No ge-
A second unexplained feature of antide- netic variations have been found in the coding
pressant drug action is that the maximum region of the SERT gene in depressed pa-
clinical benefit is not seen until treatment has tients, but a number of studies have found
been continued for several weeks (10). Al- that certain variants in a polymorphic region ,
though a significant improvement in HAM-D flanking the 5' region or in the second intron
scores can sometimes be detected after 2 are associated with depressive illness, anxiety-
weeks of treatment, it takes 4-6 weeks to ob- related personality traits, or suicidal alcohol-
tain the maximum response. Boyer and Feigh- ism (6).
ner (16) performed a meta-analysis of six trials
to determine the predictive value of nonre- 2.3 Adverse Side Effects
sponse to medication early in a clinical trial.
They found that patients who failed to achieve 2.3.1 introduction. The antidepressants
at least a 20% reduction in HAM-D scores at include a wide range of compounds with differ-
any point during the first 4 weeks of a study ing modes of action (Table 8.2). It is not sur-
had less than a 5% chance of becoming a "re- prising to find that they display a plethora of
sponder," as defined by a 50% or more reduc- differing side effects (18, 19). These range
tion in HAM-D score by the end of the 6-week from adverse effects that can be unpleasant
trial. The authors concluded that a full 6 but relatively harmless, to rarer and often un-
weeks' trial of antidepressant medication is predictable serious adverse reactions. Partic-
usually not justified if patients fail to respond ularly in the older, so-called first-generation
during the first 4 weeks. A number of explana- antidepressants, these can be life threatening.
tions have been proposed to explain the de- Because depressed patients are often suicidal,
layed clinical response to antidepressant it is not surprising that these drugs were often
drugs, and these are discussed below. implicated in deaths resulting from inten-
Depression is often associated with anxiety tional overdose (20,21). Some antidepressants
or other forms of psychiatric disorder, and the caused rare, idiosyncratic adverse effects that
2 Clinical Use
were, nevertheless, so severe as to lead to from pulmonary and cerebral edema. Despite
withdrawal of the drugs from the market (e.g., the relatively low risk of food interactions, a
nomifensine, zimelidine). wide range of foodstuffs is prohibited to paz
tients taking MA0 inhibitors, including ma-.
2.3.2 Monoamine Oxidase (MAO) Inhibi- ture cheeses, meat or yeast extracts, mature
tors. The first-generation MA0 inhibitors fish, pickled fish, smoked foods, and broad
tended to cause hypotension (often causing bean pods. Because of the long-lasting inhibir
dizziness), headache, and mild anticholinergic tion of MA0 caused by these irreversible en-
effects such as dry mouth, constipation, zyme inhibitors, the dietary precautions have
blurred vision, and difficulty in micturition. to be maintained for at least 14 days after ces-
Phenelzine (19) can cause mild sedation, but sation of drug treatment. These restrictions
tranylcypromine (23)is more likely to act as a make these drugs unpopular with both pa-
psychostimulant with mild amphetamine-like tients and physicians. At least as important as
properties, thereby leading to agitation and the food-interaction risk is that of drug in-
insomnia (18). These effects are dose depen- teractions. MA0 inhibitors are dangerous to
dent and tend to lessen in severity with time. use in conjunction with a number of other
In fact, although antidepressants are not typ- clinically important drugs (18, 24). Serious
ically associated with abuse potential, 16 of 21 interactions (usually hypertensive crises)
case reports of antidepressant abuse involved may occur with pethidine, levodopa, sympa-
tranylcypromine (22). More serious adverse thomimetic amines such as amphetamine
effects are related to the fact that the first- and ephedrine, and other antidepressants
generation MA0 inhibitors are compounds including the tricyclics and SSRIs.
that irreversibly inhibit the enzyme in the The second-generation MA0 inhibitor mo-
brain and other organs. With chronic drug clobemide (15)was designed to lessen the risk
treatment, inhibition of enzyme activity is cu- of the food and drug interactions seen with
mulative and may become almost complete. earlier MA0 inhibitors. Moclobemide selec-
The enzyme is abundant in the liver, where it tively targets one form of the enzyme, mono-
serves the function of detoxifying a variety of m i n e oxidase A, leaving monoamine oxidase
pharmacologically active organic amines that B in the liver active and capable of detoxifying
are absorbed from the diet. tyramine and other dietary vasoactive amines
Inhibition of liver MA0 leaves the patient (25). Moclobemide is also a reversible inhibi-
vulnerable to the so-called wine-and-cheese tor of the enzyme, so its effects are not cumu-
syndrome, with adverse cardiovascular effects lative and are more rapidly reversible on ter-
caused by absorption of such vasoactive mination of drug treatment. Adverse effects
amines as tyramine into the general circula- are few and infrequent, with dropouts because
tion [for review, see Blackwell et al. (23)l. The of side effects in clinical trials uncommon.
syndrome can include severe headache and Nausea, insomnia, headache, and dizziness oc-
hypertension and may lead to cerebral hemor- curred in some patients taking the drug, and
rhage and death. Although this is a real risk, it others experienced agitation and restlessness
seems likely that fears of MAO-food interac- (25, 26). Despite the improved selectivity of
tions may have been grossly exaggerated. Pare moclobemide, there have been a small number
(24) reviewed the evidence in 1985 and noted of reports of hypertension when moclobemide
that despite the widespread use of MA0 inhib- was combined with tyramine-rich foods (27).
itors in the previous decade there had only Moclobemide exhibits fewer adverse drug in-
been 17 reports of food interactions with teractions than first-generation compounds,
phenelzine (19) and none of these proved fa- although combination with SSRIs is not rec-
tal. With tranylcypromine (23) seven deaths ommended because of the danger that a "sero-
had been reported, but in only two of these tonin syndrome" could result (28). Moclobe-
could a definite relationship with diet be mide does, however, appear to be safe when
established. given in conjunction with sympathomimetic
MA0 inhibitors when taken alone in over- amines, found in many common cough and
dose can be fatal, with death usually resulting cold remedies. The drug appears to be safe if
Antidepressants
zymes. Consequently, other drugs that induce has, however, been reported in six patients
such enzymes or compete with the tricyclic an- taking citalopram (4) (37), although the cause
tidepressants for metabolism may alter their of death was disputed (38). The symptoms of
actions. Calcium channel blockers, cimetidine, SSRI overdose include nausea, agitation, sei-
phenothiazines, haloperidol, methylphenidate, zures, and sometimes loss of consciousness
glucocorticoids, oral contraceptives, and most (18).The relative safety of the SSRIs has led to
SSRIs may inhibit the metabolism of the tricy- their being prescribed more freely than the
clics. This will tend to exacerbate the adverse earlier antidepressants, and their use in a
side effects of the tricyclics. Conversely, carba- number of indications in addition to the treat-
mazepine, phenytoin, barbiturates, primidone, ment of major depression (Table 8.4).
and alcohol may induce liver cyhchrome p450 There are, however, some hazards associ-
enzymes and accelerate metabolism, making tri- ated with the use of these drugs. SSRIs de-
cyclics less effective (8,19). crease serotonin uptake from the blood by
platelets. Because platelets cannot synthesize
2.3.4 Serotonin-Selective Reuptake Inhibi- serotonin, which is involved in platelet aggre-
tors. Since their introduction in the mid- gation, SSRIs may impair platelet function. A
1980s SSRIs have become the most widely case-controlled study found that the risk of
used of all antidepressants. This is largely be- gastrointestinal bleeding was three times
cause of their improved safety and tolerability greater in SSRI users than in controls (39).
in clinical use. Although the SSRIs are no This conclusion was confirmed by a retrospec-
more efficacious or rapid in onset of action tive cohort study of 317,824 SSRI users, which
than the tricyclics, they lack most of the seri- emphasised that the risk of gastrointestinal
ous toxicity and adverse side effects associated bleeding was particularly important for el-
with the first-generation drugs. The relative derly patients (40).
absence of cardiac toxicity makes the SSRIs The SSRIs exhibit only low affinities for
relatively safe in overdose (36). Fatal overdose muscarinic and most other monoamine recep-
Antidepressants
tors (Table 8.6). Consequently at therapeutic Given concurrently with TCAs they may cause
doses they are relatively free from the cholin- serious adverse effects. Fluvoxamine may
ergic side effects associated with the TCAs, raise levels of caffeine and theophylline, and
and are less likely to cause sedation and fluoxetine can interfere with the metabolism
drowsiness or hypotension. Instead of promot- of clozapine, cyclosporin, and tefenadine (18,
ing weight gain, the SSRIs tend to suppress 46). SSRIs should never be given with MA0
appetite and this can lead to weight loss. The inhibitors because a fatal "serotonin syn-
most common side effect in the acute use of drome" has been reported with fluoxetine in
SSRIs is nausea, which clinical trial data indi- this combination (18).
cate affects about 20% of patients taking flu-
oxetine (9),fluvoxamine (101, paroxetine (l8), 2.3.5 Other Agents
and citalopram (4) (18). The risk of nausea is Bupropion. Bupropion (3)is a weak inhibi-
reduced if the SSRIs are taken with a meal. tor of dopamine reuptake that has not been
Most SSRIs tend to cause CNS "activation," widely used outside the United States as an
leading to insomnia, agitation, or anxiety. Par- antidepressant. It has received a new world-
adoxically, paroxetine (18)tends to cause se- wide lease on life as an effective means of
dation and drowsiness to such an extent that treatment for tobacco smoking cessation (47).
in several countries the drug is prescribed It has little sedative, cholinergic, hypotensive,
with a warning not to drive (18). All SSRIs or cardiotoxic properties (48). There are some
tend to cause sexual dysfunction, including adverse side effects, however, related to the
loss of libido, erectile dysfunction, and delayed ability of this drug to enhance dopaminergic
or absent orgasm. Both men and women are function. These include insomnia, agitation,
affected, and the incidence of these side effects nausea, weight loss, and sometimes psychosis
is quite high. Although initial clinical trial (49). The drug decreases seizure threshold
data suggested that only a small proportion of and so should not be given to those at risk of
patients suffered sexual dysfunction, more re- seizures (49). In overdose acute toxicity is less
cent reports suggest that these side effects serious than that seen with tricyclics (50). Bu-
may occur in as many as 50-70% of patients propion (3) should not be given with MA0 in-
taking SSRIs, and 30-40% of all those on an- hibitors, levodopa, or dopaminergic receptor
tidepressant medication of any kind (41, 42). agonists (49).
Patients are reluctant to volunteer informa- Venlafmine. Venlafaxine (26) is an inhibi-
tion on sexual dysfunction, but when asked tor of serotonin and norepinephrine uptake,
specifically a truer picture emerges. but unlike the TCAs it has little affinity for
A debate has raged for several years over muscarinic, histamine, or alpha-adrenergic
the alleged association between the use of receptors. Consequently, it does not exhibit
SSRIs and the occurrence of suicidal thoughts the cholinergic, sedative, or hypotensive side
and suicide. However, meta-analysis of the effects seen with the earlier compounds (18,
available clinical data has failed to show any 19). Nevertheless, the side-effect profile in-
such association (43, 44). Nevertheless, the cludes headache, dry mouth, sedation, and
UK Medicines Control Agency as recently as constipation in up to 15% of patients (51).
October 2000 advised manufacturers of SSRIs During the first 2 weeks of treatment as many
to include a warning in patient information as 30% of patients receiving the drug may ex-
leaflets about the possibility of suicidal perience nausea (52).At high doses close mon-
thoughts when they begin taking the prod- itoring of blood pressure is needed, given that
ucts, before the antidepressant effects become the drug tends to cause hypertension (52).
apparent (45). Venlafaxine (26) appears to have little cardiac
SSRIs are metabolized by cytochrome p450 toxicity and seems to be safe in overdose, al-
enzymes in the liver. Most SSRIs inhibit though seizures have been reported (53).
CYP2D6, fluvoxamine (10) inhibits CYPlA2, Trazodone. Trazodone (24) is a weak inhib-
and fluoxetine (9) inhibits CYP3A4. Conse- itor of serotonin uptake and is an antagonist
quently, these drugs may interfere with the at 5-HT and alpha,-adrenergic receptors (Ta-
metabolism of a number of other agents. ble 8.5). These properties appear to be related
Table 8.6 Affinities of Antidepressant Drugs for Human Monoamine Receptors"
Generic Name Histamine H l Muscarink a,-Adrenergic a,-Adrenergic Dopamine D2 5-HT,, 5-HT,
Amitriptyline
Amoxepine
Bupropion
Citalopram
Clomipramin
Desipramine
Dothiepin
Doxepine
Fluoxetine
Fluvoxamine
Imipramine
Lofepramine
Maprotiline
Nefazodone
Nortriptyline
Paroxetine
Protriptyline
Reboxetine
Sertraline
Trazodone
Trimipramine
Venlafaxine
"Equilibrium dissociation constants are nanomolar. Data were obtained from binding studies using the following: Histamine HI: 3H-doxepin (29) or 3H-pyrilamine (35);
Muscarinic: 3H-quinuclidinylbenzilate; a,-Adrenergic: 3H-prazosin; a,-Adrenergic: 3H-rauwolscine; Dopamine D2: 3H-spiperone;5HT1,: 3H-8-OH-DPAT;5-HT,: 3H-ketanserin.
Data are from Refs. 29,30, and 35. The four compounds with the highest &ty for each receptor are highlighted in bold type. For a more complete and up-to-date summary of drug
interactions with neurotransmitter receptors see http://pdsp.cwry.edu/pdsp.asp.
Antidepressants
to the side effects of sedation and hypotension from 1 to 12 h according to both the drug and
(leading to dizziness) seen with the drug, and the individual. Most of these compounds have
common at high doses (54). Trazodone lacks long half-lives, and many are metabolized by
cardiac toxicity, although dysrhythmias have demethylation in the liver, to yield biologically
been reported (18); the drug appears safe in active desmethyl metabolites, which further
overdose. Trazodone sometimes causes an un- extend their duration of action (62,63) (Table
usual type of sexual dysfunction, that can in- 8.7). For example, imipramine (11)is metabo-
clude increased libido, priapism, and sponta- lized to desipramine (6). In the case of lofepra-
neous orgasm (55, 56). These symptoms, mine (12), its metabolite, desipramine (61,
plays an important role in the overall actions
although rare, are dramatic and have received
of the drug, in that desipramine has a consid-
considerable attention. Combination with
erably longer elimination half-life than that of
SSRIs or MA0 inhibitors should be avoided
the parent compound (27,34) (Table 8.7). The
because of the risk of "serotonin syndrome" drugs are extensively metabolized in the liver
(18). by demethylation, hydroxylation, and gluc-
Nefazodone. Nefazodone (16) is chemically uronide conjugation of the hydroxy metabo-
related to trazodone but acts in a different lites. The lipid-soluble drugs are thus con-
manner, largely through inhibition of seroto- verted to water-soluble conjugates that are
nin uptake and antagonism at 5-HT, recep- readily excreted by the kidney (64). There is
tors. Adverse effects are mild and infrequent. increased renal clearance in children and de-
They include sedation, dry mouth, and dizzi- creased clearance in older people, factors that
ness in around 10% of patients (57). The drug need to be taken into account in determining
causes less hypotension than does trazodone, optimum dosage levels. Clearance is also re-
and is unlikely to cause sexual dysfunction duced in patients with compromised liver or
(18).It is considered safe to use in epilepsy and kidney function.
there appears to be no overdose risk (18). Ne- There is considerable individual variation
fazodone is a potent inhibitor of cytochrome in the metabolism of the tricyclics, and this is
p450 CYP3A4 and so should not be given with largely attributed to genetically determined
alprazolam, astenizole, terfenadine, cisapride,
-
differences in liver enzymes. Some 7 to 9% of
or cyclosporin (58). Caucasians are classified as "slow metaboliz-
Reboxetine. Reboxetine (21) is a norepi- ers," measured by the rate of hydroxylation of
nephrine-selective reuptake inhibitor that the drug debrisoquin. This is caused by ge-
lacks affinity for most of the monoamine re- netic polymorphism in the cytochrome p450
ceptors. It thus does not exhibit the typical enzyme CYP2D6 (65). This enzyme plays an
side-effect profile of the tricyclics. Neverthe- important role in the aromatic hydroxylation
less, side effects include increased sweating, of tricyclic antidepressants. The tricyclic
postural hypotension (leading to dizziness), drugs have a narrow therapeutic window, so
dry mouth, constipation, blurred vision, impo- individual variations in drug metabolism can
tence, and dysuria. Tachycardia and urinary be important in determining the correct ther-
retention have also been reported (59). There apeutic dose and avoiding toxic overdose (66,
is no evidence of cardiotoxicity and sexual dys- 67).
function seems to be rare. In contrast to some
of the earlier tricyclics that are sedative, re- 2.4.2 SSRI. All the SSRIs are well absorbed
boxetine is nonsedating and can cause insom- and most have long half-lives, compatible with
nia (60, 611.. their use as once-a-day drugs (68-70). The for-
mation of biologically active desmethyl metab-
2.4 Pharmacokinetics olites is again a factor in prolonging the dura-
tion of action of some of these drugs. This is
2.4.1 Tricyclic Antidepressants. The tricy- particularly important for fluoxetine (91,
clic antidepressants are, by and large, well ab- which is metabolized in part to form norfluox-
sorbed after oral administration, although etine, an active metabolite that has a half-life
time to peak plasma concentration can vary of 4-16 days (71, 72). The desmethyl metabo-
2 Clinical Use 497
lite of sertraline (22), although less potent the individual enantiomers may be metabo-
than the parent drug (681, also has an ex- lized and eliminated differently (70, 77).
tended half-life (73, 74). The desmethyl me-
tabolite of citalopram (4) is formed in rela- 2.4.3 M A 0 Inhibitors. The MA0 inhibi-
tively small amounts and appears to tors are rapidly absorbed and are extensively
contribute less importantly (34, 75). The long degraded by first-pass metabolism in the liver.
duration of action of fluoxetine (9) and sertra- For the irreversible enzyme inhibitors phenel-
line (22) make long drug-free periods neces- zine (19) and tranylcypromine (23) the elimi-
sary when switching patients to other drugs. nation half-lives are relatively unimportant,
All of the SSRIs are metabolized by cyto- given that enzyme inhibition persists for
chrome p450 CYP2D6 in the liver, so individ- many days after the drug has been eliminated.
ual genetically determined differences exist in A minimum of 7-14 days is needed after stop-
rates of drug clearance and there is the poten- ping treatment with these drugs before it is
tial for interaction with other drugs that are safe to switch to other agents. The reversible
metabolized by this enzyme (69, 70). Citalo- MA0 inhibitor moclobemide (15) is rapidly
pram (4) and fluvoxamine (10) are also sub- absorbed and extensively metabolized in the
strates of p450 CYP 2C19, which exhibits a liver. It has an elimination half-life of approx-
particularly high rate of genetic polymor- imately 12 h (78). Because enzyme inhibition
phism in Asians (76). The pharmacokinetics of is reversible, the time to recover after stopping
fluoxetine and citalopram are complicated by moclobemide treatment is much shorter,
the fact that they are racemic compounds and 16-24 h (79).
Antidepressants
2.4.4 Other Antidepressants. The other an- It was not until high specific activity
tidepressant drugs in Table 8.7 are rapidly ab- tritium-labeled radioactive catecholamines
sorbed and vary in their half-lives, with some became available in the late 1950s, however,
requiring multiple daily dosing. Trazodone that experiments could be performed using
and nefazodone (16)are metabolized in part to quantities of monoamine small enough to
m-chlorophenylpiperazine (m-CPP; (28), a mimic the very low concentrations of epineph-
compound that acts as an agonist at some se- rine or norepinephrine normally encountered
rotonin receptors (80). The metabolite may, in body fluids. When the first experiments
thus, contribute to the biological action of were performed in the Axelrod laboratory at
these drugs. m-CPP (28) also has a longer the National Institutes of Health with 3H-epi-
half-life than the parent drugs and readily nephrine (85) and later with 3H-norepineph-
penetrates the CNS. rine (861, they yielded an unexpected result.
The norepinephrine selective uptake inhib- Although in laboratory animals most of the
itor reboxetine (21) is rapidly and completely injected dose of labeled catecholamine was
absorbed, and is metabolized mainly by the rapidly metabolized (mainly by COMT), a sub-
cytochrome p450 3A4; because it does not in- stantial proportion of the injected monoamine
teract with CYP 2D6 there is less risk of inter- (30-40%) was removed from the circulation
actions with other drugs (81-83).Mirtazepine by a rapid uptake into tissues, where it re-
(14) also shows little interaction with p450 cy- mained for some time unchanged. A key obser-
tochrome isozymes and there is only a low risk vation was that the uptake of 3H-norepineph-
of drug interactions (84). Mirtazepine is a rine into the heart was virtually eliminated in
racemate, and the two enantiomers are elimi- animals in which the sympathetic innervation
nated at different rates, with a twofold higher had been destroyed by surgical removal of the
rate of elimination of the (S)-enantiomer than superior cervical ganglion (87). This led Hert-
of the (R)-enantiomer (84). ting and Axelrod (88) to propose that the re-
uptake of norepinephrine by the same nerves
from which it had been released might repre-
sent a novel mechanism for inactivating this
3 PHYSIOLOGY AND PHARMACOLOGY
neurotransmitter.
The discovery of norepinephrine uptake
3.1 Monoamine Transporters was followed by the finding that similar but
distinct transporters were involved in the in-
3.1 .I Discovery. The majority of both old activation of 5-HT and do~amine,and that
and new antidepressants act by virtue of their similar mechanisms existed for the inactiva-
ability to inhibit monoamine transporter tion of the amino acid neurotransmitters
mechanisms in the brain. The concept that GABA, glycine, and L-glutamate (89, 90). Re-
neurotransmitters are inactivated by uptake search interest has focused on these mecha-
of the released chemical into the nerve termi- nisms, including in recent years the identifica-
nal from which it had been released or into tion and cloning of the genes encoding the
adjacent cells is less than 40 years old. Before transporter proteins involved and the develop-
this it was generally assumed that the inacti- ment of knockout strains of genetically engi-
vation of norepinephrine and the other mono- neered mice lacking one or other of these gene
amine neurotransmitters after their release products. The family of neurotransmitter
from nerves was likely to involve rapid enzy- transporters has turned out to be far more
matic breakdown, akin to that seen with ace- extensive than previously imagined, with
tylcholinesterase. The degradation of mono- more than 20 different members (for review,
amines by the enzyme monoamine oxidase see Ref. 91).
was known early on, and in the 1950s a second
enzyme catechol-0-methyl transferase (COMT) 3.1.2 Monoamine Transporters. The nor-
was discovered and was thought to play a key epinephrine transporter (NET) was cloned by
role in inactivating norepinephrine and other Pacholczyk et a]. in 1991 (92) and this soon led
catecholamines. to the discovery of other related members of
3 Physiology and Pharmacology
the transporter gene family. Separate trans- sively in monoaminergic neurons SERT is ex-
porters exist for serotonin (SERT) and dopa- pressed in both neurons and glia.
mine (DAT).The monoamine transporters are
dependent on sodium and chloride ions for 3.1.3 Drugs as Inhibitors of Monoamine
their function. They use the electrochemical ~ r a n s ~ o r t e r sfar
. ~ ~the most important
gradient of sodium between the outside and group of CNS drugs that target the NE and *
inside surfaces of the cell membrane to pro- serotonin neurotransmitter transporters (i.e.,
vide the thermodynamic energy required to NET and SERT, respectively) is the tricyclic
pump neurotransmitters from low concentra- antidepressants and their modern counter-
tions outside the cell to the much higher con- parts. The discovery by the Axelrod group in
centrations inside the cell. Chloride ions ac- 1961 (94) that imipramine (11)potently inhib-
company the entry of neurotransmitter and ited the uptake of norepinephrine led to the
sodium, and there is a net movement of posi- first understanding of the mechanism of ac-
tively charged ions into the cell, although not tion of the first-generation tricyclic antide-
in sufficient amounts to appreciably alter the pressants. After the discovery of the serotonin
resting membrane potential of the cell. uptake system in the brain it soon became ap-
The vesicular neurotransmitter trans- parent that the classical tricyclic drugs imip-
porters represent another family (91) whose ramine (11)and amitriptyline (1)were potent
function is to maintain the very high concen- as inhibitors of both NE and 5-HT uptake (Ta-
trations of monoamine and amino acid neu- ble 8.2). This reinforced the monoamine hy-
rotransmitters in storage vesicles. They use pothesis of depression as a monoamine-defi-
the proton gradient that exists across the ciency state, and stimulated much further
vesicular membrane as the motive force. The research in the pharmaceutical industry to
vesicular monoamine transporters (VMAT) discover new inhibitors of monoamine uptake.
recognize serotonin, dopamine, norepineph- The debate as to whether inhibition of either
rine, epinephrine, and histamine. VMAT-1 NE or 5-HT was the more important in con-
is present chiefly in amine-containing endo- ferring antidepressant efficacy has swung one
crine and paracrine cells in peripheral or- way and the other over the past 40 years and
gans, whereas VMAT-2 is the predominant there is no definitive answer to this question.
form found in monaminergic neurons in the An early effort to improve the selectivity of
CNS. It is also expressed in the histamine- antidepressants was made in the 1970s by sci-
containing cells of the stomach, and in the entists at the Ciba-GeigyCompany in Switzer-
adrenal medulla and in blood cells. The Na+/ land (now Novartis), who developed the selec-
C1'-dependent transporters and the vesicu- tive NE uptake inhibitor maprotiline (13)
lar transporters are membrane proteins con- (95). This proved to be clinically effective as an
sisting of a single polypeptide chain of 5-600 antidepressant but it was not a great success
amino acid residues, with a 12 a-helical mem- commercially and had few clear advantages
brane-spanning domain (91). The molecular over the classical TCAs. This idea was also
mechanisms underlying the function of the swept away by the wave of enthusiasm for se-
neurotransmitter transporters remain un- rotonin-selective reuptake inhibitors (SSRIs)
clear. Unlike flux through an open ion chan- that started with the success of fluoxetine (96,
nel, there must be a gating cycle every time 97). Table 8.8 summarizes the affmities of cur-
solute is transported, although the exact mo- rently used antidepressants on cloned human
lecular details of this are not understood. No monoamine transporters expressed in tissue
doubt selective mutations of amino acids in culture cell lines (98). The availability of the
the transporter molecules will throw light on human transporter proteins for screening rep-
these questions in the future (93). resents a considerable advance. Although
Immunocytochemical and in situ hybrid- there are many published accounts of the ef-
ization techniques have been used to study the fects of antidepressants on monoamine trans-
cellular distribution of the transporters (91). porter mechanisms, most of these employed
Whereas NET and DAT are expressed exclu- animal tissues and there are few reported
Antidepressants
metabolites may indirectly activate noradrener- the importance of serotonin. There is some ev-
gic mechanisms (see Section 5.4 below). The idence that NET-selective drugs are subtly
compound has had little success as an antide- different in their clinical profiles from those of -
pressant, but has been approved in the United SSRIs. Healy and McMonagle (106) have sug-
States and Europe as an aid to smoking cessa- gested that these drugs affect overlapping
tion (47). clinical domains. They suggest that NET-se-
.-@
What are we to make of these twists and lective agents tend to promote levels of energy
turns? How can drugs that are selective NE re- and interest, whereas SSRIs affect impulse
uptake inhibitors be equally effective as those control and both categories of drug treat
that selectively target SERT? In practice it is mood, anxiety, and irritability.
The molecular mechanisms in the brain
difficult to know how selective the monoamine
that are triggered by the antidepressants,
uptake inhibitors are in vivo. None of the anti-
however, remain obscure. The fact that all
depressants is completely selective for either
drugs require a period of several weeks before
NET or SERT. The SSRIs have some affinity for they become fully effective suggests that they
NET, and some (e.g., paroxetine) are quite po- modify gene expression in the brain and that
tent inhibitors of NET (102). In some cases the the resulting altered biochemical state takes a
formation of active metabolites alters the drug long time to become stabilized. Many theories
selectivity profile. Thus the nonselective com- have been proposed, including alterations in
pound imipramine (11)and the partially NET- the expression of alpha- and beta-adrenergic
selective compound lofepramine (12) are exten- receptors, changes in transcription factors
sively metabolized to desipramine (61,a highly and/or neurotrophic factors, and even mor-
potent and selective NE reuptake inhibitor. phological alterations in the connectivity of
Similarly, whereas amitriptyline (1)has little se- monoaminergic nerves.
lectivity for either NET or SERT, the metabolite It is possible that antidepressant drugs
nortriptyline (17) is a selective NET inhibitor. It have other targets in addition to their actions
seems likely that both NET-selective agents and at cell surface monoamine transporters. Al-
SSRIs exert their effects through some common Damluji and Kopin (107) have described a
final pathway in the brain. Perhaps the SSRIs novel amine uptake process in peptide-con-
a d indirectly to modulate noradrenergic func- taining hypothalamic neurons, which they.
tion (103,104). Experimental data from animal named "transport-P." Like the vesicular
experiments using microdialysis probes showed transporters this process is driven by a proton
increased levels of extracellular norepinephrine gradient, but it is distinct from the vesicular
transporters in being insensitive to reserpine,
in rat hippocampus aRer chronic treatment
but sensitive to a variety of tricyclic antide-
with paroxetine (18)(103). The original mono-
pressants at micromolar concentrations (108).
amhe hypothesis of depression as formulated It is not clear, however, what role if any trans-
by Schildkraut in 1965 (105) stated: port-P plays in the inactivation of the mono-
amine neurotransmitters.
"Some, if not all, depressions are associated with Some antidepressants, notably mazindol
an absolute or relative deficiency of catechola- and bupropion (3), inhibit the dopamine
mines, particularly norepinephrine, at function- transporter (DAT) as well as NET or SERT.
ally important adrenergic receptor sites in the
The DAT is best known, however, as one of the
brain. Elation conversely may be associated with
principal sites of action of the psychostimu-
an excess of such amines."
lant drug cocaine. Mice that are genetically
engineered to knock out the expression of the
European opinion currently seems to be DAT gene are profoundly hyperactive and fail
swinging back in support of the view that an to show any further stimulation of activity in
upregulation of noradrenergic function may response to cocaine or (+)-amphetamine
be the key element underlying the efficacy of (109). Such animals, nevertheless, will con-
antidepressant drugs (103, 104), but most tinue to self-administer cocaine (110), sug-
American researchers continue to emphasize gesting that the rewarding properties of the
Antidepressants
3.2.2 5-HT, Receptors. The exact mecha- (e.g., Ref. 125). Mechanistically, then, it is rea-
nism of action of some antidepressants is cur- sonable that 5-HT, antagonists display anti-
rently unknown. For example, the atypical anti- depressant activity. Ritanserin (33)is a newer
depressant trazodone (24) is a weak 5-HT
reuptake inhibitor, whereas nefazodone (16) is a
weak inhibitor of both 5-HT and NE reuptake
(Table 8.7). Inhibition of neurotransmitter re-
uptake does not seem to account for the antide-
-
pressant actions of these two agents. Both bind
at 5-HT, receptors with high CK, 25 nM) af-
finity (30) and are 5-HT, antagonists (122) (Ta-
ble 8.6). A plausible mechanism of action for
these drugs is that they enhance noradrenergic
and serotonergic function by their ability to
block presynaptic 5-HT receptors that normally (33) Ritanserin
exert an inhibitory effect on monoamine release
in the brain, or by blockade of postsynaptic example of a 5-HT, antagonist. In humans,
5-HT, receptors (see below). Mianserin ritanserin has been found to be as effective as
("Tolvan") (27) is a nonselective 5-HT, and amitriptyline (I), and superior to trazodone
(241, as an antidepressant (126). The SSRI flu-
oxetine (9) is metabolized to norfluoxetine
(34). (-1-Norfluoxetine retains antidepres-
(27) Mianserin
5-HT, antagonist with antidepressant activity sant activity and displays only slightly lower
(123);it binds with high affinity (Ki < 10 nM) at affinity for 5-HT,, receptors than it displays
5-HT, receptors (124). Hence, trazodone (24), for the 5-HT transporter (127). Results from
nefazodone (16), and mianserin (27) represent an animal behavioral model predictive of anti-
atypical antidepressants that have in common a depressant activity (the forced swim test) sug-
high a i t y for 5-HT,,receptors and 5-HT2an- gest that compounds that activate 5-HT,, re-
tagonist action. It might be noted that certain ceptors have antidepressant-like profiles
tricyclic antidepressants also bind at 5-HT, re- (128). m-Chlorophenylpiperazine (mCPP; (28),
ceptors; imipramine (111, desipramine (61,nor- which has significant affinity as an agonist at
triptyline (17), and maprotiline (13), for exarn- 5-HT,, receptors, was among the compounds
ple, bind with submicromolar Ki values (124) that were positive in this test. Because mCPP
(Table 8.6). is an important and long-lasting metabolite of
5-HT receptors are upregulated in depres- both trazodone (24) and nefazodone (16), it
sion. Hence, agents that downregulate 5-HT may also contribute to their antidepressant
receptors might be of benefit in the treatment profiles.
of this disorder. According to receptor adapta-
tion theory, neurotransmitter antagonists 3.2.3 5-HT,, Receptors. 5-HT,, receptors
should upregulate neurotransmitter recep- have been implicated as playing roles both in
tors. However, paradoxically, 5-HT2 antago- depression and in anxiety (129). Postsynaptic
nists generally downregulate 5-HT, receptors 5-HT,, (partial) agonist effects may be more
Antidepressants
important for antidepressant action, whereas studies. These agents are full agonists at pre-
agonist effects at presynaptic 5-HT,, recep- synaptic 5-HT,, receptors, but partial ago-
tors may be more important for antianxiety nists at postsynaptic 5-HT,, receptors (126).
activity (130-132). Furthermore, postsynap- An alternative approach is to develop postsyn-
tic 5-HT,, receptors have been shown to be aptic 5-HT,, agonists with greater efficacy
hypersensitive in depressed patients, whereas than that of those currently available.
presynaptic receptors are hyposensitive (133). Flesinoxan (38),an example of such an agent,
Also, electroconvulsive therapy has been dem- is currently in clinical trials. A newer agent of
onstrated to upregulate cortical 5-HT,, recep- this type is exemplified by (39) (1391, which
tors (134). binds at 5-HT,, receptors with high aMinity
Certain long-chain arylpiperazines (LCAPs) (Ki = 1 nM).
(135) have been demonstrated to possess both A problem associated with many antide-
anxiolytic and antidepressant actions. For ex- pressants is their delayed onset of action.
ample, the anxiolytic agents buspirone (351, Agents typically require 1-3 or more weeks
gepirone (36) (136),and ipsapirone (37) (137) before effects are realized. It has been hypoth-
showed antidepressant activity in clinical esized that the delay might be related, in part,
to the initial elevation in synaptic 5-HT levels,
which reduces the firing of serotonergic neu-
rons by activating autoreceptors, mainly of
the 5-HT,, subtype (139). During treatment
with antidepressants these autoreceptors are
desensitized and proper firing of 5-HT neu-
rons is restored; many believe this to be one of
the key changes elicited by antidepressant
(35) Buspirone
drugs of many different categories. 5-HT,, se-
rotonin receptors are found both presynapti-
cally and postsynaptically. Agents that behave
as antagonists at presynaptic 5-HT,, recep-
tors ( i . . , somatodentritic autoreceptors)
could, in theory, shorten the time of onset of
those antidepressants that act by increasing
synaptic levels of serotonin. In animal studies
the combination of acute treatment with an
(36) Gepirone SSRI together with a 5-HT,, antagonist led to
a larger increase in 5-HT release, as predicted
(140). Although no 5-HT,, antagonists are
available for human use, the beta-blocker pin-
do101 has appreciable affinity as an antagonist
at 5-HT,, receptors (K, value of approxi-
mately 10 nM). To date, the results of 15 pla-
cebo-controlled clinical trials, involving some
(37) Ipsapirone 800 patients, using pindolol in treating de-
(38) Flesinoxan
3 Physiology and Pharmacology
pression have been published (141, 142). Pin- tagonist with high affinity for the 5-HT trans-
do101 significantly accelerated the onset of ac- porter (K, = 12 nM)(1491, whereas compound
tion of SSRIs in five out of seven trials (42) displays somewhat higher affinity for
designed to test this concept. The combination
of pindolol with fluoxetine (9), for example,
reduced the median period required to obtain
a clinical response (50% reduction in baseline
score) from 29 to 19 days (142). A similar ac-
celeration of rate of onset has been shown with
a combination of pindolol and paroxetine (181,
and citalopram (4) (143-146 and references
therein). A role for P-adrenergic involvement
in these actions of pindolol has been ruled out
(147).
(44) Adatanserin
Antidepressants
nM,respectively)and displays reduced aMinity pressant perspective are the 5-HT, receptors.
for a,-adrenergic receptors (Ki => 1000 nM) 5-HT, receptors are known to be involved in
but high affinity for dopamine D2 receptors the release of several neurotransmitters, in-
(Ki= 2.7 nM), whereas adatanserin (44) binds cluding norepinephrine, serotonin, and dopa-
at 5-HT,, and 5-HT, receptors with compa- mine, and there are preclinical data indicating
rable affmity (Kivalues of 1 and 73 nM,respec- 5-HT, antagonists might play a role in anxiety
tively), but displays substantially reduced af- and, to a lesser extent, in depression (reviewed
finity for dopamine D2 receptors (Ki = 166 in ref. 158). It might be noted that mirtazepine
nM).Both agents are 5-HT,, partial agonists, (14), although its antidepressant properties
and adatanserin (44) has been demonstrated have been attributed primarily to its antago-
to be a 5-HT,, antagonist and is being devel- nist action at a,-adrenergic receptors and
oped as an antidepressant (153). 5-HT, serotonin receptors, is also a 5-HTIA
agonist and a 5-HT, antagonist (159).
3.2.5 Other 5-HT Receptors. Although so- Two of the most recently discovered popu-
matodendritic serotonin receptors are of the
-
lations of 5-HT receptors are the 5-HT, and
5-HT,, type, terminal autoreceptors are of 5-HT, receptors. Of interest is that certain
the 5-HTIDllBtype. These latter receptors are typical and atypical antidepressants, as well as
involved in the modulation of serotonin re- typical and atypical antipsychotic agents, bind
lease, and blockade of these receptors has been at both populations of receptors (160, 161). A
proposed as a possible means of developing role for either receptor population in the ac-
novel antidepressants or of enhancing the ef- tions of these agents has yet to be firmly estab-
fects of SSRIs (reviewed in Refs. 154, 155). lished, but the nanomolar affinity of the
SSRIs require 1 or more weeks before their agents for these receptors has heightened in-
antidepressant actions are evident; this delay terest in them.
corresponds to the time required to desensi-
3.3 Alpha Adrenergic Receptors
tize 5-HT,, and 5-HTlDlIBreceptors (156).
Hence, a 5-HT,,,, autoreceptor antagonist Although several antidepressant drugs have
might hasten the onset of effects of an SSRI by weak affbity for alpha-adrenergic receptors
mimicking its desensitizing action. Compound (Table 8.6) only one compound is thought to
(45), a 5-HT,,,, antagonist, has been demon- owe its antidepressant activity to such an in-
strated to augment citalopram-induced effects teraction. This is mirtazepine (14), which has
in rat ventral hippocampus and provides some nanomolar affinity as an a,-adrenergic recep-
support for this concept (157). tor antagonist (162, 163). The a,-adrenergic
A population of receptors that has yet to re- receptors are located presynaptically on both
ceive extensive investigation from an antide- noradrenergic and serotonergic nerve endings
3 Physiology and Pharmacology
and cell bodies, and exert an inhibitory effect The first-generation MA0 inhibitors
on monoamine neuronal firing and mono- phenelzine (19)and tranylcypromine (23) act .
mine release. Administration of mirtazepine as substrates for MAO-A and MAO-B but are
to animals has been reported to increase the converted by the enzyme to highly reactive in-
spontaneous rate of firing of noradrenergic termediates that then react irreversibly with
neurons in rat locus coeruleus, and serotonin the enzyme to cause an irreversible inhibition,
neurons in the raphe nucleus (164), and to of activity. Recovery of MA0 activity after ex-
increase levels of 5-HT release in hippocam- posure to these MA0 inhibitors requires the
pus (165),although this was not confirmed in synthesis of new enzyme protein, a process .
another publication (166). Clinical trials have that takes some weeks to completely restore
shown that mirtazepine is equivalent to ami- activity (173, 174). A clinical antidepressant
triptyline and other tricyclics in antidepres- response is associated with an inhibition of
sant efficacy (167). Some of these studies platelet MA0 activity of approximately 80%,
showed a clinical improvement within the first and measurement of platelet MA0 activity
week of treatment (167).Mianserin (27) is an- can be used to monitor treatment dose re-
other agent whose effects, at least in part, gimes (175).
might involve a,-adrenergic antagonism A new series of MA0 inhibitors are selec-
(168). Many adrenergic agents possess an im- tive for MAO-A and cause reversible inhibi-
idazoline ring. Imidazolines have been found tion of the enzyme, thus leaving MAO-B in the
to bind at nonadrenergic imidazoline binding liver intact to detoxify dietary amines, and
sites and this has led to speculation that such showing rapid recovery of enzyme activity af-
sites might play a role in depression (see Sec- ter discontinuation of drug treatment. The
tion 5.4). only drug of this type so far available for hu-
man use is moclobemide (15).
3.4 Monoamine Oxidase 3.5 Other Proposed Mechanisms of Action
Monoamine oxidase (MAO) is an enzyme - lo- Although many lines of evidence point to a
cated in the outer mitochondria1 membrane. common mode of action of antidepressant
As its name implies, it catalyzes the oxidative drugs by an enhanced release of the mono-
deamination of a varietv" of monoamines. The m i n e norepinephrine and serotonin in the'
enzyme is particularly abundant in liver, brain, many questions remain unanswered. If
where it serves to detoxify a variety of amines the monoamines themselves were responsible
that are absorbed from the diet; these include for regulating mood, why do depressed pa-
the vasoactive monoamines tyramine, octo- tients not feel an improvement immediately
pamine, phenylethanolamine, and phenyleth- after receiving the first dose of antidepres-
ylamine. The enzyme is also present in mono- sant? The fact that the clinical response is
mine-containing neurons, where it serves to delayed by several weeks suggests that the im-
regulate levels of cytoplasmic monoamine mediate effects of the drugs on monoaminer-
neurotransmitters (169). There are two forms gic mechanism in turn trigger longer-term
of MAO: MAO-A and MAO-B (170). Both are changes in the brain, probably involving alter-
present in the liver, and in most monoaminer- ations in gene expression. One possibility al-
gic neurons, although MAO-A is predominant ready discussed is that the early effects of in-
in norepinephrine and dopamine-containing creased monoamine release are counteracted
neurons, and MAO-B in serotoninergic cells. by brain mechanisms that downregulate
Both forms of the enzyme have a wide and monoamine release in response to the imme-
overlapping range of substrates, but MAO-A diate effects of the drugs. Thus, receptor de-
shows some preference for the catecholamines sensitization is required before the full effect
norepinephrine and epinephrine, and sero- of the antidepressants on monoamine release
tonin, and MAO-B for tyramine, phenyl- can be seen.
ethylamine, phenylethanolamine, and benzyl- However, there are other alternative expla-
mine. - Both enzymes metabolize dopamine nations for the delayed clinical response.
and tryptamine (169-171). There have been many studies of the neuro-
Antidepressants
chemical changes caused in animal brain by mechanisms could provide a novel approach to
chronic treatment with antidepressants. One antidepressant drug discovery in the future
of the changes elicited by many antidepres- (186).
sants is a downregulation in the expression of Another possibly important change in re-
P-adrenergic receptors (176-178). This is of ceptor sensitivity associated with chronic
interest because one of the most consistent treatment with antidepressants concerns re-
findings in depressed patients has been that ceptors for glucocorticoids. Antidepressant
P-adrenergic receptors are upregulated in pe- treatment produces an improvement in the
ripheral lymphocytes and in the brains of sui- function of the hypothalamic-pituitary axis, in
cide victims (179). It was proposed that the which several neuroendocine responses are
downregulation of P-receptors represents a blunted in depressed patients (187, 188).
marker of antidepressant efficacy (176, 177).
The validity of this concept was soon chal-
lenged, however, because it was found that the 4 HISTORY
newer SSRIs did not consistently downregu-
late P-adrenergic receptors, and citalopram
4.1 Discovery of the First Antidepressants
(4)actually caused an increase (146). p-Adren-
ergic receptors are coupled to cyclic AMP for- Before 1954, except for the use of electrocon-
mation but, although the receptors may be vulsive therapy, there were no effective treat-
downregulated by antidepressant drugs, other ments for depression. The two major classes of
components of cellular signaling that are reg- antidepressants, the monoamine oxidase in-
ulated by cyclic AMP are upregulated, notably hibitors and inhibitors of monoamine trans-
the cyclic AMP response element protein port, were discovered by accident in the 1950s.
(CREB), a prominent transcription factor in The drug iproniazid used for the treatment of
the brain (180).Another way of increasing lev- tuberculosis was found to have a mood-elevat-
els of cyclic AMP is to inhibit its degradation ing property (189) and clinical studies by
by phosphodiesterases. George Crane and Nathan Kline in the United
The compound rolipram is a phosphodies- States showed it to be effective in treating ma-
terase inhibitor and has been found to have jor depression (190, 191). Its actions were
clinical antidepressant activity (181). Al- traced to its ability to inhibit the monoamine-
though rolipram is not well tolerated because degrading enzyme MA0 (192). Although this
it causes severe nausea. it is ~ossiblethat in- and other subsequently developed MA0 inhib-
hibitors with more selectivity k r phosphodies- itors proved highly effective in the treatment
terase subtypes could be of future interest. In of depression, the possible dangers associated
animals chronic treatment with SSRIs leads to with their use led to their being largely re-
increased expression of the PDE4A and placed by the safer inhibitors of monoamine
PDE4B subtypes (183). Among the many transport. The first examples of the latter
genes that may be regulated by CREB are drugs to be widely used were imipramine (11)
those encoding neurotrophic factors (183). An in Europe and amitriptyline (1)in the United
important new finding is that chronic treat- States. Imipramine was synthesized originally
ment with antidepressants leads to increased by the Swiss company Geigy as a chlorproma-
expression of the neurotrophic factor BDNF zine-like molecule with potential as an anti-
(brain-derived neurotrophic factor) in rat hip- psychotic drug. The Swiss psychiatrist Roland
pocampus, and this may underlie the recent Kuhn, however, found it to be an effective an-
finding that chronic antidepressant treatment tidepressant (193, 194). On the other side of
causes a proliferation of progenitor cells in the the Atlantic, Merck first made amitriptyline
rat hippocampus (180, 184, 185). Neuroimag- also as a chlorpromazine-like molecule. It was
ing and postmortem histological studies have shown subsequently to be an antidepressant
reported reductions in neuronal and glial den- by Frank Ayd (195). For a detailed and enter-
sities in dorsolateral prefrontal cortex in pa- taining account of the history of the discovery
tients with mood disorders (185a,b). Com- of antidepressant drugs see Healy's The Anti-
pounds that targeted neurotrophic factor depressant Era (1997) (196).
4 History
ter, or to enjoy the opera more! A whole liter- 5-HT and NE reuptake, whereas the second-
ature was spawned around the drug, most ary amines are typically more selective at in-
famously in Peter Krmer's book, Listening to hibiting NE reuptake. In vivo, however, one of
Prozac (200). A new market was even found the major routes of metabolism of the tertiary
for the drug in the treatment of depression in amines is by demethylation to a secondary
companion animals. By the turn of the cen- amine.
tury, sales of Prozac were earning Lilly in ex- Doxepine (8) is related in structure to the
cess of $2 billion annually. antipsychotic agent pinoxepin (47). However,
5 STRUCTURE-ACTIVITY RELATIONSHIP
AND METABOLISM
(48) Loxapine
Sertraline and fluoxetine undergo meta- tonin, and most other receptors, brofaromine,
bolic demethylation. Unlike the metabolites of interestingly, binds at the 5-HT and NE trans-
most other SSRIs, the desmethyl metabolite of porters with modest affinity (IC,, values of
fluoxetine, norfluoxetine (341, retains the abil- 150 and 500 nM) (207). For comparison, the
ity to inhibit serotonin reuptake (205). Hence, half-life for disappearance of MAO-A inhibi-
fluoxetine takes longer to achieve steady-state tion in brain is phenelzine (19), 11 days; tra-
levels, and it retains activity after metabolism. nylcypromine (23)' 2.5 days; brofaromine
The half-life of fluoxetine is approximately 1 (54), 12 h; and moclobemide (15)' 6 h (re-
day, but elimination of norfluoxetine is pro- viewed in ref. 207). Another advantage of RI-
longed (7-15 days) (205). In addition to its ac- MAS over the older MA0 inhibitors is that a
tions as an SSRI, norfluoxetine also binds at shorter washout time is required; whereas the
5-HT, receptors (127). older MA0 inhibitors typically require about 2
weeks for washout, the washout period for mo-
5.2 Monoamine Oxidase Inhibitors clobemide is about 48 h (208). This is an im-
portant consideration when switching antide-
Because of undesirable side effects associated
pressant therapies from a MA0 inhibitor to,
with monoamine oxidase inhibitor therapy
for example, a TCA or SSRI.
(see section 2.3.2), pharmaceutical companies
Moclobemide undergoes multiple routes of
nearly abandoned research on new analogs in
metabolism with formation of a lactam (55)
the 1960s. The early MA0 inhibitors were
nonselective and irreversible. Today, efforts
toward the development of monoamine oxi-
dase inhibitors are focused on selective
MAO-A or MAO-B inhibitors. Selective
MAO-B inhibitors are being examined in the
treatment of, for example, schizophrenia, Alz-
heimer's disease, and Parkinson's disease. (54) Brofaromine
MAO-B inhibitors might be effective in the
treatment of depression, but relatively little
work has been done in this area. Selegiline
(53) or (-)-deprenyl, a selective irreversible
tine levels are significantly elevated in de- (235,236) and Pierre Fabre [milnacipran (51)l
pressed patients relative to those in healthy (237,238). The Danish company NeuroSearch
controls and that treatment with bupropion has taken one step further and is developing a
normalized these levels (228, 229 and refer- triple inhibitor of norepinephrine/serotonin
ences therein). Peripheral norepinephrine and dopamine uptake with GSK (NS2389).
neurons possess nonadrenergic imidazoline Another idea is to separate the individual en-
binding sites that mediate inhibitory effects antiomers of existing racemic antidepressant
on the release of norepinephrine (230). Plate- drugs, in the hope that the individual isomers
let I, receptors are also downregulated after may show some improvement over the parent
antidepressant treatment; this has led to con- drug. A number of existing antidepressants
jecture that elevation in brain imidazoline re- are racemates, which include fluoxetine (9),
ceptors might lead to greater inhibition of nor- citalopram (41, mirtazepine (14), mianserin
epinephrine release (229). (27) and reboxetine (21). Lundbeck appears to
Another curious agent is tianeptine (69).In have been successful in developing the active
vitro, neither tianeptine nor any of its major (S)-enantiomer of citalopram (4), escitalo-
pram, which is reported to be slightly more
efficacious and to have fewer side effects and a
faster onset of action than that of the parent
compound (239, 240). This approach will not
always succeed, however. Lilly's attempt to
develop (R)-fluoxetinefailed because the com-
pound caused small increases in cardiac QT
intervals (241).
The ability of pindolol to accelerate the on-
set of action of antidepressant drugs has
(69) Tianeptine
prompted a search for more selective antago-
nists or partial agonists acting at the 5-HT,,
metabolites has any effect on monoamine re- receptor. The compound EMD 68843, from
uptake, release, or neurotransmitter binding; Merck KgaA, combines activity as an SSRI
the biochemical effects (acute or chronic) of with a partial agonist profile at the 5-HT,,
this agent in vivo indicate enhanced serotonin receptor, and is in development as an antide-
uptake in the cortex and hippocampus (230, pressant (242,243). The compound YM-35 992
231). (40) from Yamanouchi, which combines an
Tianeptine has now been evaluated in over SSRI profile with blockade of 5-HT,, recep-
3000 patients and has been found to be at least tors, represents another variant on this theme
as effective as the TCAs (e.g., amitriptyline, (244).
imipramine, maprotiline), SSRIs (e.g., fluox- In view of the good clinical safety profile of
etine, sertraline), and mianserin (231, 232). the reversible MAO-A-selective MA0 inhibi-
tor moclobemide (15), it is worth noting that a
6 RECENT DEVELOPMENTS A N D second reversible MA0 inhibitor befloxatone
THINGS TO COME (61)is in development (245). In animal studies
a combination of befloxatone with pindolol
greatly accelerated the action of the MA0 in-
6.1 Monoaminergic Drugs
hibitor in increasing serotonergic neuron dis-
All of the existing antidepressant drugs act charge in rat brain (246), suggesting that this
through monoaminergic mechanisms, and combination may be of clinical interest.
further refinements are still possible in this
6.2 N M D A Receptor Antagonists
arena. The concept of developing dual norepi-
nephrinelserotonin uptake inhibitors, claimed Since the early 1990s evidence has accumu-
for venlafaxine, has been adopted by other lated to show that a variety of drugs that block
companies. Compounds with this profile are the glutamate receptor of the NMDA subtype
being developed by Eli-Lilly [duloxetine (50)l have antidepressant-like profiles in behavioral
6 Recent Developments and Things to Come
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?
:
i
f Antianxiety Agents
KEVIN S. CURRIE .
Neurogen Corporation
Branford, Connecticut
Contents
1 Introduction, 526
1.1The Anxiety Disorders, 526
2 Clinical Applications, 527
2.1 Current Medications, 527
2.1.1 Monoamine Oxidase Inhibitors and
Tricyclic Antidepressants, 528
2.1.2 Benzodiazepines, 528
2.1.3 Buspirone, 531
2.1.4 Serotonin Reuptake Inhibitors, 531
2.2 Adverse Effects, 532
2.3 Drug Metabolism, 533
2.3.1 Benzodiazepines, 533
2.3.2 Buspirone, 536
3 Physiology and Pharmacology, 537
3.1 GABA, 537
3.1.1 The GABAA/BenzodiazepineReceptor
Complex, 538
3.2 Serotonin, 541
3.2.1 5HT-1 Receptors, 542
3.2.2 5HT-2 Receptors, 542
3.2.3 5HT-3 Receptors, 543
3.2.4 GABAISerotonin Interactions, 543
3.3 Norepinephrine, 543
3.4 Neuropeptides, 544
3.4.1 Cholecystokinin, 544
3.4.2 Corticotrophin-Releasing Factor (CRF),
544
3.4.3 Neuropeptide Y, 545
3.5 Glutamate Receptors, 546
4 History, 546
4.1 Discovery of the Benzodiazepines, 546
5 Structure-Activity Relationships, 548
5.1 Animal Models of Anxiety, 548
5.2 Benzodiazepines, 549
5.2.1 1,4-Benzodiazepine-2-Ones: General
Trends, 549
Burger's Medicinal Chemistry and Drug Discovery 5.2.2 1,2-Ring-Fused 1,4-Benzodiazepines,
Sixth Edition, Volume 6: Nervous System Agents 552
Edited by Donald J. Abraham 5.2.3 Heteroaryl[el[1,4]Diazepin-2-Ones,554
ISBN 0-471-27401-1 O 2003 John Wiley & Sons, Inc. 5.2.4 Bis-Heteroaryl[a,f][1,4]Diazepines,554
Antianxiety Agents 2
job interviews). Exposure to the feared situa- rates of 9-13% in the general population have
tion induces pronounced anxiety, and the sit- been reported (16), but this can increase to
uation is either totally avoided or is endured over 70% in high risk populations (e.g.,combat -
only with extreme distress. The avoidance be- veterans, serious accident victims) (17).
haviors and associated anxiety cause signifi- Specific phobias are perhaps the most fa-
cant disruption of the patient's normal social miliar of the anxiety disorders, and are char-
and occupational functioning. SP has been acterized by a disproportionate fear of an ob- '
classified into two subtypes: public speaking ject (e.g., spider, snake) or situation (e.g.,
only and generalized social phobia (11).Re- flying, receiving an injection). When the stim-
cent epidemiological studies reveal lifetime ulus is confronted it elicits an intense anxiety -
prevalence rates around 10% (12), making so- reaction, which the sufferer recognizes to be
cial phobia one of the most common anxiety excessive but is unable to moderate. Prophy-
disorders. lactic use of anxiolytics for predictably stress-
The essential feature of panic disorder ful situations is helpful, but in the long term,
(PD) (13) is the occurrence of repeated, unex- medication is less successful than behavioral
pected panic attacks. There is a marked worry therapy in the treatment of specific phobias.
about the consequences of the attack and the Anxiety disorders are chronic conditions
possibility of having a future attack. The per- and drug therapy may need to continue for
sistent anxiety evoked by the panic attacks months or even years, particularly for the
causes major behavioral changes and intru- more intractable disorders such as OCD and
sion into normal life. Around 50% of panic dis- PTSD. Response rates are often incomplete,
order patients also suffer from agoraphobia. A and relapse upon discontinuation of treat-
lifetime prevalence of 3.5% has been estimated ment is common. For disorders with multiple
(3), and a high comorbidity with depression symptoms (e.g., PTSD) only some of the symp-
and other anxiety disorders is observed. toms are relieved. Coexistence of anxiety with
Obsessive-compulsive disorder (OCD) in- other mental disorders, particularly depres-
volves recurrent obsessions and compulsions, sion (18,19),is very common and such comor-
which are severe enough to cause marked dis- bidity is predictive of a poorer treatment out-
tress and major functional impairment. The come.
sufferer is aware that the obsessions and com-
pulsions are unreasonable, but is powerless to 2 CLINICAL APPLICATIONS
stop them. Obsessions are recurrent, un-
wanted thoughts or images, whereas compul- 2.1 Current Medications
sions are repetitive acts or rituals. The indi-
vidual typically feels compelled to perform Throughout history, humankind has used a
compulsions to alleviate the anxiety associ- variety of different agents to relieve anxiety.
ated with an obsession, or to prevent the oc- The oldest one of these is probably alcohol,
currence of some dreaded event. The lifetime which has served as an anxiolytic for thou-
prevalence of OCD is estimated at 2.3% (14). sands of years and is still widely used for the
In posttraumatic stress disorder (PTSD) self-medication of anxiety today. In the past
(15),a characteristic set of symptoms develops century, a number of CNS-depressant medica-
following exposure to an event that induces tions have been introduced, beginning with
extreme-fe& or terror (e.g., war, rape). These ethanol surrogates such as chloral hydrate.
symptoms include a persistent and intrusive The barbiturates, carbarnates (e.g., mepro-
reexperiencing of the event through flash- bamate), and bromide salts were also used, al-
backs or nightmares, and an intense distress though these agents were associated with a
at exposure to cues that are reminiscent of the significant sedative effect, as well as more se-
event. The sufferer deliberately avoids such rious drawbacks such as the risk of depen-
stimuli and may become detached, with- dency, high toxicity in overdose, and a poten-
drawn, and emotionally numb. Additional tially fatal interaction with alcohol.
symptoms include insomnia, impaired concen- At present, a number of drugs with various
tration, and unprovoked anger. Prevalence mechanisms of action are available for the
Antianxiety Agents
8
(1) Diazepam (2) Chlordiazepoxide (3)Clorazepate
(4) Lorazepam
0
(5) Alprazolam (6)Oxazepam
v
(9) Buspirone
(7) Bromazepam (8)Clonazepam
CI CF3
(10) Paroxetine (11) Sertraline (12) Fluvoxamine
der, although other high potency benzodiaz- alprazolam requires higher doses than those
epines (e.g., lorazepam) have shown a similar for GAD, reflecting the greater resistance of
effect. Lower potency BZs (e.g., diazepam) re- panic disorder to pharmacotherapy. There is
quire higher doses for panic disorder, and even little evidence for the efficacy of benzodiaz-
2 Clinical Applications
-i
/
F
(13) Fluoxetine (14) Citalopram (15) Venlafaxine -
epines in OCD or PTSD, and none has been in patients who have recently used benzodiaz-
approved specifically for these disorders. In epines (53). Buspirone is the only member of
addition to the most common anxiolytic ben- the azapirone class to be marketed in the
zodiazepines shown in Table 9.1, a number of United States, although tandospirone (16) is
others are available (51), including pinaz- registered in Japan.
epam, camazepam, 2'-chlorodesmethyldiaz-
epam, cloxazolam, ketazolam, and oxazolam.
The use of these agents is much less wide-
spread, however, and they are not available in
the United States. Benzodiazepines have the
potential to produce withdrawal symptoms
and dependency upon prolonged administra-
tion. As a result they are indicated primarily
for the short-term relief of anxiety and are
increasingly not recommended for chronic 2.1.4 Serotonin Reuptake Inhibitors. Although
dosing. The increasing awareness and nega- there have been no launches of novel first-indica-
tive reaction to the withdrawal, dependency, tion anxiolytics since buspirone in 1986, the last 10
and abuse liabilities of the benzodiazepines years have seen a @cant shift in the treatment
has led to a significant reduction in their use, of anxiety disorders. During this time it has be-
despite their proven efficacy. come increasingly clear that the selective sero-
tonin reuptake inhibitors (SSRIs), origmally
2.1.3 Buspirone. The dominance enjoyed launched as antidepressants, are effective in a
by the benzodiazepines is illustrated by the range of anxiety disorders (54-56). This finding
fact that no new anxiolytics were launched un- has coincided with the realization of the with-
til buspirone (9) reached the market in 1986. drawal and dependency liability of the benzodiaz-
Buspirone is a member of the azapirone class, epines, meaning that the SSRIs have comparable
and produces its therapeutic effects through efficacy with an improved safety protile. The net
partial agonism at 5HT-1A receptors. Buspi- result is that the SSRIs are now considered first-
rone is indicated and widely used for GAD, but line treatments for panic disorder, OCD (57), so-
has failed to show significant efficacy in other cial phobia (58),and PTSD, and are gaining popu-
anxiety disorders (52). Although perhaps less larity for GAD. Unlike the benzodiazepines or
consistently effective than the BZs, buspi- buspirone, the SSRIs have the added advantage of
rone's improved safety profile provides the effectively treating depression, which is frequently
main point of differentiation between the two comorbid with anxiety disorders (59).
therapies. Buspirone causes less sedation, mo- Paroxetine (10) (60), is the most widely
tor and cognitive impairment, and does not used SSRI in the treatment of anxiety. It is
appear to be associated with any withdrawal approved for the treatment of OCD and panic
syndrome. Drawbacks include a delayed onset disorder, and is currently the only SSRI indi-
of action (several weeks), and a reduced effect cated for use in social phobia and GAD. Parox-
Antianxiety Agents
etine has also shown efficacy in one small trial though they also act at muscarinic acetylcho-
for specific phobia (61).Fluvoxamine (12) (62) line receptors, histamine H1 receptors, and
is used in OCD, and was the first SSRI to gain al-adrenergic receptors. Common anticholin-
approval for this indication. Fluoxetine (13) ergic side effects include dry mouth, constipa-
(631, and sertraline (11)(64,65) are also indi- tion, urinary retention, and blurred vision.
cated for OCD. In addition, sertraline (66, 67) Blockade of adrenergic receptors can cause or-
is approved for treatment of panic disorder thostatic hypotension and dizziness, and ac-
and recently became the first and only medi- tions at histamine receptors lead to sedation
cation approved for PTSD. In the United and weight gain. The therapeutic effect of the
States, citalopram (14) (68) is indicated for TCAs does not appear until after 2-4 weeks of
use in OCD and has been used to prevent panic therapy, and during this period there can be
attacks for several years in Europe. Parox- an initial exacerbation of anxiety symptoms,
etine and sertraline are marketed as single en- which may lead to patient termination of ther-
antiomers, whereas citalopram and fluoxetine apy. The unfavorable side-effect profiles of the
are available as racemic mixtures, and fluvox- MAOIs and TCAs have largely relegated these
arnine is achiral. Venlafaxine (15) is a dual drugs to last resort cases where other medica-
serotonin and norepinephrine reuptake inhib- tions have failed.
itor (SNRI) that has recently been approved As a class, the benzodiazepines have dem-
for use in GAD (69, 70). The major drawback onstrated a remarkable safety profile and
of the SSRIs in comparison to the BZs is their their high tolerability has been a major factor
delayed onset of action. As with their antide- in their widespread acceptance and phenome-
pressant effect, it takes 2-4 weeks before an- nal success. The most commonly reported side
xiolytic efficacy is seen and this can cause pa- effects with benzodiazepine anxiolytics are
tients to discontinue the drug. In this daytime sedation ("hangover" effect), sleep
scenario, the benzodiazepines can be a useful disturbances, cognitive impairment, motor in-
adjunct to anxiolytic therapy with SSRIs. The coordination, and ataxia. The latter symptoms
immediate anxiolysis produced by the benzo- are of greater importance in the elderly, who
diazepine serves as a bridge until the efficacy constitute a significant population of BZ users,
of the SSRI appears after 3-4 weeks, and the in whom they are associated with an increased
BZ is then tapered off slowly to avoid with- incidence of falls and fractures (71). Many pa-
drawal, leaving the SSRI as a long-term mono- tients develop a tolerance to these side effects
therapy. over time, however, while rarely developing
tolerance to the anxiolytic effects. The great-
2.2 Adverse Effects
est problems with the benzodiazepines are
The monoamine oxidase inhibitors are associ- withdrawal symptoms, dependency liability,
ated with a number of undesirable side effects and abuse potential, the separation of which is
including weight gain, postural hypotension, not always obvious (72). Abrupt discontinua-
sexual dysfunction, and insomnia. The most tion of benzodiazepine therapy commonly
serious side effect is the risk of tyramine-re- leads to a "withdrawal syndrome" character-
lated hypertensive crisis, often referred to as ized by insomnia, anxiety, fatigue, irritability,
the "cheese effect," which can be fatal. To light-headedness, headache, and gastrointes-
avoid this situation patients taking MAOIs tinal upset. These symptoms must be differen-
must limit their tyramine intake, and the re- tiated from rebound phenomena (i.e., recur-
strictive diet required to accomplish this leads rence of the original anxiety), which may also
to low patient compliance. A similar interac- occur upon termination of therapy. Short-act-
tion occurs when switching patients from ing benzodiazepines (e.g., lorazepam) tend to
MA01 to SSRI therapy, and a minimum have more severe withdrawal symptoms than
2-week washout period before commencement those of long-acting ones (e.g., diazepam) be-
of SSRI therapy is essential to allow MA0 lev- cause of the more pronounced fluctuations in
els to return to normal. The therapeutic ef- blood levels.
fects of the TCAs derive from their inhibition To minimize the risk of withdrawal effects.
of serotonin and norepinephrine uptake, al- benzodiazepine therapy is usually discontin-
2 Clinical Applications
ued by a gradual tapering of the dose. Never- however, and the symptoms typically resolve
theless, an estimated 25% of patients find after a few days. Like buspirone, the SSRII
withdrawal difficult and fail to discontinue SNRI class has a delayed therapeutic effect
therapy (73, 741, and such patients may be and a potentially serious drug interaction with
considered to have a low dose dependency on MAOIs.
benzodiazepines. On the other hand, high 2.3 Drug Metabolism
dose dependency, and abuse, is characterized P
by drug-seeking behaviors and the consump- The metabolic profile of anxiolytic agents has
tion of elevated, nontherapeutic doses. The important ramifications for their clinical use.
risk of withdrawal and dependency increases This section discusses the metabolism of the -
with dose and length of treatment, but clinical benzodiazepines and buspirone in some detail.
experience shows that anxious patients have The metabolism of the SSRIs, TCAs, and
little tendency to escalate the dose of benzodi- MAOIs is covered in the chapter on antide-
azepines, and such abuse is typically found in pressants.
patients who have a history of alcohol and/or
substance abuse (75, 76). It is noteworthy, 2.3.1 Benzodiazepines. To varying de-
however, that substance abuse is often comor- grees, all benzodiazepines produce five clinical
bid with anxiety disorders. The increasing effects: anxiolysis, sedation, muscle relax-
public and medical concern over the depen- ation, seizure protection, and memory impair-
dency and abuse liabilities of benzodiazepines ment. In terms of the pharmacology underly-
has considerably reduced their use, and many ing these effects there is little to choose
countries now have restrictions on their dis- between the benzodiazepines, in that they all
pensation. This may be an overreaction, how- act as nonselective agonists at the benzodiaz-
ever, which in many cases has likely led to the epine receptor (BZR). The classification of
substitution of benzodiazepines by less effec- benzodiazepines into therapeutic indications
tive medications. Recently, it has been argued is therefore based largely on their pharmaco-
that the risk-to-benefit ratio of benzodiaz- kinetic profile. Thus, benzodiazepines with
epines be reassessed and their clinical utility long half-lives and/or long-lived active metab-
reevaluated (77, 78). olites are used as anxiolytics, whereas those
Buspirone is a well-tolerated drug, the with short half-lives are more appropriately
most commonly reported side effects being used as sedative-hypnotics. Elimination of the
transient dizziness, light-headedness, head- benzodiazepines is by hepatic metabolism and
ache, and gastrointestinal disturbances. excretion of conjugated metabolites in the
Other limitations of buspirone are its delayed urine (80-83). During metabolism, many ben-
onset of action (few days to a few weeks) and a zodiazepines give rise to metabolites that of-
significant drug interaction with MAOIs. ten have comparable pharmacological activity
The SSRIs (paroxetine, fluoxetine, sertra- to, but a longer half-life than, the parent com-
line, fluvoxamine, citalopram) and SNRI (ven- pound. The metabolic profile is therefore an
lafaxine) have an impressive side-effect pro- important determinant of the ultimate in vivo
file, and this has contributed to their biological activity.
widespread use. Possible adverse effects in- In general the first, and most rapid, step in
clude nausea, insomnia, and agitation, but 1,Cbenzodiazepine metabolism (Fig. 9.2) is
these are generally manageable and diminish the oxidative removal of the N-1 alkyl sub-
over time. More significant is the association stituent, if present (82).Benzodiazepines with
-
of the SSRIs with sexual dysfunction. in both a methyl group at N-1 are the most stable and
men and women. These effects are longer last- the rate of dealkylation increases with the size
ing, and can occur in up to 40% of patients of the N-1 alkyl group. For example, the deal-
(79). A withdrawal syndrome has also been kylation half-life for diazepam is 20 -30 h, but
observed with the SSRIs, characterized by " diz- for prazepam, which bears a cyclopropyl-
ziness, headache, and irritability upon abrupt methyl group at N-1, this half-life is approxi-
discontinuation. This is much less serious mately 1 h (82).Substituents elsewhere in the
than that observed with benzodiazepines, molecule have little impact on the rate of deal-
Antianxiety Agents
Chlordiazepoxide Diazepam --
I Dealkylation I
+
NP-desmethyl
chlordiezepoxide
demoxepam*
\
45h #
1-2 h
Clorazepate ----+ desmethyl diazepam*, Alprazolam Bromazepam
I Hydroxylation I
1-hydroxymethyl 3-hydroxy Lorazepa,,,
Oxazepam* Temazepam* alprozolam bromazepam
5-15 h I 10-20 hl 1-2 h l Short 1 10-20 hl
1 Glucuronidation I
kylation. Following dealkylation, the next step disease, making these drugs more suitable for
is hydroxylation at the 3-position. The hy- use in elderly patients and those with liver
droxylation half-lives (typically approaching dysfunction. The limited structural diversity
or exceeding 100 h) are longer than the deal- of the benzodiazepines means that the same
kylation half-lives, resulting in an accumula- active metabolites can be generated from sev-
tion of the pharmacologically active N-1 deal- eral different drugs. For example, oxazepam,
kylated metabolites, so these compounds which is marketed as an anxiolytic in its own
contribute significantly to the overall biologi- right, is generated in uivo from diazepam, clo-
cal activity. The rates of hy droxylation are rel- razepate, prazepam, and chlordiazepoxide. In
atively independent of the substituent in the the case of the triazolo- (e.g., alplrazolam) and
C-ring, with the exception of bromazepam. imidazo- (e.g., midazolam) benzodiazepines,
The 3-hydroxy metabolites are then glucu- hydroxylation of the C-1 methyl group is the
ronidated and excreted in the urine. The glu- principal metabolic pathway.
curonides are pharmacologically inactive, and Alprazolam (5) is rapidly absorbed after
this fact has been exploited in drugs such as oral dosing, reaching peak plasma levels in
oxazepam, lorazepam, and temazepam. As less than 1 h with an oral bioavailability ex-
3-hydroxybenzodiazepines, these agents are ceeding 90% (86). Alprazolam has a lower pro-
glucuronidated directly and their clinical use tein binding (68%)than that of most benzodi-
is not complicated by the formation or accu- azepines (87), reflecting the increased polarity
mulation of active metabolites. Further, the imparted by the triazole ring. Although the
rate of glucuronidation (84, 85) appears to be principal metabolite l-hydroxymethylalprazo-
much less impacted than the rate of dealkyla- lam is a potent BZR ligand (Ki = 4.2 nM) (881,
tion or hydroxylation (82) by aging or by liver it does not contribute significantly to the clin-
2 Clinical Applications
ical effect because it is rapidly conjugated and pared to other benzodiazepines, has a rela-
plasma levels never exceed 10% of the parent tively high free fraction (30%).Bromazepam is
drug (89). The elimination half-lives of alpra- metabolized principally by hydroxylation at.
zolam and 1-hydroxymethylalprazolamare 9 the 3-position. The pyridyl ring at position 5
to 12 h and 1 to 2 h, respectively. facilitates this reaction, the hydroxylation'
Chlordiazepoxide (2) is completely ab- half-life for bromazepam being considerably
sorbed upon oral administration and reaches shorter (t,,, = 8-19 h) than that for other benr
peak plasma concentrations in 1-2 h (90).As a zodiazepines (94).A second pathway opens the
highly lipophilic molecule it is 94% bound to diazepine ring to give the pharmacologically
plasma proteins (91) and readily penetrates inactive 2-(2-amino-5-bromobenzoy1)pyridine-
the brain, with CSF levels paralleling un- (95). Bromazepam metabolism does not pro-
bound plasma levels (92). Chlordiazepoxide duce any metabolites with a longer half-life
has a mean half-life of 15 h. It is metabolized than that of the parent compound.
first by oxidative removal of the N-2 methyl Clonazepam (8) is almost completely ab-
group (to give N-desmethylchlordiazepoxide), sorbed after oral dosing (96) with an average
followed by hydrolysis to the lactam (demox- T,, of 2-4 h. As with other 7-nitro benzodi-
epam), and reduction of the N-5 oxide to give azepines, the major metabolic pathway for
desrnethyldiazepam. All of these metabolites clonazepam is reduction of the nitro group,
are pharmacologically active. acetylation of the resulting amine, and elimi-
Clorazepate (3)is readily decarboxylated in nation of the acetamide. Hydroxylation of
the stomach to generate desrnethyldiazepam, clonazepam or of 7-amino clonazepam to give
which is then rapidly absorbed with peak the 3-hydroxy derivatives represents minor
plasma levels occurring 0.5 to 2 h postdose. metabolic pathways. The elimination half-life
Clorazepate itself is poorly absorbed (93) and of clonazepam is 20-30 h (97), and no active
acts only as a prodrug of desmethyldiazeparn. metabolites are produced.
Diazepam (1)is completely and rapidly ab- Lorazepam (4) is well absorbed, with oral
sorbed following oral administration, reach- bioavailabilty close to 100% after a typical 2
ing peak plasma levels after 30-90 min. As a mg dose, and peak levels are obtained within
result of its lipophilic character it is highly 2 h of administration. Lorazepam is conju-
protein bound (98-99%)and is widely distrib- gated at the 3-hydroxy group and the resulting
uted, readily crossing the blood-brain barrier inactive glucuronide is excreted in the urine
and placenta, and also passing into breast with a mean elimination half-life of approxi-
milk. Metabolism of diazepam gives rise to mately 14 h (82).
three active metabolites. N-1 demethylation Oxazepam (6)is formed during the metab-
produces desmethyldiazepam, which has a olism of many other benzodiazepines, but its
longer half-life than that of diazepam own metabolic profile is relatively simple. Like
(40-200 h compared to 20-30 h) (82).On mul- lorazepam, the major metabolic pathway is
tiple dosing, plasma desrnethyldiazepam lev- glucuronidation at the 3-hydroxy group fol-
els exceed diazepam levels. Hydroxylation of lowed by urinary excretion. Up to 80% of the
desrnethyldiazepam then produces oxazepam dose is recovered from the urine as the gluc-
(6), which is conjugated and eliminated as the uronide. The mean half-life of oxazepam is ap-
glucuronide. Direct hydroxylation at the 3-po- proximately 9 h (98).
sition of diazepam yields temazepam, which is As previously mentioned, the choice of ben-
also directly conjugated and excreted. Of these zodiazepine for a given indication is governed
metabolites, desmethyldiazepam contributes in large part by its plasma half-life. This half-
more to the overall pharmacodynamic effect life, however, does not always predict the du-
because of its longer half-life and resultant ration of the pharmacological effect. Using se-
accumulation. dation (99) or psychomotor impairment (100)
After oral dosing bromazepam (7) is well as endpoints, lorazepam was shown to have a
absorbed, with a bioavailability of 84-98% and longer lasting effect than that of diazepam,
a rather variable T,, of 0.5 to 8 h (94). It has even though its plasma half-life is about half
an elimination half-life of 8-19 h and, com- as long. For diazepam, the rate of elimination
Antianxiety Agents
from the CSF is equivalent to the rate of elim- 5'-hydroxybuspirone plasma levels (both free
ination from plasma (101), but lorazepam has and conjugated) are up to six times greater
a longer half-life in CSF than in plasma. than buspirone levels, indicating the impor-
Lorazepam has a greater affinity for the BZR tance of this metabolic pathway. Plasma levels
than does diazepam (lorazeparn, Ki = 1 nM; of 1-PP are approximately fourfold greater
diazepam, Ki = 27 nM) and dissociates from than those of buspirone (105). 5-Hydroxybus-
the receptor more slowly (102). Thus the re- pirone shows no biological activity, in keeping
lease of lorazepam from the receptor has been with the structure-activity relationship (SAR)
suggested as the rate-limiting step in its elim- for this series of compounds as it relates to
ination from CSF, resulting in a longer phar- affinity for the 5HT-1A receptor (see section
macodynamic effect than would be expected 5.4.1); however, 1-PP has up to 20% of the
based on the plasma half-life. potency of buspirone in the Vogel anticonflict
model (105). It also has some affinity (IC,, =
2.3.2 Buspirone. Buspirone is completely 95 nM) for the adrenergic a2 receptor, al-
absorbed after oral dosing, but exhaustive though it has no activity at the 5HT-1A recep-
fist-pass metabolism limits the absolute bio- tor. Given its significantly higher plasma and
availability to only 4% (103). The pharmacoki- brain levels relative to those of buspirone,
netics are linear and metabolism is not satu- 1-PP may contribute to the overall anxiolytic
rated in the therapeutic dose range (10-40 effect.
mg) (104). First-pass metabolism is decreased Until recently,- . it was believed that 1-PP
when buspirone is taken after a meal but ab- was the only buspirone metabolite to possess
sorption is not, leading to increased drug ex- any biological activity. Under the assumption
posure (105). This effect is also seen with that buspirone was the active agent, dosing
other drugs that undergo extensive first-pass schedules were designed to optimize buspi-
metabolism (106).Age has little impact on bu- rone exposure at the expense of metabolites.
spirone pharmacokinetics, but hepatic cirrho- Bristol-Myers Squibb, the manufacturer of
sis extends the elimination half-life by 50% buspirone, recently carried out clinical studies
(105). The mean half-life of buspirone in nor- on a buspirone transdermal patch (108) with
mal subjects ranges from 2 to 11 h, averaging the aim of increasing buspirone levels, and
at around 4 h, and this short half-life accounts hence the anxiolytic effect, by circumventing
for one of buspirone's drawbacks, that is, the first-pass metabolism. Surprisingly, no signif-
need for three times daily dosing. Buspirone is icant activity was seen with this delivery sys-
a lipophilic compound and is highly bound to tem, prompting a reevaluation of buspirone
plasma proteins (>95%), predominantly albu- and its metabolites. Remarkably, almost 15
min (105). years after the launch of buspirone, it was dis-
The extensive oxidative metabolism of bu- covered that 6-hydroxybuspirone (18) not
spirone, mediated by CYP450 3A4, produces a only possessed anxiolytic activity, but is ap-
number of hydroxylated metabolites including parently responsible for most, if not all, of bus-
1-(2-pyrimidiny1)piperazine (1-PP), 5'-hy- pirone's clinical effects (109). (18)has signifi-
droxybuspirone (17) and 6-hydroxybuspirone cant affinity for the 5HT-1A receptor (Ki = 57
(18) (107). After a 20-mg dose of buspirone, nM), approaching that of buspirone (Ki = 31
nM). Furthermore, (18) is the second most
abundant human metabolite of buspirone,
having blood levels 40 times greater than that
of buspirone and severalfold greater than that
of 1-PP after oral dosing. The initial preclini-
cal work on buspirone revealed no in vivo an-
xiolytic activity for (18)in animal models, but
more recently, using the rat pup ultrasonic
vocalization model, (18) was found to have
(17) Rl = OH, R2 = H comparable activity to that of buspirone (ID,,
(18) Rl = H,R2 = OH = 0.13 and 0.10 mgbg, respectively) (109).
3 Physiology and Pharmacology
cluding those for barbiturates, neurosteroids, ranged round a central chloride channel (Fig.
picrotoxinin, inhalation anesthetics, and eth- 9.3). To date, 21 receptor subunits have been
anol (118). cloned (six a, four P, four y, one 6, one E , one T ,
Studies have shown a reduced density of one 8, and three p), although the E and p sub-
lymphocyte BZ receptor sites (119) and plate- units appear to be associated with GABA,
let BZ sites in GAD patients, which was in- rather than GABA, receptors. Immunohisto-
creased following diazepam treatment (120). chemical studies indicate that the a,P2/3, and
These peripheral receptors are pharmacologi- y2 subunits are most frequently coassembled
cally distinct from the central GABA, recep- to form GABA, receptors in uiuo, with a stoi-
tors, so the relevance of these studies to anxi- chiometry of 2 ~ ~ 2 P : l (129).
-y Even with this
ety is unclear. More relevant may be the study restriction, the differential combination of
of central BZR activity in anxiety patients. Re- these subunits could potentially lead to thou-
duced BZR sensitivity has been shown in GAD sands of receptor subtypes. Fortunately, the
patients by measuring saccadic eye move- GABA, receptor population appears to be
ments (121, 122). This effect was even more dominated by only a handful of receptor sub-
pronounced in patients with panic disorder types, differentiated mainly by the identity of
(123), but was absent in OCD (124). These the a subunit. These subtypes have been
studies suggest that there may be a reduced shown to possess a distinct but regionally
BZ receptor function in some anxiety disor- overlapping distribution within the brain
ders. (128, 130). a1-containing subunits are the
most abundant, accounting for over 50% of
3.1 .I The GABAJBenzodiazepine Receptor all GABA, receptors, and are distributed
Complex. The GABAAreceptor (125-127) is a throughout the brain (131-133). a2 and a3
transmembrane protein belonging to the su- subtypes together constitute a further 30-
perfamily of ligand gated ion channels (128). 35% of GABA, receptors, with a2 isoforms lo-
The receptor consists of five subunits ar- cated in limbic structures and a 3 receptors
i
:I
3 Physiology and Pharmacology
!
found mainly in the cortex (134). a5 subtypes (Ambien) has implicated the a1 subtype in se-
represent only 5% of the total GABA, receptor dation, whereas the localization of the a 5 sub-
population and are localized in the hippocam- unit in the hippocampus suggests its involve-.
pus (135). ment in memory processes. The presence of (112.
As shown in Fig. 9.3 the GABA, receptor and a 3 subtypes in limbic structures points to
has a topology characteristic of the nicotinoid a role for these subtypes in anxiety and emo-
superfamily of receptors. Each subunit spans tional behavior. Recent work with transgeniiz
the membrane four times, and the second animals is confirming, for the first time, some
transmembrane domain of each subunit forms of these theories (142). Mutation of the critical
the wall of the ion channel. The GABA binding His-102 of the a subunit to an arginine abol-
sites, of which there are two per receptor, are
ishes the binding affinity of benzodiazepines
located at the two homologous alp interfaces.
(143) at the mutated subtype, but does not
Point mutation studies have identified several
key amino acid residues on both subunits that affect the response to GABA. Mice expressing
contribute to GABA binding (1361, and both al, a2, or a 3 receptors containing this knock-in
GABA sites must be occupied to induce open- mutation develop normally and show no overt
ing of the chloride channel. The benzodiaz- anxious phenotype, arguing against the long-
epine receptor site (BZR) (137) is located at held notion that there are endogenous ligands
the interface of the a- and y-subunits, and for the BZR that regulate anxiety.
there is one BZR per GABA receptor (129). Evaluation of these animals in behavioral
Point mutation and photoaffinity labeling ex- paradigms shows that the sedative and amnes-
periments have revealed at least 11amino acid tic effects of diazepam are mediated through
residues in the a- and y-subunits that contrib- the a1 subtype (144-146). Although the a1
ute to ligand binding and efficacy at the BZR subtype appears to mediate sedation, the sleep
(138),the most significant of which is the his- latency and sleep pattern were unchanged in
tidine at position 102 (101 in rat) of the a-sub- the mutant mice, indicating that different
unit. The a1-, a2-, a3-, and a5-containing re- subtypes mediate the sedative and hypnotic
ceptors carry a histidine at position 102 and effects of diazepam (147). Conditional knock-in
are benzodiazepine sensitive, but the a4 and mice wherein the a2 and a 3 subtypes are ren-
a6 subtypes, which bear arginine at this posi- dered benzodiazepine insensitive have also
tion, are benzodiazepine insensitive (139). been generated. In the elevated plus maze and
The y2 subunit is more abundant in the brain lightldark choice models, a loss of diazepam
than y l or y3 subunits and is important for anxiolytic activity is observed in the a2
ligand binding, given that the GABA, recep- knock-in animals, but not with the a 3 knock-
tors of mice lacking the gene for y2 subunits ins, implicating the a2 subtype in the media-
are insensitive to benzodiazepines (140). The tion of benzodiazepine anxiolytic effects (148).
nature of the P-subunit has a lesser influence Given this evidence, it is tempting to speculate
on BZR pharmacology (141). This fact, cou- that the ideal anxiolytic, one that is clinically
pled with the consistent expression of y2 sub- effective but free from sedation, amnesia, de-
units in various subtypes, means that the pendency, and withdrawal effects, will be real-
identity of the a -subunit is the major deter- ized in a compound possessing the appropriate
minant of BZR pharmacology (128, 135). Ac- subtype selectivity. It is probable, however,
cordingly, the dominant benzodiazepine-sen- that a given subtype is associated with more
sitive GABA, subtypes in the brain are often than one clinical effect. Indeed the a2 subtype,
referred to simply as the al, a2, a3, and a5 although clearly associated with the anxiolytic
GABAAreceptors. actions of diazepam, has also been shown, us-
Along with an increased understanding of ing a2 and a 3 knock-in mice, to contribute to
the molecular diversity and anatomical local- the myorelaxant actions of diazepam (149).
ization of different GABAAreceptor subtypes Thus, simple subtype selectivity alone may
has come the notion that specific clinical ef- not be enough to yield a true side effect-free
fects may reside in specific subtypes. The se- anxiolytic. More fruitful may be the combina-
lective affinity of the hypnotic agent zolpidem tion of subtype selectivity and partial ago-
Antianxiety Agents
nism, and this hypothesis underlies the most response is modulated by a given ligand is de-
promising area of current anxiolytic research. r~endenton the GABA concentration used in
A
Ligands at the BZR are allosteric modula- the assay. When this variability is superim-
tors of GABA function, and they have no effect posed on the subunit heterogeneity and differ-
in the absence of GABA. This allosteric mod- ential anatomical distributions of GABA, re-
ulation can be positive (enhancement of ceptors a complex picture emerges, but it is
GABA function, increased neuronal inhibi- one that offers numerous possibilities for im-
tion) or negative (inhibition of GABA func- proved therapeutic modulation of central
tion, reduction in neuronal inhibition). Li- GABA function.
gands effecting maximum positive modulation Based on SAR data for a variety " of struc-
are known as full agonists, or as compounds t u r d classes of ligands, several models of the
with high intrinsic efficacy; that is, they pro- benzodiazepine binding site have been ad-
duce the maximal response at low receptor oc- vanced (151-162). These models, although
cupancy. This profile is characteristic of the sharing many common features, have gener-
classical benzodiazepines. Ligands that induce ally been slightly different for inverse ago-
maximum negative modulation are full in- nists, antagonists, and agonists, as well as for
verse agonists, whereas antagonists have no diazepam-sensitive and diazepam-insensitive
effect on GABA function. In between these ex- subtypes. The pharmacophoric descriptors for
tremes, on a continuum of intermediate effi- all these models have now been incorporated
cacy, lie partial agonists and partial inverse into an inclusive pharmacophore model for
agonists. Partial agonists can be defined as li- the BZR (163) that rationalizes ligand-recep-
gands that induce a smaller response than tor interactions at the molecular level for in-
that of a full agonist, at the same receptor oc- verse agonists, antagonists, and agonists and
cupancy (Fig. 9.4). In principle, a partial ago- for all major subtypes (d-a6) (Fig. 9.5). This
nist might never produce the maximum poten- model identifies three important interactions
tiation of a full agonist, even at 100% receptor that anchor the ligand through hydrogen bond
occupancy. Because anxiolysis requires less donor (HI, H2) and hydrogen bond acceptor
receptor occupancy than sedation and other (Al) points on the receptor. L1, 2, 3, and Di
side effects (150),a number of partial agonists represent lipophilic regions, the occupation of
have been investigated as potential nonsedat- which influences the affinity, efficacy, and se-
ing anxiolytics. The modulation of GABA re- lectivity of the ligand. S1, S2, and S3 are re-
sponse can span a tremendous range, from gions of steric intolerance that restrict the size
increases in GABA-induced C1- current ap- of BZR ligands. Region LDi appears to be
proaching 800% to inhibition of 60%. It should larger in a1 subtypes than in the others, and
be noted that the degree to which the GABA occupation of this region may thus confer a1
3 Physiology and Pharmacology
LP
n
I
I
LDi , S1, S2, 53 = regions of steric repulsion.
,, I
L--4
0 I
I
' \ I
Figure 9.5. Diazepam (1)(heavy line) and CGS9896 (67) (dotted line) fitted to a schematic repre-
sentation of the inclusive pharmacophorelreceptor model for the BZR. Adapted from Ref. 163, copy-
right Overseas Publications Association, NV, with permission from Taylor and Francis Ltd.
selectivity (161, 162). Occupation of region L3 originate in the raphe nucleus (168). They in-
(e.g., by the C-5 phenyl ring of the benzodiaz- nervate a number of brain regions including
epines) is commonly associated with agonist the limbic system, and in particular the septo-
activity. Recently, the inclusive pharmaco- hippocampal system and the amygdala. It has
phore model has been integrated with data been proposed (169) that anxiety is a result of
from point mutation and photoaffinity-label- excessive serotonin activity in these specific
ing experiments to provide an enhanced pic- brain regions. The serotonergic neurons aris-
ture of the benzodiazepine binding site (164). ing from the dorsal raphe nucleus innervate
Another study has used behavioral endpoints the periacqueductal gray (PAG)and the arnyg-
for different ligands to construct a model of dala, and these may regulate adaptive responses
ligand-BZR interactions that contribute to an to acute stress (166). On the other hand, 5HT
anxiolytic effect (165). neurons originating in the median raphe nu-
cleus, which innervate the hippocampus, me-
3.2 Serotonin
diate resistance to chronic stress and failure of
Both depression and anxiety appear to be as- this pathway may contribute to depression.
sociated with a dysregulation of serotonergic Through their widespread blockade of the
function at some level, a hypothesis supported serotonin transporter, the SSRIs are known to
by the clinical efficacy of the SSRIs in both increase serotonin levels at virtually every
disorders. On the other hand, the benzodiaz- synapse in the CNS, yet the exact neuronal
epines have no clinical utility as antidepres- pathways and receptors through which their
sants. It has therefore been suggested that, anxiolytic effect is ultimately produced re-
although serotonin has a role in both disor- main unclear (166). To date, 15 different 5HT
ders, different serotonergic pathways and re- receptor subtypes have been identified (170).
ceptor subtypes are responsible for the modu- Of these, seven subtypes have been implicated
lation of anxiety and depression (166, 167). to a greater or lesser degree in anxiety: the
Some of the major 5HT neuronal pathways 5HT-lA, 5HT-lB/D, 5HT-2A/B/C, and 5HT-3
Antianxiety Agents
receptors (171, 172). A survey of preclinical lytic effect in the mouse lightldark box model,
research into serotonergic agents as potential and this effect was attenuated by a 5HT-1B
anxiolytics (173) shows that 50% of studies agonist and also by the benzodiazepine antag-
over the past two decades involve 5HT-1A li- onist flumazenil (185). The latter phenome-
gands, whereas 5HT-2 and 5HT-3 compounds non suggests a functional interaction between
account for 15 and 13% of studies, respec- 5HT-1BP receptors and the GABA system in
tively, giving a rough guide to the presumed the modulation of anxiety. Indeed, the 5HT-
importance of each receptor in anxiety. 1 B P heteroreceptor is known to exert an in-
hibitory influence on the release of a number
3.2.1 5HT-1 Receptors. Of all the seroto- of neurotransmitters, including GABA (186).
nin receptors implicated in anxiety, the Thus, antagonists at the 5HT-1B/D receptors
5HT-1A receptor (174) has been the subject of may produce an anxiolytic effect, at least in
the most study. The only marketed anxiolytic part, through a secondary enhancement of
acting directly on a serotonin receptor is bu- GABA function. 5HT-1B knockout mice (187)
spirone (91, a 5HT-1A partial agonist. The exhibit behaviors consistent with reduced
5HT-1A receptor is a member of the G-pro- anxiety in some animal models but not in oth-
tein-coupled receptor family, and is found in ers (188-190). 5HT-moduline is an endoge-
brain structures known to be involved with nous tetrapeptide that influences serotonergic
emotional behavior, such as the hippocampus, activity through antagonism at 5HT-1Brecep-
amygdala, raphe nuclei, and hypothalamus. In tors (191, 192). Centrally administered 5HT-
the raphe nucleus, 5HT-1A receptors are pre- moduline desensitizes 5HT-1B receptors, re-
synaptic autoreceptors, whereas in the other sulting in increased serotonin release (193).
brain regions they are postsynaptic. Activa- Consistent with this observation, specific
tion of the postsynaptic receptors leads to neu- 5HT-moduline antibodies have shown anti-
ronal inhibition in some limbic structures. anxiety effects in animal models (194), sug-
Activation of presynaptic autoreceptors sup- gesting the involvement of this peptide in the
presses the firing rate of serotonin raphe neu- regulation of anxiety. Clearly, further study is
rons, thereby reducing serotonin turnover in needed to fully characterize the role of 5HT-
the terminal fields (175, 176). Both modes of 1BD receptors in anxiety and to ascertain the
action are presumed to contribute to the ther- potential, if any, of 5HT-1BP antagonists as
apeutic effects of buspirone (177). Mice lack- anxiolytic drugs.
ing the 5HT-1A receptor have been generated
by homologous recombination (178, 179), and 3.2.2 5HT-2 Receptors. It has been sug-
these animals show an overt anxious pheno- gested that the pathology of anxiety disorders
type in several anxiety models, supporting a may involve a hypersensitivity of central
role for the 5HT-1A receptor in the regulation 5HT-2 receptors (171). This is supported by
of anxiety. the anxiogenic effect of the nonselective
5HT-1BP receptors, best known as the site 5HT-2 agonist m-chlorophenylpiperazine (m-
of action of popular antimigraine drugs such CPP) in animal models of anxiety (195). Fur-
as sumatriptan, have also been associated ther, administration of m-CPP, which is the
with anxiety (180), although the evidence for principal metabolite of the antidepressant ne-
their involvement is less compelling than that fazodone, elicited anxiety in panic disorder pa-
for the 1A subtype. The 5HT-1/BP receptors tients (196) and induced a transient exacerba-
are found in the basal ganglia, hippocampus, tion of symptoms in OCD patients (197).
and cortex (1811, where they exist as autore- Together, these data raise the possibility of
ceptors on serotonergic neurons and as het- 5HT-2 receptor antagonists as anxiolytics. All
eroreceptors on nonserotonergic neurons. three of the 5HT-2 subtypes (2A, 2B, 2C) have
5HT-1Breceptor agonists show an anxiogenic been associated with anxiety to some degree,
effect in various animal models (1821, includ- the 2A and 2C isoforms being the most fre-
ing the elevated plus maze (183) and social quently linked.
interaction (184)tests. Furthermore, selective In the CNS, 5HT-2A and 2C receptors are
5HT-1BP antagonists have shown an anxio- found postsynaptically on nonserotonergic
3 Physiology and Pharmacology
neurons (198). 5HT-2C receptors are found in ing norepinephrine, dopamine, and CCK
the choroid plexus, cerebral cortex, hippocam- (211). Overall, 5HT-3 antagonists display er-
pus, and amygdala (199), and this localization ratic efficacy in animal models of anxiety and -
is supportive of a role in affective disorders. As have failed to demonstrate a significant anxi- :
well as in anxiety, 5HT-2C receptors have olytic effect in the clinic (173,208).
been implicated in depression (200) and in the
-v
modulation of feeding behavior (201). 5HT-2A 3.2.4 GABAISerotonin Interactions. The un-
receptors are more widespread, with higher certainty surrounding the relative roles of
levels in cortical regions (198), and antago- GABAergic and serotonergic pathways in the .
nists at this subtype may also possess antipsy- regulation of anxiety is compounded by the
chotic activity. In behavioral studies there is interactions of the GABA system with the se-
i considerable inconsistency, in that 5HT-2 an-
tagonists have been shown to produce anxio-
rotonergic system. The inhibitory influence of
benzodiazepines on serotonin turnover in the
a lytic effects, no effects, or even anxiogenic ef- brain was first noted over 30 years ago (212)
I
fects in various animal models (202). and led to the theory that the therapeutic ef-
Administration of a nonselective 5HT-2 an- fects of the BZs may be mediated, in part, by
tagonist (mianserin) or a selective 5HT-2BlC their potentiation of GABAergic inhibition of
antagonist, but not a selective 5HT-2A antag- 5HT neuronal firing and subsequent reduc-
onist, produced anxiolytic effects in animal tion in serotonin release.
models, implicating the 2B/C subtypes in anx- Genetically modified mice lacking the
iety modulation (203). The available evidence 5HT-1A receptor are inherently anxious, and
paints a confusing picture of 5HT-2 receptors are insensitive to the anxiolytic effect of diaz-
in anxiety, and knowledge of the exact func- epam (213)in the elevated plus maze and open
tion and potential clinical applications of the field tests. In addition, there is altered GABA,
various 5HT-2 subtypes remains incomplete. receptor expression in the brains of these
Further behavioral studies with subtype-se- 5HT-1A knockout mice. Expression of the a2
lective ligands would help delineate the prop- subunit was reduced to 59% of wild type levels
erties of the 5HT-2 subtypes, but the high de- in the amygdala, and to 47% of wild-type levels
gree of homology among the 5HT2 receptors in the cortex, whereas no reduction in a 3 ex-
(204, 205) has made this approach difficult. pression was found. In addition, no subunit.
The pharmacology of this class of receptors is. reduction of any kind was found in the hip-
further complicated by the unusual down- pocampus and raphe nucleus. Given that the
regulation of 2A and 2C receptors upon expo- knockout mice lack both pre- and postsynaptic
sure to antagonists (206). 5HT-1A receptors, the relative influence of
each on GABA, receptor expression remains
3.2.3 5HT-3 Receptors. 5HT-3 receptors unclear. These results, and the fact that ben-
are best known as the site of action of clinically zodiazepines are known to mediate fast inhib-
useful antiemetics, such as ondansetron (Zof- itory transmission in the amygdala by interac-
ran). Although 5HT-3 receptors are found pre- tion with GABA, receptors (214), point to an
dominantly in the medulla oblongata (2071, intimate relationship between the 5HT-1A re-
their presence, albeit at lower levels, in the ceptor and GABA* a2 receptors. This is par-
amygdala and hippocampus raises the possi- ticularly interesting in light of recent work
bility of some role in emotional behaviors such with genetically altered mice containing mu-
as anxiety (208, 209). In rats, 5HT-3 antago- tations in GABA, receptor subtypes (148),
nists have shown anxiolytic effects when in- which suggests that the anxiolytic effect of di-
jected into the amygdala but not when injected azepam is mediated specifically by the a2 sub-
into the dorsal raphe nucleus (210). A similar unit.
anatomical pattern was observed for the anx-
3.3 Norepinephrine
iogenic effects of 2-methyl-5-hydroxytrypta-
mine, a 5HT-3 receptor agonist. Central The majority of noradrenergic neurons in the
5HT-3 receptors may influence the release of brain are located in the locus ceruleus (LC),
other neurotransmitters in the brain, includ- and electrical stimulation of this region in an-
Antianxiety Agents
also located in brain regions associated with equate evidence that excessive CRF secretion
anxiety, including the LC and the amygdala. or activity is associated with at least some of
The modulatory effects of CRF are mediated the anxiety disorders (233,234,2581,and CRF -
through CRF-1 and CRF-2 receptors, both receptor antagonists are therefore valid anxi- :
members of the GPCR superfamily. CRF-1 re- olytic targets.
ceptors are the dominant form in the CNS
and hence the most frequently associated 3.4.3 Neuropeptide Y. Of the neuropep-'
with neuropsychiatric disorders such as anxi- tide Y (NPY)receptors identified to date, Y-1is
ety, depression, and stress disorders (226, the most strongly linked to the regulation of
232-234). CRF also interacts with the soluble anxiety. This receptor is found in high densi- - -
CRF-binding protein, which influences CRF ties in the brain, particularly in the cortex,
neurotransmission as well as the ability of thalamus, and amygdala, and Y-1 receptors in
CRF to activate the HPA axis (235). the latter structure are believed to mediate
Application of CRF directly into the LC the anxiolytic effect of NPY (226,259,260). In
stimulates neuronal firing, enhances NE re- rats, administration of antisense oligonucleo-
lease (236, 237), and produces anxiogenic ef- tides corresponding to the Y-1 receptor signif-
fects in behavior models (238, 239). Alprazo- icantly increased anxiety in behavioral models
lam reduces CRF concentrations in both the compared to normal controls (261). Postmor-
LC and the amygdala (2401, suggesting that tem examination of the brains of the treated
suppression of CRF stimulation of noradren- animals revealed a 60% decrease in Y-1 recep-
ergic neurons in the LC may contribute to the tors and no change in Y-2 receptors.
anxiolytic effect of the benzodiazepines. In NPY has shown an anxiolytic effect in sev-
various rodent models, including the elevated eral animal models (262, 263), although the
plus maze (2411, social interaction (242), and mechanism through which this anxiolysis is
Geller-Seifter conflict (243) paradigms, ad- produced is unclear. In rats, the anxiolytic ef-
ministration of CRF elicits behaviors consis- fect of NPY could be blocked by treatment
tent with an anxiogenic effect (244,245). CRF with an adrenergic a2 antagonist (idazoxan),
also potentiates the acoustic startle reflex, and but not by an a1 antagonist (prazosin) or a
this potentiation can be blocked by treatment benzodiazepine receptor antagonist (flumaze-
with chlordiazepoxide (246). Genetically al- nil) (262, 264). These results might suggest a
tered mice that overproduce CRF have been selective interaction between NPY and norad-
described and these animals exhibit a height- renergic transmission, and indeed NPY is ex-
ened state of anxiety in animal models (247, tensively colocalized with norepinephrine in
248). Conversely, mice lacking the CRF-1 re- the CNS (265). Yohimbine, an adrenergic a2
ceptor display reduced anxiety in many behav- antagonist known to increase anxiety, signifi-
ioral models (249, 250). The behavioral re- cantly increases circulating NPY levels in hu-
sponses to CRF in animals are therefore man subjects, again suggesting that the anxi-
suggestive of a role for CRF in human anxiety ety-modulating effects of NPY are, in part,
disorders. This proposal is supported by the related to noradrenergic transmission (266).
elevated cerebrospinal fluid levels of CRF Other clinical data regarding this proposal are
found in OCD (251) and PTSD (252,253) pa- inconclusive, however. Low CSF levels of NPY
tients, although this was not the case in panic have been correlated with higher anxiety
disorder (254) or GAD (255) patients. Simi- scores among depressed patients (267), yet
larly, urinary free cortisol levels were no dif- higher levels of NPY were found in panic dis-
ferent in social phobia patients compared to order patients than those in normal controls
those of normal controls (256), suggesting (268). Another study failed to find any differ-
normal function of the HPA axis in this ences in plasma NPY levels between normal
disorder. volunteers and patients with panic disorder or
The fact that CRF enhances the behavioral social phobia (269). Although current evi-
responses to stressful situations in animals in- dence suggests a linkage to the noradrenergic
dicates that it may play a role in the develop- system, NPY is an abundant transmitter in
ment of anxiety in humans (257). There is ad- the CNS and interactions with a variety of sys-
Antianxiety Agents
tems are likely. For example, NPY is also fre- vated plus maze test (279,280). Furthermore,
quently colocalized with GABA and soma- NMDA was able to substitute as a discrimina-
tostatin, offering further pathways through tive stimulus in pigeons trained to recognize
which NPY may modulate anxiety. In mice, the anxiogenic benzodiazepine receptor in-
chronic treatment with benzodiazepine recep- verse agonist P-CCE (281). The BZR antago-
tor agonists has been shown to increase Y-1 nist flumazenil blocked the ability of P-CCE,
receptor expression in the amygdala (270). Ad- but not NMDA, to act as a cue, suggesting that
ditionally, it has been suggested that NPY anxiety is the common stimulus produced by
could reduce anxiety by counteracting the both compounds.
anxiogenic effects of stress-induced CRF re- The foregoing evidence suggests that the
lease from the amygdala (271). excitatory glutamate system could have a com-
plementary role to that of the inhibitory
3.5 Glutamate Receptors
GABA system in anxiety. Thus activation of
The glutamate system is responsible for most glutamate receptors or inhibition of GABA re-
of the brain's excitatory neurotransmission. ceptors is anxiogenic, whereas inhibition of
Glutamate interacts with a number of recep- glutamate receptors or activation of GABA re-
tors, including the NMDA, AMPA, kainate, ceptors is anxiolytic.
and metabotropic glutamate receptors. The
latter receptor is coupled to G-proteins,
4 HISTORY
whereas the others are directly linked to ion
channels. In addition to the ion channel, the
The anxiolytic agents currently on the market
NMDA receptor contains a number of modu-
owe much to serendipity as well as rational
latory sites, including sites for glycine and glu-
design in their genesis. With the exception of
tamate. Just as enhancement of GABA's in-
buspirone, all first indication anxiolytics were
hibitory action decreases anxiety, it might be
introduced before 1975, before many of the
expected that inhibition of glutamate's excita-
modern techniques of medicinal chemistry
tory functions would produce the same effect.
such as radioligand binding assays, molecular
Indeed, the direct injection of NMDA antago-
modeling, and pharmacokinetic screening
nists into discrete brain regions has provided
were introduced into common practice.
evidence that functional antagonism of spe-
cific populations of these receptors produces
4.1 Discovery of the Benzodiazepines
an anxiolytic effect. Competitive antagonists
(272), as well as glycine site antagonists and The benzodiazepines represent the single
partial agonists (273, 2741, are active in the most important advance in the treatment of
elevated plus maze when microinjected into anxiety, and the story of their discovery is a
the dorsal periaqueductal gray matter truly remarkable one (282,283). In the 1950s,
(DPAG). In addition, intrahippocampal ad- a major research effort was the search for new
ministration of competitive and uncompeti- tranquilizers that would overcome the short-
tive NMDA antagonists and glycine partial comings of the medications in use at the time,
agonists was reported to significantly increase such as the barbiturates and the phenothia-
punished responding (275, 276). Complemen- zines. In the chemistry laboratories at Hoff-
tary to these results indicating that blockade man-La Roche in New Jersey, Dr. Leo Stern-
of glutamate receptors is anxiolytic, other bach approached this task by turning to a
studies shave shown activation of NMDA re- series of compounds, the "benzoheptoxdia-
ceptors to be anxiogenic. Thus NMDA in- zines" (191, which he had previously investi-
creases distress calls in the rat pup isolation gated as dyestuffs during his research at the
model (2771, decreases social interaction in Jagiellonian University in Krak6w, Poland.
rats, and decreases the time spent on the open Sternbach considered, then rejected, the pos-
arms of the elevated plus maze (2781, behav- sibility of making modifications to known nat-
iors characteristic of increased anxiety. Addi- ural or synthetic tranquilizers and, having no
tionally, injection of glycine into the DPAG knowledge of any discrete receptor target, he
produced an anxiogenic response in the ele- decided to pursue the benzoheptoxdiazines
4 History
(2) Chlordiazepoxide
These models are classified into two major stable in acid solution, hydrolyzing to the lac-
groups: those based on unconditioned behav- tam (261, which possessed superior anxiolytic
ior and those based on conditioned behavior.
The former tests employ responses controlled
by operant conditioning procedures, and are
typified by the traditional Geller-Seifter and
Vogel conflict tests, whereas the latter models
rely on the natural aversive reactions of ani-
mals to novel stimuli, such as an unfamiliar
environment (elevated plus maze) or another
animal (social interaction). Although it is im-
perative to use a variety of animal models,
tests that do not involve unnatural responses
or punishment are increasingly favored. potency. In general, a carbonyl group is the
optimum substituent at the 2 position, the
5.2 Benzodiazepines
analogous 2-amino derivatives being some-
what weaker. Thus the basic side chain, the
In the study of compounds acting at the BZR, introduction of which formed the basis of the
modern researchers have at their disposal an original research strategy, turned out to be
array of powerful in vitro assays that provide a unnecessary, indeed detrimental, to the de-
detailed pharmacological profile of any new li- sired activity. An exception to this generaliza-
gand prior to in vivo behavioral testing. This tion is medazepam (27), which lacks any sub-
environment is in sharp contrast to that which
produced most of the classical 1,Cbenzodiaz-
epines. The clinical success and structural
novelty of chlordiazepoxide and diazepam
sparked a massive chemical investigation of
this class of compounds, but in the 1960s and
1970s no in vitro assays existed for the phar-
macological profiling of these new compounds.
Indeed their mode of action was a mystery, the
benzodiazepine receptor had yet to be discov-
ered, and the concepts of intrinsic activity and
subtype selectivity were many years away. stituent at position 2. In humans, medazepam
Nevertheless, the productivity of this period is metabolized to give a number of pharmaco-
was remarkable, with thousands of analogs logically active benzodiazepines, however, in-
being prepared and examined for tranquiliz- cluding diazepam, desmethyldiazepam, and
ing activity. Consequently, many of the key oxazepam (287,288),all of which contribute to
SAR observations on the benzodiazepines are the anxiolytic effect. Quazepam (Doral, 28) ex-
to be found in the older chemical literature
(for reviews, see 285, 286.) These SAR obser-
vations were initially built around i n vivo test-
ing for tranquilizing and anticonvulsant activ-
ity rather than pharmacological properties
such as affinity and intrinsic efficacy.
emplifies another variation at the 2 position, tution with fluorine or chlorine is allowed.
where the benzodiazepin-2-one has been con- These substituents strongly potentiate the an-
verted to the thiolactam. The reverse lactam xiolytic effects, however, giving compounds
of diazepam (29)shows comparable activity in significantly more potent than the unsubsti-
anticonvulsant tests (289). tuted analogs (282,290). Although much less
common than the C-5 phenyl benzodiaz-
epines, some heteroaryl derivatives, of which
bromazepam (7) is an example, have shown
significant biological activity. Bromazepam is
available as an anxiolytic and a sedative and is
reported to have a similar in vivo activity pro-
file to that of diazepam (291). Other heterocy-
cles at C-5 (e.g., pyrimidine, pyrazine) show
less activity than that of the 2-pyridyl group
(290). Within the 1,4-benzodiazepine tem-
plate, the effects of favorable substituents in
different parts of the molecule appear to be
Some of the fundamental structure-activ- synergistic. Flunitrazepam (31),which com-
ity relationships established in the early years
of benzodiazepine research have held true for
virtually all benzodiazepines prepared since
then. It was quickly found that methylation of
chlordiazepoxide at N-1 enhanced the potency
severalfold (282). N-Methyl benzodiazepines
are typically more potent than the N-H ana-
logs, and substituents larger than methyl
generally diminish activity. In the A-ring,
electronegative substituents such as halogen
(especially chlorine and bromine), nitro, or tri-
fluoromethyl at position 7 are required for sig-
nificant biological activity (290). Electron-re-
leasing groups (e.g., methyl, methoxy) at this
position decrease activity, as does substitution bines the optimal pharmacophoric groups at
of any kind at position 6, 8, or 9. An aromatic N-1, C-2, C-7, and C-5 (2921,is among the most
substituent at C-5 is usually required for use- active of all the benzodiazepines. Flunitraz-
ful biological activity, although tetrazepam epam (Rohypnol) is a powerful sedative-hyp-
(30) provides a rare example where this is not notic with significant memory-impairing and
the case. The C-5 phenyl ring is not tolerant of muscle-relaxant effects, all of which are
structural modification, and only ortho substi- strongly potentiated by alcohol. In recent
times, these effects have earned Rohypnol no-
toriety as a "date-rape" drug, and have re-
sulted in its removal from the market in many
countries.
3-Hydroxybenzodiazepines represent ac-
tive metabolites of many clinically useful ben-
zodiazepines (e.g., diazepam), and conse-
quently a large number of s-substituted
derivatives have been prepared and evaluated
as anxiolytics in their own right. These com-
pounds often show comparable potency to that
of the parent benzodiazepines, but have a dra-
matically different metabolic profile. In hu-
mans, diazepam gives rise to several active 3-oxazepam hemisuccinyl ester produced po-
metabolites that contribute to buildup of ac- tentiations of GABA induced chloride flux al-
tive agent in the blood over time. One of these most twofold greater than that of the 3-(R).
metabolites, oxazepam, is itself a marketed enantiomer (299). Likewise the 3-(S) enantio--
anxiolytic (293). Oxazepam is directly conju- mer of 3-methyldesmethyldiazepam is twice
gated at the 3-hydroxy group and is excreted as potent as, and is a stronger agonist (GABA
in the urine as the pharmacologically inactive shift = 2.7) than, the 34R) isomer GAB^
glucoronide (294,295). By virtue of this small shift = 1.5) (300). This stereochemical bias is
structural change, the clinical use of oxaze- believed to arise from a preferential stabiliza-
pam is not complicated by the buildup of active tion of conformation (33) by the 345) antip-
metabolites, a factor known to contribute to
the "hangover" effect seen with some benzo-
diazepines. Other 3-hydroxybenzodiazepines
in clinical use include lorazepam Ativan (4),
and the hypnotics temazepam (Restoril) and
lormetazepam (Loramet).
The &position, by way of the hydroxy
group, has been modified in an attempt to in-
crease the water solubility of benzodiazepines,
which are generally highly lipophilic com-
pounds. For example, the amino ester deriva- odes, in which the substituent occupies a
tive (32) of lorazepam had excellent water sol- quasiequatorial orientation (301). This con-
formation is believed to be preferred by the
receptor (3021, and is disfavored for the 3-(R)
enantiomers. The commercially available
3-hydroxybenzodiazepines are marketed as
racemic mixtures because their rapid race-
mization in uiuo renders a chiral formulation
impractical and unnecessary.
The N-4 atom may be replaced with a car-
bony1 group, as in clobazam (Frisium, 34),
show comparable activity to that of the parent midazolam (41). Midazolam is a highly potent
compounds. In general, ksubstitution in the compound possessing strong hypnotic, anxio-
triazolobenzodiazepine series is detrimental
to activity (285, 318).
Imidazo[l,2-al[1,4lbenzodiazepines gener-
ally show inferior biological activity to that of
the analogous triazolo [1,2,4][4,3-a] deriva-
tives, but some success has been achieved with
a related series of amino-substituted imida-
zolone derivatives. This series is exemplified
by loprazolam (40), which possesses potent
5.2.3 Heteroaryl[el[l,4lDiazepin-2-Ones.A
number of chemical classes with anxiolytic ac-
tivity have been produced by replacement of
the fused phenyl ring of the 1,4-benzodiaz-
epines with heterocycles. Ripazepam (46)
Table 9.3 Preferred Profiles of Partial Agonists and Profiles of Reference Drugs
a1 a2 a3 a5 Reference
Preferred profile <20% >30% >30% <20% 344
Preferred profile <10% nd 40->60% nda 345
Alprazolam (5) 326% 355% 787% 264% 127
Diazepam (1) 186% 290% 568% 157% 127
Lorazepam (4) 328% 233% 421% 181% 127
Bretazenil (83) 40% 37% 76% 64% 127
"nd. not disclosed,
GABA. This coupling, and hence affinity en- been an intensive effort to identify "anxio-
hancement, is stronger for full agonists than selective" compounds that selectively modu-
for partial agonists, and is nonexistent for an- late GABA function at specific receptor sub-
tagonists. Thus the GABA shift (ratio of IC,, types. Although they are attractive proposals,
without GABAK!,, with GABA) is loosely partial agonism or subtype selectivity on their
predictive of the relative degree of intrinsic own may not be enough to produce the desired
efficacy. This estimate is based on comparison clinical profile. A given subtype (e.g., a2) may
with known full agonists such as diazepam, be associated with more than one clinical ef-
which has a GABA shift of 2.2-2.9. Accord- fect, depending on receptor activation, and so
ingly, inverse agonists have values less than 1,
a full agonist selective for this subtype would
full agonists have values of 2 or more, and par-
be of little value. Similarly, a nonselective par-
tial agonists lie between 1 and 1.5 (339,340). A
tial agonist may be unable to differentiate be-
more accurate and meaningful assay is the di-
rect measurement of the potentiation of tween effects requiring low activation of only
GABA-induced chloride currents by a BZR li- one subtype. In recent times both these ap-
gand. This is determined in single oocytes ex- proaches have coalesced into the search for
pressing the desired recombinant receptor subtype-selective partial agonists, and this ef-
subtype, using a two-electrode voltage-clamp fort dominates the current medicinal chemis-
technique (341-343). A fixed concentration of try and preclinical work in the area. With in-
GABA is applied (typically enough to produce creasing knowledge of the physiological roles
10-20% of the maximal current), and the po- of GABA, subtypes guiding the subtype selec-
tentiation of this current by the drug is mea- tivity profile, the next challenge in this ap-
sured over a full dose-response range. The proach is the determination of the "right
maximal potentiation (%) and the EC,, value amount" of partial agonism. Based on preclin-
describe the efficacy and the potency, respec- ical animal models, some companies have re-
tively. This assay directly measures the recep- vealed their preferred partial agonist profile
tor activation by the ligand, unlike the TBPS for anxiolytics (344,345) (Table 9.3). It is clear
and GABA shift assays that provide only rela- from Table 9.3 that these profiles are dramat-
tive efficacies. ically different from those of the classical ben-
A second approach to improve the side-ef- zodiazepines, and are more selective than the
fect profile has grown from the increasing prototypical partial agonist bretazenil.
knowledge of the diversity of GABA, receptor An exhaustive review of the non-benzodi-
subtypes. Each of the four major diazepam- azepine ligands at the BZR is beyond the scope
sensitive subtypes exhibits a distinct, al- of this chapter, and the following section fo-
though perhaps overlapping, pharmacology, cuses only on those templates that have pro-
anatomical distribution, and physiological duced advanced anxiolytic candidates. In addi-
hnction. Recent work with conditional tion to the compounds described below, a wide
knock-in animals strongly implicates the a1 range of small molecules bind with high affin-
and a2 subtypes in the mediation of the seda- ity to the BZR. The reader is invited to consult
tive and anxiolytic actions of diazepam, re- recent reviews on this topic for more extensive
spectively (144-149). Accordingly, there has coverage (346-351).
5 Structure-Activity Relationships
5.3.2 p-Carbolines. The isolation of the po- a to the oxygen, but not P or y to the oxygen,
' tent BZR ligand p-CCE (53) from human suggesting a well-defined and limited li- .
pophilic pocket in the receptor (354). In the
C-6substituted p-carbolines this change -
causes a greater loss in affinity, possibly be-
cause of a steric interaction between the C-3 ?.
and C-4 groups and consequent disruption of
hydrogen bonding at N-2 (356). A 3-amino
substituent abolishes binding affinity (356) -
because of insufficient lipophilic interaction
(53) X = COzEt (P-CCE) with the receptor and the existence of the
(54) X = C02CH3(P-CCM) amino group in the imino tautomer, which
(55) X = C02-n-Pr would prevent hydrogen bond formation with
(56) X = alkoxy N-2.
The size of the substituent at position 6
urine in 1980 (352) prompted a great deal of also has a major impact on the intrinsic effi-
interest in p-carbolines as possible endoge- cacy, but less so on receptor affinity. Thus
nous ligands for the BZR. Although the pres- compound (57) has an IC,, value of 0.5 nM for
ence of p-CCE in urine was subsequently
shown to be an artifact of the extraction pro-
cedure (3531, interest in this series continued
because of their high potency and unusual
pharmacology. p-CCE and p-CCM (54) were
the first compounds to exhibit inverse ago-
nism at the BZR, and p-carbolines have be-
come the most widely studied class of BZR li-
gands after the benzodiazepines themselves.
The pyridyl nitrogen atom is essential for
affmity and is presumed to participate in a
critical hydrogen bonding interaction with the
receptor (354,355). A free NH at position 9 is
also required, as affinity is drastically reduced
in the N-methyl analogs (356). Computer the BZR, antagonized the anticonvulsant ef-
modeling suggests that this loss of affinity re- fects of diazepam, and showed no anticonvul-
sults from a negative steric interaction be- sant effects of its own, consistent with an an-
tween the N-methyl group and the receptor tagonistic profile (359). The corresponding
rather than the removal of a hydrogen bond n-propyl compound (58)has an IC,, value of 8
donor site. Most of the early p-carbolines were nM, and at 20 mgkg showed comparable effi-
inverse agonists but, through appropriate cacy to that of diazepam (2.5 mg/kg) in the
modification of substituents, compounds span- elevated plus maze. Further, (58) showed no
ning the entire continuum of intrinsic activity myorelaxation activity and antagonized diaz-
up to full agonist can be obtained. Increasing epam-induced myorelaxayion, indicating par-
the size of the 3-substituent increases the in- tial agonist properties. Increasing the size of
trinsic activity. Thus extending the ester alkyl the 6-substituent further gives ZK93423 (59),
group from methyl (54) to n-propyl (55) which behaves as a full agonist. Modeling
changes the profile from inverse agonist to an- studies suggest that the influence of the 6-sub-
tagonist (357). The p-carboline ester group is stituent on intrinsic efficacy derives from its
hydrolytically labile and it can be replaced full occupation of a lipophilic pocket (L3) of
with alkoxy groups (56) to enhance stability the receptor. The partial agonist (58)only par-
and water solubility" (358). The rank order of tially occupies this region compared to the full
potency is methoxy < n-butyloxy < ethoxy < agonist (59). Moving the 6-benzyloxy group
n-propyloxy (356,357). Branching is tolerated from the 6- to the 5-position lowers the activ-
Antianxiety Agents
ity to that of a partial agonist (360, 361), fur- symptoms of withdrawal, tolerance, and de-
ther underlining the importance of this inter- pendency (364-367), although the latter
action in the agonist response. The C-5 phenyl symptoms have been observed in other studies
ring of the benzodiazepines is also believed to (368). In clinical trials, abecarnil was found to
confer agonist activity by interacting with the be well tolerated (369) and effective in GAD at
L3 region (see Fig. 9.5). Agonist and inverse doses of 3-9 mg per day with minimal side
agonist p-carbolines probably bind at the effects or discontinuation symptoms (370),al-
same site, but have slightly different interac- though the therapeutic effect diminished after
tions with various receptor regions. The pres- 6 weeks of dosing (371). At higher doses, how-
ence of a methoxyrnethyl substituent at C-4 is ever, side effects typical of a full agonist were
proposed to favor the "agonist" alignment of observed including sedation, amnesia, and un-
the ligand through formation of a hydrogen steady gait. Subsequent studies have shown
bond with the receptor. This substituent does that abercarnil potentiates GABA-induced
indeed confer agonist properties, as evidenced chloride currents at the a 3 subtype to the
by the anxiolytic, anticonvulsant, and myore- same degree as the full agonist flunitrazepam
laxant activities of (59). The corresponding (372), and to the same degree as diazepam in
4-ethyl analog, which lacks this hydrogen rat cerebellar slices (373). These results sug-
bonding capability, is five- to 10-fold weaker at gest that abercarnil possesses neither the op-
the BZR and is devoid of any such agonist ac- timal subtype selectivity nor the optimal par-
tivity (356). The affinities of numerous p-car- tial agonist properties for a side effect-free
boline derivatives at recombinant receptor anxiolytic, and its clinical development has
subtypes reveal nonselective binding to a l , been terminated.
a2, a3, and a5 subtypes (356). In the absence
of efficacy measurements at these subtypes, 5.3.3 Cyclopyrrolones. The cyclopyrrolones
the clinical relevance of even the best selectiv- were among the earliest non-benzodiaz-
ity (~20-fold) remains questionable. epine structures shown to have high affinity
The conversion of the ethyl ester of (59)to for the BZR. The best-known member of this
an isopropyl ester gives abercarnil (60) the series is zopiclone (611, which is available as a
5.3.4 Pyrazoloquinolines. The pyrazoloquino- increasing size (H, methyl, ethyl, n-propyl, n-
line series, originally developed at Ciba-Geigy, butyl) in the thiophene ring leads to a shift in
provides highly potent BZR ligands that span efficacy from inverse agonist, through partial
the entire continuum of intrinsic efficacy from agonist, to agonist (394). Incorporation of un-
full inverse agonist to full agonist. The freely substituted heterocycles at N-2 (pyrazine,
rotating phenyl ring at N-2 plays a critical role pyridine) maintains affinity and gives inverse
in determining the intrinsic efficacy of the agonist compounds (395). The influence of the
molecule, and minor changes in this ring in- N-2 aromatic ring on the intrinsic activity is in
duce dramatic shifts in efficacy. In anticonvul- keeping with molecular modeling studies,
sant and anticonflict tests CGS8216 (65) behaves which place this ring in a lipophilic binding
pocket (L2) responsible for agonist activity
(396) (see Fig. 9.5) and identify N-1 and the
C-3 carbonyl group as the key hydrogen bond
acceptors. Differential occupation of the L2 re-
gion by various substituents is proposed to
produce varying degrees of agonism.
In the A-ring, small substituents (e.g., OH,
OCH,, C1, ethynyl) are tolerated at all four
positions (395, 396), and have less impact on
intrinsic activity than that of substituents in
the N-2 phenyl ring. Large substituents are
tolerated only at the 8-position, as indicated
by the high affinity of the benzyloxy derivative
(68) (396). The other three positional isomers
the BZR, having intermediate affinity at a2 both BZRs (405). Removal of the methylene
and a 3 sites and low affinity for a5 sites. The linker between the carbonyl group and the
original anxiolytic imidazopyridine research imidazo ring, or extension of this linker to two
strategy was based on al-selective ligands carbons, dramatically reduces affinity at both
(399), but it is now believed that the a2 sub- BZRs (405). This suggests that there is a crit-
type mediates the anxiolytic effects of BZR li- ical spatial relationship between the two hy-
gands and so a1 selectivity is clearly a nonop- drogen bond acceptor sites (the imidazole ni-
timal profile for an anxiolytic. The picture is trogen and the carbonyl oxygen) that cannot
further complicated by the fact that alpidem be disrupted. Indeed, most BZR receptor mod-
has an affinity for the peripheral benzodiaz- els demand the formation of two hydrogen
epine receptor (PBZR) comparable to that for bonds at specific locations.
the central BZR (Ki = 1-28 and 0.5-7 nM,re- Molecular modeling studies have suggested
spectively) (400). Further, the BZR antagonist that the acetamide side chain of alpidem is
flumazenil completely blocks the anxiolytic more important for PBZR binding than for
and anticonvulsant effects of benzodiaz- CBZR binding, whereas the hydrogen bonding
epines, but only partially blocks the same ac- capability of the imidazole nitrogen is more
tions of alpidem (401). Thus it is possible that important for CBZR than for PBZR affinity
the anxiolytic actions of alpidem may result (407). The a1 selectivity of imidazopyridines
from a combination of direct action at the cen- has also been rationalized through conforma-
tral BZR and an indirect action at the periph- tional analysis and molecular calculations
eral BZR (402-404). Alpidem progressed into (408). Zolpidem is selective for BZl(a1) sites,
clinical trials, where it was shown to be a n whereas saripidem (71) has equal affinity for
Antianxiety Agents
5 Structure-Activity Relationships
EtO
F
4T N
\
C02Et Y
CH3
0
-
(82)
tagonize the sedative and myorelaxant effects heterocycles have been investigated as poten-
of benzodiazepines. Clinical trials showed tial replacements for the metabolically labile
bretazenil to be an effective anxiolytic, but ester group, of which the 3- and 5-alkyl-1,2,4-
also revealed side effects including sedation, oxadiazole derivatives appear to possess the
amnesia, and performance impairment. The best affinity (420). As a general rule, the ox-
development of bretazenil was discontinued in adiazole derivatives have a higher intrinsic ef-
1997, and the results with this compound sug- ficacy than that of the corresponding ester an-
gest that nonselective partial agonism alone alogs. The SAR of the 3-substituent closely
may not be sufficient to provide a side effect- parallels that of the 3-substitent in the p-car-
free anxiolytic. bolines, suggesting that this region of both
Many members of the imidazobenzodiaz- templates occupies the same binding pocket
epine class display potent affinity for diaze- (420). The substitution pattern in the phenyl
pam-insensitive (DI) subtypes (a4, a6) as well ring also has implications for DSDI affinity
as the diazepam-sensitive (DS) subtypes ( a l , and selectivity. Substitution with electron-
a2, a3, a5) of the BZR, and numerous SAR withdrawing groups (e.g., halogens) at posi-
studies have examined the requirements for tion 7 enhances affinity for the DS subtype
DSDI selectivity. Overall, these studies sug- more than for the DI subtype, whereas 8-sub-
gest that the binding sites for DS and DI sub- stitution preferentially enhances affinity for
types are similar and that selectivity is gov- the DI subtype (421-423). The net result of
erned by structural modifications at positions 7-substitution is therefore an enhancement of
3,7,and 8 of the imidazobenzodiazepine struc- DS selectivity. Substitution at C-8 and C-9 of
ture (84). DS subtypes are responsible for the the phenyl ring dramatically reduces affinity
typical benzodiazepine actions (anxiolysis, se- for DS and DI isoforms (422). Among the four
dation, etc.), and DI subtypes have been impli- DS subtypes, a lipophilic substituent at C-8
cated in mediating some of the behavioral ef- has been found to confer selectivity (4251, but
fects of ethanol (417-419). no selectivity for subtypes implicated in anxi-
olysis (a21 has been reported.
(86) Y = NR2
(87) Y = alkoxy
(88) Y = alkyl, aryl at concentrations up to 1 and reduces it
thereafter, returning to baseline at around 10
carbamate (87),and arnide (88)derivatives. In (429).Given that the TBPS assay was run
this series the 3-oxadiazole and 3-aryl deriva- at 5 and the chloride current assay at 0.5
tives were equipotent. The 3-phenyl derivatives pM, the latter method gives a higher, but
were active in physical-dependency models, more relevant, result. This phenomenon is
whereas the oxadiazoles generally were not, irn- limited specifically to the piperazine ureas and
plicating the 3-phenyl group in the mediation of thus appears to be intimately related to the
this side effect (426). The nature of the N-5 sub- presence of a basic nitrogen in the urea moi-
stituent has little impact on affkity (generally ety. Other ureas, amides, carbamates, and the
4 5 nM) but strongly influences intrinsic effi- imidazoquinoxalin-4-ones show no biphasic
cacy. The amide and carbamate derivatives properties. This phenomenon may result from
tended to be full agonists, the degree of agonism the interaction of ligands such as (89) with
increasing with the size and lipophilicity of the two binding sites at the BZR, one high affinity
carbamate group (337, 428), whereas the urea and one low affinity (426). Such behavior led
analogs usually gave partial agonist profiles. In to the hypothesis that ureas such as (89)
addition, the urea derivatives showed more con- would antagonize their own agonistic effects
sistent in vivo activity (anticonvulsant) than at high concentrations and so limit their abuse
that of the others. As with the quinoxalin-4-one liability, although acute dependency studies
series, no useful subtype selectivity was ob- have failed to support this theory.
served in terms of affinity or efficacy (TBPS Molecular modeling studies show that the
shift). Nevertheless, several compounds in this imidazoquinoxalin-4-ones, despite their obvi-
Antianxiety Agents
ous structural similarity to the imidazobenzo- among simple phenyl piperazines and azapi-
diazepines (e.g., 831, adopt quite different rone derivatives. 1-Pyrimidinylpiperazine,the
three-dimensional conformations (428). For parent piperazine of buspirone, has negligible
the imidazoquinoxalines (e.g., 86) the N-5 affinity for 5HT-1A receptors and l-phe-
substituent lies out of the plane of the rigid nylpiperazine has only weak affinity (-500
tricyclic nucleus, and may occupy the same ag- nM)(433, 4341, but appropriate substitution
onistic receptor pocket as the C-5 phenyl of the of the phenyl ring produces phenylpiperazines
benzodiazepines and the piperazine side chain with potent 5HT-1A activity. Substitution at
of zopiclone (61). Such a n orientation ac- the 2- or 3-position is generally favorable for
counts for the strong influence of this group on 5HT-1A affinity, but 4-substitution is not, and
the intrinsic efficacy. a C1 or CF, substituent at the 3-position en-
hances affinity twofold over that of 1-PP (434).
5.4 5HT-1A Ligands
2-Methowhenyl is consistently one of the
A number of chemical classes are known to most active substitution patterns among sim-
provide potent 5HT-1A ligands (174, 430) in- ple aryl piperazines. A methoxy group at the 2-
cluding the aminotetralins (e.g., 8-OH or 3-position increases affinity by three- and
DPAT), indolealkylamines (serotonin mim- 1.5-fold, respectively, relative to that of l-phe-
ics), ergolines (e.g., LSD), and arylpiperazines. nylpiperazine, and the 4-methoxy derivative is
In terms of potential antianxiety agents, by far inactive (433). The 2,3-dimethoxy compound
the most important of these chemotypes are is weaker (1pik0 than either of the monome-
the arylpiperazines, also referred to as the thoxy derivatives, but constraining the me-
azapirones. In 1986 buspirone (9) became the thoxy groups into a ring system such as ben-
first azapirone to be approved for the treat- zodioxan increases affinity dramatically (40
ment of GAD, and tandospirone (16) has re- nM). The 2-methylbenzodioxene derivative
cently been launched for the same indication, (90) is more potent still, with an affinity (5
although only in Japan. Other azapirones to
reach late-stage clinical trials (431) for anxiety
are gepirone, flesinoxan, and ipsapirone, al-
though development of the latter two com-
pounds has been discontinued. Buspirone it-
self has potent affinity (IC,, = 31 nM) for the
5HT-1A receptor, but also has significant do-
pamine D2 receptor binding (IC,, = 250 nM)
(432).Indeed, buspirone was originally inves-
tigated as an antipsychotic and the dopami-
nergic properties were initially believed to un-
derlie its unexpected anxiolytic effects. Many nM)comparable to that of &OH-DPAT. 2,3-
azapirone compounds possess significant D2 Fusion of other rings such as furan, thio-
and adrenergic a1 activity in addition to the phene, and phenyl gave similarly active com-
desired 5HT-1A affinity, and much of the me- pounds. These results indicate that the
dicinal chemistry work has been directed to- azapirone N-4 substituent is not required for
ward improving the selectivity for the 5HT-1A potent 5HT-1A affinity. The phenylpiperazine
receptor. Modifications of the azapirone struc- SAR also applies to N-4 substituted azapi-
ture can be broken down into three regions of rones, with 2- and &substitution favored and
the molecule: the N-1 aromatic ring, the linker 4-substitution being detrimental to receptor
between N-4 and the imide, and the imide sub- affinity (435, 436). In a bicyclohydantoin se-
structure. ries, the 3-trifluoromethyl derivative (91) was
the only compound to show any selectivity (27-
5.4.1 Azapirones. Numerous SAR studies fold) over that of a1 receptors (435). A positive
have shown that the N-1 aryl substituent relationship between the van der Wads vol-
plays a key role in determining affinity and ume of the meta substituent and the 5HT-
selectivity for the 5HT-1A receptor, both 1Na1 selectivity has been noted (437). In
5 Structure-Activity Relationships 567
(100) X = NH, Y = (C = 0)
(101) X = CH2,Y = (C = 0)
(102) X = CH2, Y = CHOH
(103) X = CH2,Y = CH2
on 5HT-1A or D, affinity, indicating that the clic piperazine linker. Sunepitron is an agonist
NH does not contribute a hydrogen bond in- at 5HT-1A autoreceptors and also has appre-
teraction (439). Likewise, reduction of the car- ciable affinity (35 nM) for a2 adrenergic recep-
bony1group in (100)to the alcohol (102) or the tors, where it acts as an antagonist (451).
methylene (103) had only minor effects on the (104) was effective in the Vogel conflict test
affinitiesfor both receptors. In contrast to its with an med of 1.78 mg/kg, ip, and a maximal
minimal effects on receptor affinity, the na- effect greater than that of gepirone and com-
ture of the linker has a significant impact on parable to that of diazepam (452). The combi-
agonist/antagonist character (450). The amide nation of 5HT-1A agonist and a2 antagonist
(100) is a full agonist, whereas the buytrophe- activities may account for the enhanced effi-
none analog (101)is a partial agonist, and the cacy relative to gepirone. Sunepitron is cur-
n-butyl compound (103) has a predominantly rently in phase I1 clinical studies for anxiety.
antagonistic profile. These studies maintained The substituent at the terminus of the
the length of the spacer at 4 atoms and showed linker chain is tolerant of a number of changes
that changes in the electrostatic or conforma- without a loss in potency. Indeed, the nature
tional nature of this chain had no effect on of this moiety provides the only point of differ-
5HT-1A or D2 affinity, but did modulate the entiation between buspirone (91, tandospi-
functional characteristics of the compounds. rone (16), gepirone (105), ipsapirone (1061,
The linker need not be a linear chain, as and zalospirone (107), five of the most clini-
Antianxiety Agents
cally advanced azapirones. In a series of buspi- from buspirone, (110) has negligible activity
rone analogs, a gradual enhancement of affin- at adrenergic and dopaminergic sites. In ani-
ity was seen as the log P of the molecule was mal models of anxiety, such as the mouse
increased through variation of the imide lightldark box and the rat social interaction
(4451, indicating a positive relationship be- test, (110) was effective at low doses (c0.5
tween the lipophilicity of this group and mgfkg, ip) and produced no sedative effects
5HT-1A receptor affinity. The imide of buspi- (453).Drug metabolism studies in rats suggest
rone may be replaced by hydantoin deriva- that, like buspirone, lesopitron may be subject
tives, as in (99) (435, 449), diketopiperazines to extensive first-pass metabolism (455).
(4371,phthaloyl groups, amides, or even a sim- Phase I studies showed lesopitron to be well
ple phenyl ring (434, 439) without disruption tolerated in human subjects up to 50 mg/day
of affinity. In the amide series, a preference for (456). In a lorazepam controlled phase I1 trial
a lipophilic group was suggested by the fact in patients with GAD, lesopitron (4-80 mg/
that the acetamide analog (108) was 25 times day), showed signs of efficacy, although a clear
endpoint was not achieved.
In the field of 5HT-1A receptor ligands ago-
nists and partial agonists abound, but pure
antagonists remain scarce. The first such com-
pound to become available was WAY-100135
(111) (4571, which incorporates a lipophilic
models including the Geller-Seifter and social iety is clearly desirable, but the 5HT-2 recep-
interaction paradigms (470). tors form a very closely related group (475)
In addition to the 5HT-2 antagonists de- and this objective has been hampered by the
scribed above, some agonists have also shown lack of subtype selective ligands.
anxiolytic effects. Org-12962 (118) is a pyri- A number of compounds with mixed
5HT-1A agonist and 5HT-2 antagonist prop-
erties have been investigated, seeking to com-
bine the anxiolytic effects of both mechanisms
(476). Molecular modeling studies have been
used to study the receptor interactions that
may contribute to 1A and 2A affinity among a
series of buspirone analogs (477). Adatanserin
(120) is a modified azapirone derivative with
dylpiperazine derivative with agonist activity
at 5HT-2A and 2C receptors that is undergo-
ing clinical investigation for anxiety and de-
pression. In a DPAG stimulation model of
panic in rats, (118)was reported to have anti-
panic efficacy intermediate between that of
clonazepam and fluoxetine (471). The 5HT-2B
agonist BW-723C86 (119), whose structure is affinity for 5HT-1A sites (Ki= 1 nM), where it
acts as a partial agonist, and 5HT-2 sites (Ki =
66 nM)where it acts as an antagonist (478-
480). It is an effective anxiolytic in a range of
anxiety models (481,482) predictive of clinical
anxiolysis. In a pigeon conflict model adatan-
serin was the only mixed 5HT-1A agonistl
5HT-2 antagonist to show significant effects,
and elicited larger anxiolytic-like effects than
those of either a 5HT-1A agonist or a 5HT-2
antagonist alone (481). HT-SOB (121) is an-
cess in this indication. It has also been sug- The discovery of the first nonpetide CCK an-
gested that 5HT-3 antagonists may have use- tagonist, the natural product asperlicin, in
ful antianxiety properties (209,485). The vast 1985 (495) shifted the medicinal chemistry fo- -
majority of clinical data in this respect con- cus from peptides to small molecule antago- .
cerns ondansetron (122) (4861, a widely pre- nists (496,497).CCK-B receptors are the dom-
inant isoform in the brain, and antagonists at
this subtype are potential anxiolytics. Many
CCK-B antagonists also modulate gastric acid
secretion through peripheral CCK-B (gastrin)
receptors.
The most significant structural class de-
rived from the asperlicin structure is the 1,4-
benzodiazepines, exemplified by L-365,260
(123). The most striking aspect of the SAR in
6 CURRENT DEVELOPMENTS
AND THINGS TO COME
(1.7 nM), potent functional antagonism (IC,, An ideal anxiolytic would be orally efficacious
= 82 nM) in vitro, and excellent selectivity for with a rapid onset of action, and be devoid of
the CRF-1 receptor (566). This compound has side effects such as tolerance, withdrawal, de-
a favorable pharmacokinetic profile in several pendency, sedation, motor and cognitive im-
species and has a greater oral bioavailability in pairment, interaction with CNS depressants,
the dog than that of DMP-904. (142) was less and toxicity in overdose. Such an agent would
effective than DMP-904 in the rat brightldark be effective in different anxiety disorders and
open field test, however, with a med of 3 mgkg would treat all the core symptoms of each dis-
(po) and a maximally effective dose of 10 order with a high response rate. Despite the
mgkg (po). Although the maximal effects of progress made in anxiolytic research, the cur-
DMP-904 and DMP-696 in the situational rently available antianxiety agents fall some
anxiety test were slightly smaller than those way short of this perfect profile. This fact, cou-
of 20 mgkg (po) chlordiazepoxide (57,63, and pled with the prevalence of the anxiety disor-
72%, respectively), the CRF antagonists ders, their impact on society, and the conse-
showed no sedative or ataxic effects at doses quent market size, ensures a continued
up to 100 mg/kg (po) (565, 566), indicating a intense interest within the pharmaceutical in-
greatly improved side-effect profile compared dustry in the development of new anxiolytics
to that of the benzodiazepine. Replacement of that come closer to the ideal profile. Currently,
the substituted phenyl ring with pyridine there are more biological mechanisms under
(567),in an effort to improve water solubility, investigation as potential targets than ever
culminated in R-121919 (143),a potent CRF-1 before (569). The most advanced of these ap-
antagonist with good brain penetration and proaches are those involving the GABA, sero-
reasonable bioavailability. (143) recently com- tonin, CCK, and CRF neurotransmitter sys-
pleted phase I clinical studies showing prelim- tems, all of which have produced clinical
inary signs of antidepressant and antianxiety candidates (570 -572).
6 Current Developments and Things to Come
-
As already discussed, anxiety constitutes a the regional expression of these receptors,
complex group of emotional disorders, the de- techniques such as positron emission tomog-
1 velopment, regulation, and pharmacomodula- raphy (PET) scanning may reveal the involve- -
tion of which involves a number of the brain's ment, or otherwise, of each in human anxiety.
8 major neurotransmitter systems. A clearer To design serotonergic anxiolytics, it is impor-
5
1 understanding of the influence of each of these tant to gain a fuller understanding of the var-
[ systems on the genesis, development, and ious 5HT receptors at the molecular level, par- *
!
treatment of anxiety is central to the discovery ticularly in the case of closely related
! of new and improved anxiolytics. In this re- subgroups such as the 5HT-1BiDand 5HT-2A/
: spect, molecular biology, genetics, and phar- B/C receptors. The involvement of neuropep- -
macology are throwing light on the intricate tide systems in anxiety is less well established
neurobiology of anxiety. Transgenic animal than GABA or serotonin, but antagonists at
i studies, for example, are identifying for the CRF-1 and CCK-B receptors have shown
; first time the individual GABA,/BZR receptor promising results and have exciting clinical
subtypes that mediate the anxiolytic and other potential. As the neurobiology of each path-
effects of the benzodiazepines. With the a2 way implicated in anxiety is investigated, ev-
subtype already implicated as mediating the ery result seems to bring more questions. It is
anxiolytic effects of diazepam, future work to increasingly obvious that one neurotransmit-
determine the subtypes involved in serious ter, far less one receptor or receptor subtype,
benzodiazepine side effects such as with- does not act in isolation in regulating emo-
drawal and dependency will be of tremendous tional behavior. The complex interrelation-
value in the design of truly selective and side ships between all of the neurotransmitter sys-
effect-free GABAergic anxiolytics. Things will tems associated with anxiety makes it
not be as simple as engineering a subtype-se- extremely difficult to extricate the true caus-
lective compound, however, because it is likely ative mechanism. Nevertheless, the existence
that a given subtype will participate in more of so many receptor targets that are able to
than one clinical effect. The a2 subtype, for modulate anxiety in some fashion offers nu-
example, is known to contribute to both the merous possibilities for new drug develop-
anxiolytic and myorelaxant actions of diaze- ment.
pam. Thus, to obtain real clinical selectivity, a With over 40 years of outstanding clinical
degree of partial agonism must be superim- success behind it, and a continuing research
posed on the subtype selectivity. "What is the effort today, the modulation of GABAergic
right subtype selectivity?" and "What is the transmission through the BZR remains the
right degree of partial agonism?" are ques- mechanism most likely to provide the next ad-
tions that are only now being posed in the vance in the treatment of anxiety. The efficacy
clinic. of the benzodiazepines as antianxiety drugs is
The serotonin system is intimately in- without question,-but the current challenge is
volved in anxiety, and up to seven 5HT recep- the im~rovement
* of what has become increas-
tor subtypes have been implicated to varying ingly regarded as an undesirable side-effect
degrees. Different 5HT receptors and receptor profile. The subtype-selective partial agonist
subtypes may play overlapping, or even oppos- approach holds the most promise in this re-
ing, roles in emotional behavior, and the same gard, but the extent of partial agonism re-
receptor may have different functions in dif- quired to maintain efficacy and optimize the
ferent brain regions. The sheer complexity of therapeutic index remains an open question.
this transmitter system is underlined by the Thus far, anticipated optimal profiles for anxi-
fact that in over 30 "years of active research olysis in humans have been extrapolated from
linking serotonin and anxiety, only one anxio- animal studies, and a number of studies with
lytic acting directly on 5HT receptors (buspi- partial agonists currently in progress should
rone) has been launched. From a better under- soon reveal the applicability, or otherwise, of
standing of the role of each implicated subtype these profiles in the clinic. The most advanced
will surely come improved serotonergic anxio- candidate is pagoclone (64), currently in
lytics (573, 574). As molecular biology reveals Phase 111 trials, which has already demon-
Antianxiety Agents
strated efficacy in panic disorder. In a placebo around a213 selective partial agonist 1,2,4-
controlled trial pagoclone was found to be ef- triazolopyridazines (e.g., 146) suggests the
ficacious in GAD at low doses (0.3-1.2 mg/day)
while causing minimal sedation (575). This
could not be replicated in larger trials, how-
ever, and Pfizer is no longer pursuing this
compound. Other compounds in the clinic in-
clude NGD91-3 (Neurogen) (576), NS-2710
(144) (Neurosearch)(577-580),and RWJ-51204
(81).SL-65,1498 (145) (Sanofi-Synthblabo)is
5HT-2 antagonists (adatanserin) as anxiolytiw however, and their application in anxiety re-
has been discontinued in recent years, perhaps mains to be validated in the clinic.
reflecting that a fuller understanding of the
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C.ANTHONY ALTAR .
i
E
i
ARNOLD R. MARTIN
Department of Pharmacology
College of Pharmacy
j
; University of Arizona
j Tucson, Arizona
i
r
: ANDREW THURKAUF
j Department of Medicinal Chemistry
: Neurogen Corporation
Branford, Connecticut
Contents
1 Introduction, 600
2 Clinical Applications, 602
2.1 What Is Schizophrenia?, 602
2.2 Biochemical Basis for Schizophrenia, 602
2.2.1 Dopamine, 602
2.2.2 Serotonin, 603
2.2.3 Glutamate, 603
3 Antipsychotic Agents, 604
3.1 Therapeutic Indications, 604
3.1.1 Schizophrenia and Psychoses, 604
3.1.2 Other Indications, 605
3.2 Adverse Effects and Precautions, 605
3.2.1 Extrapyramidal Symptoms, 605
3.2.2 Tardive Dyskinesia, 607
3.2.3 Neuroleptic Malignant Syndrome, 607
3.2.4 Seizures, 608
3.2.5 Sedation, 608
3.2.6 Cognitive Impairment, 608
3.2.7 Anticholinergic Effects, 608
3.2.8 Cardiovascular Effects, 609
3.2.9 Sexual Side Effects and
Hyperprolactinemia, 609
3.2.10 Weight Gain, 610
3.2.11 Hematologic Side Effects, 610
Burger's Medicinal Chemistry and Drug Discovery 3.2.12 Hepatic Effects, 610
Sixth Edition, Volume 6: Nervous System Agents 3.2.13 Dermatologic Side Effects, 611
Edited by Donald J. Abraham 4 Animal Models of Efficacy and Side Effects, 611
ISBN 0-471-27401-1 O 2003 John Wiley & Sons, Inc. 4.1 Use of Animal Models, 611
Antipsychotic Agents
1F (10) often present psychotic symptoms of un- rotransmission was suspected. This was based
treated paranoid psychosis (11).Increases in on the psychotogenic and hallucinogenic prop-
E D, receptors (15, 16, 342, 343) in the ventral erties of the partial serotonin 5-HT2, receptor
1 striatum of the schizophrenic brain and in- agonist, lysergic acid diethylamide (LSD) (179
: creases in amphetamine-stimulated dopamine and on reports of abnormal CSF and circulat-
I
release in the striata of schizophrenic patients ing levels of serotonin in schizophrenics (1%
(14, 707) provide clinical evidence for height- The validity of the latter finding fell into ques-
ened limbic striatal dopamine transmission in tion as these and subsequent measurements
schizophrenia. It is not surprising therefore of cerebrospinal fluid (CSF) or peripheral se:
-
that essentially without exception, all drugs rotonin activity in schizophrenics were incon-
that treat schizophrenia are potent DJD, re- sistent and probably of no relevance to CNS
ceptor antagonists (12). A preferential antag- - function. Clinical trials with ei-
serotonergic
onism of serotonin 5HT2, or D4 receptors, or a ther 5-HT precursors or depleting agents were
more rapid dissociation from the dopamine D2 also inconclusive (19, 20).
receptor (699) may account for the atypical Noting the structural resemblance of sero-
profile of drugs such as quetiapine, aripipra- tonin to the hallucinogenic indoles dimethyl
zole, olanzapine, and clozapine. Most of typi- tryptamine (DMT)and bufotenin, researchers
cal and atypical antipsychotics occupy proposed that psychotic symptoms were
7040% of striatal D, (and probably D, recep- caused by these or similar compounds gener-
tors) at clinically effective doses (13). The an- ated in schizophrenics by the abnormal trans-
tipsychotic ineffectiveness of drugs that share methylation of endogenous indoleamines (21).
many receptor features of effective antipsy- Unfortunately, studies were unable to confirm
chotics except D, antagonism, including increases in methvlated
" indole amines in the
5HT,, antagonism (666) (see Section 8.2.1.1), plasma or CSF of schizophrenic patients ver-
implicates the D, receptor in the prime mech- sus controls (22). The transmethylation hy-
anism of their action. On the other hand, in- pothesis is also questioned by the recognition
creased CNS levels of dopamine are not an that LSD-induced psychosis differs significant
absolute predictor for psychosis; many other ways from the signs and symptoms of schizo-
agents that stimulate dopamine release such phrenia (23).
as ethanol or opiates do not produce the affec- Renewed interest in serotonin's role in
tive states seen in schizophrenia. Even the ef- schizophrenia was initiated by three major
fects of amphetamine are not reliably psy- findings. First, clozapine, thioridazine (24),
chotogenic. Lower doses can enhance focused and newer atypical antipsychotics (25) were
attention and skilled motor performance, pro- found to more potently antagonize 5-HT,, re-
cesses which are diminished in advanced ceptors than D, receptors. Second, the identi-
schizophrenia. fication of 14 serotonin receptor subtypes pro-
Developmental malformations, impaired vided new candidates for antipsychotic
neuronal innervation, diminished parenchy- etiology and targets for drug development
mal mass, metabolic compromise, and dimin- (26). A 5-HT,, partial agonist activity of clo-
ished dopamine tone in the frontal cortex typ- zapine and newer atypical antipsychotics can
ify schizophrenia but have little to do with be readily reconciled with their superior clini-
dopamine receptors. The diminished tran- cal profiles. Third, allelic variations of genes
scription of genes for synaptogenesis, myeli- for the 5-HT,, receptor have been associated
nation, metabolism, and development in the with the diagnosis of schizophrenia (27) and
schizophrenic brain (8) may explain these im- the clinical response to atypical antipsychot-
pairments and are beginning to provide a ics, for example, as shown by Arranz et al. for
novel set of targets for therapeutic interven- clozapine (28).
tion.
2.2.3 Clutamate. Glutamate and its modu-
2.2.2 Serotonin. Even before the dopa- latory receptors have been implicated in
mine hypothesis of schizophrenia became es- schizophrenia and potentially in its treatment
tablished, a role for overactive serotonin neu- with novel agents (29). After identification
Antipsychotic Agents
mentia, or Huntington's disease, which are mate function would disrupt the balance be-
free of psychosis in all but the late stages. Also, tween glutamate and D, receptors, which con-
a hypothesis of enhanced glutamate toxicity is trols signaling between the basal ganglia,
inconsistent with the decreases in glutamate thalamus, and neocortex (40). A similar con-
found in schizophrenia. trol loop involving 5-HT,, and glutamate re-
A role for decreased glutamate function in ceptors has been postulated (41). The com-
schizophrenia was first proposed by Kim et al., plexity and diversity of excitatory amino acid
who found glutamate to be decreased in the receptors provides many drug targets for
CSF of schizophrenics (37). They postulated pharmaceutical intervention. No drug that
that hyperactive dopamine neurons in the combines activity at glutamate, D,, and
ventral tegmental area (VTA)stimulate D, re- 5-HT,, receptors has appeared.
ceptors, which in turn inhibits glutamate re-
lease and the activity of cortical neurons. D,
antagonism would mitigate the effects of ex- 3 ANTIPSYCHOTIC AGENTS
cessive dopamine release in schizophrenia (14,
707) and thereby disinhibit cortical glutamate 3.1 Therapeutic Indications
release.
The hypoglutamatergic hypothesis of 3.1.1 Schizophrenia and Psychoses. Anti-
schizophrenia is attractive because it is consis- psychotic drugs are primarily used to treat
tent with the lack of changes in D, number in psychotic symptoms associated with schizo-
schizophrenia, and the increases in dopamine phrenia, schizoaffective disorder, and to con-
release in schizophrenia. They are also consis- trol acute mania in patients with manic-de-
tent with the ability of the noncompetitive pressive disorder. Most antipsychotics are
NMDA antagonists PCP and ketamine to in- more effective in controlling the positive
duce behaviors reminiscent of the positive rather than the negative symptoms of schizo-
symptoms of schizophrenia in normals and phrenia. Some of the side effects associated
precipitate these episodes in patients. Ket- with their use, particularly with the use of typ-
amine was administered to schizophrenic pa- ical agents, may be mistaken for negative
tients acutely in a blinded, placebo-controlled symptoms. This confounds the assessment of
trial (31). The drug caused a dose-related ini- their effectiveness and can mislead the less-
tiation of positive psychotic symptoms that experienced practitioner to administer higher
were not blocked by haloperidol. The patients drug doses. The atypical agents are relatively
3 Antipsychotic Agents
free of neuroleptic-induced deficit symptoms ety of newer, safer agents have supplanted the
(NIDS) (42) and perhaps only for this reason use of CPZ in these indications.
seem to be superior to typical agents for treat- A number of unapproved uses of antipsy--
ing negative symptoms of schizophrenia. chotic drugs also exist. CPZ and haloperidol -
Compared with even the atypical antipsychot- were used early on to treat phencyclidine
ics, clozapine has been shown by Kane et al. to (PCP)-induced psychosis. Psychoses associ$
be effective for residual positive symptoms in ated with depression, bipolar disorder, and
the treatment of refractory patients (2, 706). Alzheimer's disease are commonly treated
With the success of the atypical agents, newer with haloperidol, risperidone, or olanzapine..
therapies are expected to show improvements Psychotic symptoms in Parkinson's disease
patients caused by levodopa and/or dopami-
in positive and at least some negative symp-
nergic agonists have been alleviated with
toms, particularly deficits in cognitive func-
quetiapine, because EPS-prone typical neuro-
tioning (43, 701). Clozapine treatment has
leptics contraindicated in Parkinson's disease.
been shown to improve several measures of
cognitive function, especially attention and 3.2 Adverse Effects and Precautions
verbal fluency, whereas other cognitive func- Antipsychotic drug therapy, particularly with
tions such as memory are impaired by cloza- typical agents, is associated with a wide range
pine (702). Risperidone may improve working of unwanted side effects (Table 10.2). Such
memory of schizophrenic patients. Overall, side effects can be troubling for patients, di-
however, the evidence shows that the im- minish compliance with their treatment, and
provement in cognitive function with typical in some cases, create serious health and safety
or atypical antipsychosis is small and of vari- risks. The most frequently observed effects re-
able clinical relevance (702, 703) (see Section sult from dose-dependent actions on the cen-
3.2.6). Similarly, atypical agents such as cloza- tral nervous system (EPS, sedation, and cog-
pine have little significant benefit on negative nitive impairment), the autonomic nervous
symptoms of schizophrenia (704, 705). system (anticholinergic symptoms) including
dry mouth, the cardiovascular system (tachy-
3.1.2 Other Indications. Some of the phe- cardia, postural hypotension, cardiac arrhyth-
nothiazines such as chlorpromazine (CPZ), mias, QT, prolongation), the endocrine sys-
triflupromazine, prochlorperazine, and per- tem (elevated prolactin release and decreased
phenazine are also used as antiemetic drugs. sexual function), and metabolism (weight
CPZ and haloperidol are effective in control- gain). The frequency and severity of dose-de-
ling intractable hiccups. Haloperidol and pendent side effects with individual antipsy-
pimozide are approved, and other antipsychot- chotic drugs can, in general, be correlated with
ics are used, to control tics associated with potencies at D,, a-1, muscarinic, and cholin-
Tourette's disorder and the agitation associ- ergic and other receptors (44-47) (see Table
ated with dementia. CPZ, thioridazine, ris- 10.2). Other adverse effects that are more se-
peridone, and haloperidol have been used in rious, less predictable, but fortunately less fre-
children to treat behavioral problems, includ- quent include tardive dyskinesia, neuroleptic
ing aggressive outbursts, hyperactivity, and malignant syndrome, blood dyscrasias, and
stereotypies associated with conduct disorder, impaired hepatic function. Dermatologic and
attention deficit hyperactivity disorder other allergic reactions and ophthalmic
(ADHD), and autism, respectively. Antipsy- changes are also less frequent.
chotics are also used to treat tics produced by
methylphenidate or amphetamine prescribed 3.2.1 Extrapyramidal Symptoms. Acute ex-
for those with ADHD. CPZ was also used long trapyramidal symptoms (EPS) consists of
ago to treat symptoms of migraine, preopera- drug-induced parkinsonism (DIP), akathisia,
tive anxiety (the first use of CPZ in man), vas- and dystonia. Because of a common link to
cular headaches, tension headaches, and as an diminished CNS dopamine function at D, re-
adjunct to control convulsions in tetanus and ceptors, DIP and idiopathic Parkinson's dis-
to treat acute intermittent porphyria. A vari- ease are indistinguishable. As in Parkinson's
Table 10.2 Receptor Binding and Side Effects of Selected Antipsychotic Drugs
Receptor Binding, pK," Side Effectsb
D2-
D ~ p a m i n e r ~ i c " ~5-HT,".'
~ ~uscarinicf~ l-Adrener& HI-~istaminic'~
Weight Anti- Orthostatic
Drug l,c m,c n,d 1,c n,e m,f n,g 1,h i n,i lj mj n,k EPS Gain Muscarinic Hypotension Sedation
Promazine 6.55 6.80 6.89 6.82 8.03 8.22 7.60 8.70 +2 +3 +2 +2
Chlorpromazine 7.60 7.72 7.74 7.15 8.37 8.59 7.55 8.05 +2 +2 +2 +3 +3
Thioridazine 7.20 7.59 7.80 7.74 8.15 8.30 7.60 7.80 +1 +3 +3 +3 +3
Mesoridazine 7.72 7.16 8.70 8.74 +1 +3 +3 +2 +3
Prochlorperazine 8.15 8.15 6.27 7.62 7.72 +3 +1 +1 +2
Trifluoperazine 8.36 8.59 7.62 6.18 7.17 7.62 6.87 7.21 +3 0 +1 +1 +1
Perphenazine 8.85 5.82 8.00 8.10 +2 +2 +1 +1 +2
Fluphenazine 8.43 9.10 7.60 5.72 7.89 8.05 7.24 7.68 +3 +1 +1 +1 +1
Thiothixene 8.35 9.35 7.44 5.54 7.96 7.96 7.43 8.22 +3 +2 +1 +2 +1
Haloperidol 8.36 8.40 8.69 7.35 6.52 4.05 5.46 8.21 7.58 4.66 5.72 5.92 +3 +1 +1 +1 +l
Pimozide 8.44 7.60 7.70 5.96 +2 -1 +2 +1 +2
Molindone 6.92 3.41 5.60 3.91 +2 0 +1 +1 +2
Risperidone 8.39 9.39 <5 8.61 7.48 +1 +1 0 +l +1
Ziprasidone 8.17 8.68 5.61 7.88 7.19 +1 0 0
Loxapine 7.15 6.35 7.55 8.31 +2 +l + 1 +2 +3
Clozapine 7.42 6.74 6.75 7.54 8.20 7.92 7.48 7.77 8.05 7.65 7.55 8.55 8.97 0/+1 +3 +3 +2 +3
Olanzepine 7.20 8.63 7.26 7.24 8.92 +1 +3 +3 +1 +1
Quetiapine 6.14 6.56 5.97 7.28 8.08 0/+1 +1 0 +2 42
ONegative logarithm of the dissociation constant, KD.
b+lmild, +2 moderate, +3 severe.
c3H-Spiperonedisplacement.
d3H-Haloperidoldisplacement.
e3H-Ketanserindisplacement.
DH-QNB displacement.
g3H-Dexetimidedisplacement.
h3H-WB4101displacement.
'3H-Prasocin displacement.
J3H-Mepyraminedisplacement.
"H-Doxepin displacement.
'Ref. 45.
"Ref. 46.
"Ref. 47.
Antipsychotic Agents
disease, the cardinal signs of DIP are bradyki- considered to be less typical variants of TD.
nesia, muscle rigidity, and resting tremor fre- TD most commonly manifests during periods
quently accompanied by a stooped posture, an of dose reduction or abrupt withdrawal of neu-
unsteady gait, seborrheic dermatitis, and ex- roleptic medication. In the early days of anti- -
cessive salivation. Akathisia is described by psychotic therapy, it was an accepted clinical
patients as an inner restlessness and an in- practice to allay the symptoms of TD by ini
ability to remain still while seated or standing. creasing the dose of a typical antipsychotic
Acute dystonia is characterized by involun- drug, or by changing from a less potent to a
tary, sustained muscle contractions that may more potent drug (e.g., CPZ to fluphenazine or _
temporarily cease, only to be repeated in a haloperidol).
slow writhing motion. Dystonic reactions may The precise biochemical mechanisms giv-
be manifested as oculogyric crises, torticollis, ing rise to TD are poorly understood, but prob-
tongue thrusting, cramping of the hands or ably include effects on GAl3Aergic neurons
arms, and laryngeal spasm. within the substantia nigra (44,261). Dopami-
EPS associated with antipsychotic therapy nergic D, receptor antagonists (i.e., typical
tends to lessen with dosage reduction. The neuroleptics) alleviate the symptoms for a
conjoint use of the same anticholinergic time, whereas dopaminergic D, receptor ago-
agents employed as adjuncts in the treatment nists and cholinergic muscarinic antagonists
of Parkinson's disease tends to ameliorate worsen the symptoms of TD. A direct correla-
DIP and dystonia, but has less effect on aka- tion between the tendency of antipsychotic
thisia. The frequency and intensity of EPS drugs to cause acute EPS in the short term
correlate with the antagonist potencies of the and the risk of TD in the long term has been
typical neuroleptics at dopaminergic D, recep- established (49). Thus, the incidence of TD is
tors. Thus, the high potency agents (like flu- much higher with typical, than with atypical,
phenazine and haloperidol) cause a higher in- antipsychotic drugs. The ability of clozapine to
cidence of EPS, while the lower potency ameliorate TD caused by other antipsychotic
agents (particularly thioridazine and mesorid-
-
drugs (50) demonstrates active protective
mine, which also antagonize muscarinic re- mechanisms of its action beyond D, antago-
ceptors) cause a lower incidence of EPS. Clo- nism.
zapine and other atypical agents generally do
not cause EPS. Dose-dependent EPS has been 3.2.3 Neuroleptic Malignant Syndrome.
reported to occur with the putative atypical Neuroleptic malignant syndrome (NMS) is a
antipsychotic drug risperidone, but only at rare, but potentially lethal, adverse effect that
levels above the recommended daily dosage of has been reported with virtually all typical an-
2-10 mg daily (48, 694). The absence of EPS tipsychotic drugs in current clinical use. NMS
seen with the atypicals has been attributed to is a complex syndrome consisting of hyper-
several mechanisms and will be discussed in thermia, frequently associated with severe
Sections 6.4 and 9.3. EPS (dystonia and parkinsonism), autonomic
nervous system instability, myoglobinuria,
3.2.2 Tardive Dyskinesia. Tardive dyskine- and increased serum creatinine phosphoki-
sia (TD) is a late-appearing and sometimes nase (CPK) levels. NMS has most frequently
irreversible syndrome that may occur after been reported in agitated male patients receiv-
prolonged treatment with antipsychotic med- ing high and rapidly escalating intramuscular
ications. It occurs more frequently in older pa- doses of antipsychotic drugs (51). Comorbid
tients and is characterized by involuntary, medical conditions, such as agitation and de-
quick, repetitive movements of the face, eye- hydration, seem to play a role in the manifes-
lids, mouth (grimaces), tongue, extremities, tation of NMS. A rechallenge of patients who
and trunk. These disturbing choreiform move- have recovered from NMS with the same or a
ments may be accompanied by slow, twisting different antipsychotic drug is generally not
movements of the body and dystonic postures. associated with a reoccurrence of NMS symp-
The appearance of late developing symptoms toms. The elucidation of the pathophysiologi-
of tardive dystonia and akathisia are generally cal basis for NMS has been confounded by the
Antipsychotic Agents
complexity of symptomology. Guerra has of- ing with their significant HI-antihistaminic
fered a hypothesis for the etiology of NMS in- potencies. Less sedation is generally seen with
volving dysregulated sympathetic nervous risperidone. Fortunately, the sedative effects
system hyperactivity (52). of these drugs tend to abate with prolonged use.
3.2.4 Seizures. The lowering of the thresh- 3.2.6 Cognitive Impairment. Studies at-
old to seizures in susceptible patients has long tempting to determine the cognitive effects of
been known to be a property of antipsychotic antipsychotic drugs in schizophrenic patients
drugs. The aliphatic phenothiazines seem to have yielded conflicting results. The use of
present a slightly higher risk than do the pi- conventional neuroleptics has shown either no
perazine phenothiazines or haloperidol. How- impairment, some impairment, or even im-
ever, the overall incidence of actual seizures
provement of cognition (56-59) and the issue
provoked by the typical antipsychotics is esti-
is even somewhat controversial for the atypi-
mated to be less than 1%,so there is some
disagreement about comparative risk (53). cal agents (702, 703). Side effects of typical
Clozapine, on the other hand, was associated antipsychotic drugs can potentially impair
by Welch et al. with a dose-related seizure in- performance in tasks intended to measure
cidence of 3% or greater (54). A correlation cognitive effects. EPS, in particular akathisia,
between seizure-inducing propensity and the caused by the more potent neuroleptics, can
central antimuscarinic potencies of antipsy- impair performance requiring motor re-
chotic drugs has been proposed (55), but other sponses. Sedation can interfere with perfor-
equally plausible mechanisms should also be mance in tasks requiring attention and vigi-
explored. In general, concern about the possi- lance. The addition of anticholinergic drugs to
ble occurrence of drug-induced seizures minimize EPS associated with conventional
should not represent a contraindication to an- neuroleptics disrupts short-term memory in
tipsychotic medication use. The risk can usu- schizophrenic patients (58). This is consistent
ally be minimized by the appropriate choice of with the well-known, adverse effects of anti-
antipsychoticagent, dosage reduction, and the cholerinergic agents on memory and cognitive
concomitant use of anticonvulsant drugs, if performance in normal persons (60). In a re-
needed. view of adverse neurobiological effects arising
from long-term use of typical neuroleptics,
3.2.5 Sedation. Conventional antipsychot- Jeste et al. (61) conclude that persistent cog-
ics cause sedation to varying degrees. Drug- nitive impairment associated with their
induced sedation can cause daytime somno- chronic use has not been clearly established.
lence and can facilitate the induction of sleep
day or night. In the early use of CPZ for the 3.2.7 Anticholinergic Effects. Antipsychotic
treatment of agitated psychotic patients, seda- drugs having relatively high affinities for
tion was initially seen as a beneficial effect. blocking muscarinic cholinergic receptors can
For the long-term treatment of schizophrenia, cause a variety of atropine-like side effects,
however, drug-induced sedation is undesir- such as dry mouth, blurred vision, constipa-
able, can contribute to negative symptoms, tion, urinary retention, and tachycardia. Such
and be mistaken for the impaired cognition annoying side effects are usually transient and
frequently associated with the disorder. Seda- seldom dangerous. Elderly patients, however,
tive effects correlate with the HI-antihista- are more susceptible to problems of constipa-
minic and possibly a,-antiadrenergic poten- tion, paralytic ileus, and urinary retention.
cies of the classical antipsychotic drugs. Thus, Patients with cardiovascular disease may be
agents with low D, potency are more sedating compromised by tachycardia induced by these
than the high potency agents, because the rel- drugs. The piperidine substituted phenothia-
ative contribution of HI and a, antagonism zines, thioridazine, and mesoridazine, and to a
are more significant relative to D, potencies. lesser extent CPZ, cause the most anticholin-
Among the atypical agents, clozapine and ergic effects among the classical antipsychot-
olanzapine are particularly sedating, in keep- ics. Clozapine and olanzapine have the highest
i
3 Antipsychotic Agents
I
incidence of anticholinergic side effects among larization warranted Caley and Cooper to rec-
r
i
the atypical antipsychotic drugs. ommend its use onlv " as a second-line
7 treatment in patients with comorbid cardio- -
3.2.8 Cardiovascular Effects. Many of the vascular risks (697). Torsade de pointes has -
. cardiovascular side effects of antipsychotic also been associated with the use of intrave-
medications result from their actions on the nous haloperidol (67). It is likely that many ofF
i autonomic nervous system. For example, the the unexplained deaths of younger patients in
; tachycardia seen with certain drugs is because Great Britain in the 1980s attributed to high
of their antimuscarinic effects (see Section dose pimozide may have been caused by its -
3.2.7). Postural hypotension caused by some arrhythmogenic effects (68).
agents is believed to be caused by a,-adrener- Torsade de pointes is known to be triggered
gic blockade. The lower potency phenothia- by hypokalemia and by the class I11 antiar-
zines, CPZ, thioridazine, and mesoridazine, rhythmic drugs that interfere with potassium
cause the highest incidence of postural hypo- channels. Potassium channel blockers prolong
tension, whereas this side effect rarely occurs the QT, interval by inhibiting the rapid com-
with haloperidol. Among the atypical agents, ponent of the delayed rectifier current, I,
clozapine and risperidone cause a higher inci- (69).Recently the human gene HERG (human
dence than do olanzapine, quetiapine, or aripi- ether-a-go-go-related gene) that encodes for a
prazole. The dizziness experienced by most protein associated with I,, has been trans-
patients is usually transient and not serious, fected into mammalian cell lines (70) and em-
especially if the antipsychotic dosage is ti- ployed to screen antipsychotic drugs for car-
trated gradually. However, postural hypoten- diotoxic effects (71-73). Haloperidol (71),
sion, possibly coupled with sedation, can pose thioridazine (72), and the otherwise promis-
a serious risk of hip fracture in elderly ing atypical antipsychotic drug sertindole (73)
. are all high affinity antagonists for the HERG
Certain antipsychotic drugs exert direct potassium channel protein. These antipsy-
quinidine-like actions on the heart (62).These chotics bear more than a casual structural re-
dose-dependent changes, which are readily ob- lationship to certain other non-cardiac drugs
served in the electrocardiograms (ECGs) of known to cause torsade de pointes arrhyth-
patients include the following: QT, prolonga- mias (63, 64), such as the HI-antihistaminic
tion, abnormal T-wave morphology, and wid- drugs, terfenadine and astemizole, and the
ening of the QRS complex. Drug-induced pro- 5-HT, agonist and prokinetic drug, cisapride.
longation of the QT, interval can trigger the
initiation of torsade de pointes, a polymorphic 3.2.9 Sexual Side Effects and Hyperpro-
ventricular tachycardia that is often fatal (63, lactinemia. Clinical evaluation of the sexual
64). Abnormal ECG effects in psychiatric pa- function effects of antipsychotic agents are
tients were first observed with the phenothia- fraught with methodological difficulties in-
zines, CPZ and thioridazine (65). Based on cluding reduced sexual performance in un-
case reports, thioridazine seems to be the medicated schizophrenics. The few well-con-
worst offender, having been implicated in sev- trolled studies that have appeared have
eral instances of sudden death attributed to involved male subjects (74, 75).
ventricular tachycardia (66; 695). A close ana- Some of the effects of antipsychotic drugs
log, mesoridazine, can also cause ventricular on sexual function have been attributed by
arrhythmias. Chlorpromazine and trifluoper- Aizenberg et al. to increased prolactin secre-
mine seem to cause fewer ECG abnormalities. tion by the anterior pituitary (74, 761, other
There is no association of alterations in QT effects may result from their specific auto-
intervals with the use of olanzapine, quetiap- nomic actions (75). Prolactin secretion by the
ine, or risperidone (695). Ziprasidone mod- anterior pituitary is tonically inhibited by the
estly prolongs the QT interval, but there is no hypothalamus, with dopamine acting as the
evidence to suggest that this leads to torsade prolactin release-inhibiting factor (PIF).
de pointes or sudden death (695). Neverthe- Thus, conventional neuroleptics cause dose-
less, its potential effects on ventricular repo- related increases in serum prolactin levels (hy-
Antipsychotic Agents
chotic drugs since the introduction of CPZ tempt to mirror specific symptoms of schizo-
(95).Mild-to-moderate increases in transami- phrenia. Of these behaviorally isomorphic
nase enzymes are frequently seen during the models (1071, the few that have face validity -
first few weeks of therapy. Such findings are likely to become particularly useful for -
rarely justify discontinuation of therapy be- identifying atypical antipsychotic drugs (105,
cause the levels usually return to normal. A 106, 108, 109).
-P
cholestatic-like jaundice, accompanied by ab- The concept of schizophrenia as a neurode-
dominal pain and idiosyncratic fever and velopmental disorder has inspired attempts to
chills, affects up to 2% of patients taking CPZ create adverse and early postnatal events in -
(95). This non-dose-dependent cholestasis animals to model the psychopathological pro-
has also been reported with haloperidol (96) cesses underlying the disorder (109, 110).
and has rarely been reported for clozapine. These neurodevelor~mental models include
prenatal malnutriGon, viral infection and
3.2.1 3 Dermatologic Side Effects. Allergic hypoxia, disrupted neurogenesis by X-ray irra-
skin reactions associated with antipsychotic diation or neurotoxins in utero, adverse post-
drugs range from urticaria and macropapular natal ex~eriential factors such as maternal de-
rashes to erythremia multiforme. Such reac- privation and social isolation, and postnatal
tions seem to be uncommon with the newer brain damage created by hippocampal, neocor-
atypical agents. Photocontact urticaria (loo), tical, or thalamic lesions (109-111).With the
photosensitivity (sunburn) (101),and deposits possible exception of maternal deprivation
in the lens (700) have been observed with CPZ and social isolation, these models have not
and other phenothiazines including mesorid- been sufficiently characterized pharmacologi-
azine. The occurrence of hyperpigmentation cally to be used for antipsychotic drug screen-
of the skin seems to be associated with higher ing.
doses of phenothiazine-type neuroleptics in
chronically treated patients (102, 103). It has 4.2 Prediction of Antipsychotic Efficacy
been suggested that light-generated free radi-
cal species, possible interacting with melanin 4.2.1 Behavioral Effects of Psychostimulants.
in the skin, may be responsible for both the The psychosis-inducing properties of the psy-
hyperpigmentation (102,103) and photosensi- chostimulants amphetamine (121) and PCP,
tivity phenomena (101). (115) in human volunteers are well known.
Both drugs also exacerbate psychotic symp-
toms in schizophrenic patients (115,121,122).
4 ANIMAL MODELS OF EFFICACY
However, the constellation of symptoms elic-
AND SIDE EFFECTS
ited by the two psychostimulants differ signif-
icantly. Amphetamine induces predominantly
Because of its genetic complexity and uncer-
positive symptoms such as paranoia in volun-
tain etiology, schizophrenia, like so many neu-
teers, and tends to worsen such symptoms in
ropsychiatric disorders, has resisted the devel-
schizophrenics. Certain negative symptoms
opment of suitable animal models. Early
may actually be improved by amphetamine
progress in the field was hampered because of
(123). PCP induces positive and negative psy-
the prevailing belief that schizophrenia was a
chotic symptoms in normal volunteers and
social or psychological disorder rather than a
worsens a broad spectrum of psychotic symp-
neurodevelopmental brain disorder.
toms in schizophrenics (122). Because of the
psychotomimetic effects of amphetamine and
4.1 Use of Animal Models
PCP, both drugs have been used as animal
After the introduction of the first neuroleptic models against which novel antipsychotic
drugs, many animal models were developed to drug action could be predicted.
screen new compounds for potential antipsy- 4.2.1.1 Behavioral Models of Dopaminergic
chotic activity. These models predict both an- Overactivity. The initial behavioral animal
tipsychotic efficacy and side effect liability in models for identifying potential antipsychotic
humans (104-106). Other animal models at- drugs were based on the dopamine theory of
Antipsychotic Agents
schizophrenia (9). The antagonism of in- treatment with clozapine, and to a more par-
creased locomotor and stereotypic behaviors tial degree, remoxipride and sertindole, re-
induced by dopamine stimulants like apomor- verses deficits in social interaction in rats
phine and amphetamine, the induction of cat- induced by PCP (109,119, 134, 135). Haloper-
alepsy, and the disruption of the conditioned idol, chlorpromazine, and risperdal are not ac-
avoidance response each result from the tive. This is in keeping with the unique effi-
blockade of Dfl, dopaminergic receptors cacy of clozapine in ameliorating negative
(108, 112). The concept of limbic selectivity symptoms in schizophrenic patients (2). The
(10, 11) further resolves these models, effectiveness of olanzapine only at higher
whereby antipsychotic activity results from doses (119,135) was confirmed by more recent
the blockade of mesolimbic D, receptors, while studies that, however, failed to demonstrate
EPS is produced by mesostriatal D, blockade. an effect for the newer antipsychotic drug
The phenothiazine derivative, thioridazine, quetiapine (136).
and clozapine were the first neuroleptics to The behavioral despair of mice forced to
exhibit some degree of functional limbic selec- swim without escape is measured by their im-
tivity on chronic administration (113,114). In- mobility during a retest under the same con-
creased locomotor activity induced by low (0.5 ditions. Immobility in the forced swim test
mgjkg) doses of amphetamine is largely medi- (FST) is reversed by antidepressant drugs
ated in the nucleus accumbens (124, 125). All (137), whereas PCP enhances the immobility.
typical and atypical antipsychotics antagonize Pretreatment with clozapine and risperidone,
hyperactivity in rats induced by low dose am- as well as the 5-HT, antagonists, mianserin
phetamine (126,127,112), making this simple and ketanserin, attenuated the PCP-induced
animal model a useful initial screen for poten- increase in immobility, whereas haloperidol
tial antipsychotic drugs. This model is not en- and antidepressants have no effect (138,139).
tirely selective, however, because drugs lack- The PCP enhancement of the FST may pro-
ing antipsychotic activity in humans, such as vide a more selective animal model for the neg-
the GABA agonist muscimol, also antagonize ative symptoms of schizophrenia.
amphetamine-induced locomotion (128). PCP and other NMDA antagonists increase
4.2.1.2 Behavioral Models of Glutamateri- cortical glutamate efflux (140, 141). This
gic Overactivity. The observation that psy- prompted the suggestion that the psychotoge-
chotic-like symptoms can be induced in hu- nic action of PCP may result from a potentia-
man volunteers by the psychomimetic drug tion of glutamatergic effects at non-NMDA re-
phencyclidine (PCP) (115) led to an interest in ceptors, in addition to their block of NMDA
the examination of PCP-induced behaviors in receptors. Several metabotropic glutamate re-
animals as models for screening potential an- ceptor subtypes regulate glutamate efflux
tipsychotic drugs (116) (see Section 8.2.2). The (142). Moghaddam and Adams (143) have
potent noncompetitive NMDA receptor antag- shown that the group I1 metabotropic gluta-
onist properties of PCP provided an additional mate agonist LY 354740 [(+)-2-amino-
hypothesis for the etiology of schizophrenia bicyclo[3.1.Olhexane-2,6-dicarboxylate] pre-
(117). This hypothesis has been tested in a vents PCP-induced locomotion and stereotypy
wide range of PCP-induced behavioral effects and prevents the disruptive effects of PCP on
in rodents, including increased locomotor and working memory. These effects were achieved
stereotyped behaviors 116-118, decreased at doses of LY 354740 that did not affect spon-
social interaction (118, 1191, and deficits in taneous locomotion or dopaminergic neuro-
prepulse inhibition (120). transmission, suggesting that metabotropic
Hyperlocomotion and stereotyped behav- glutamate agonists might represent a new
iors induced in rodents by noncompetitive class of antipsychotic drugs.
NMDA antagonists such as PCP and dizo-
cilpine are antagonized by a wide range of an- 4.2.2 Prepulse Inhibition. Abnormalities in
tipsychotics (116, 117, 129-133). PCP can information processing and attention mecha-
model in rats the negative symptomatology of nisms have long been recognized as hallmark
social interaction (119, 134, 135). Chronic characteristics of schizophrenia. Prepulse in-
4 Animal M of Efficacy and Side Effects
hibition (PPI) is the reduction-in the startle in experimental animals and in humans. Con-
reflex response produced by a weak (non- sequently, the LI paradigm has been proposed
startling) sensory stimulus given just before as an animal model of schizophrenia to predict -
the startling stimulus. PPI is a measure of antipsychotic activity (165, 169).
sensorimotor gating or information process- Amphetamine disrupts the LI response in
ing and is reliably impaired in schizophrenic rats (170,171), and this is reversed by haloper-,
patients (144, 145). Deficits in PPI are be- idol, clozapine, and sertindole (172, 173) and
lieved to reflect an inability to adequately in- the 5-HT,, antagonist MDL 100,907 (174).
hibit irrelevant sensory information leading to The 5-HT, agonist DOI, when administered -
sensory overload and cognitive fragmentation. -
in the pre-exposure phase only, disrupts LI
The cross-species nature of PPI supports its
and this disruption is prevented by haloperi-
use as an animal model with potential face,
dol, clozapine, risperidone, and MDL 100,907
predictive, and construct validity (146). PPI in
animals is impaired by the psychotomimetic (175). This disruption could be because of ef-
agents amphetamine (144), PCP (147), and fects of DO1 on state-dependent learning
other NMDA antagonists (148, 149), and by rather than on attentional processes (175).
5-HT,, agonists (150, 151). Potentiation of the LI response in rats has
Most antipsychotics, but also many non-an- been consistently demonstrated for classical
tipsychotic drugs, reverse deficits in PPI in- antipsychotics (169, 176) and for clozapine
duced by amphetamine or apomorphine (152, (169, 172, 173, 177, 178), remoxipride (178),
153). In contrast, D, receptor antagonists sertindole (179), and MDL 100,907 (174). Be-
such as haloperidol fail to reverse deficits in cause very few of the newer antipsychotic
PPI induced by PCP or dizocilpine (153-155), drugs have been investigated for their effects
while such deficits are, at least in part, re- on LI, and MDL 100907 is not an effective
versed by clozapine (156), remoxipride (157), antipsychotic, further studies with these
olanzapine (1581, and quetiapine (155). Ris- drugs are required to validate the predictive-
peridone fails to reverse the effect of PCP ness of the LI model.
(155),but it antagonizes the disruption of PPI Although LI and PPI both reflect the ability
induced by the 5-HT, agonist DO1 (150,153). of the organism to ignore irrelevant stimuli,
Rats reared in isolation after weaning also they differ in many respects. PPI is a sensori;
experience deficits in PPI (159) and decreased motor gating process that does not require
conditioning and relies on mechanisms that
social interaction. This effect has been attrib-
uted to enhanced dopaminergic activity (160). filter exteroceptive stimuli for their physiolog-
ical or cognitive significance. LI is a cognitive
Isolation rearing deficits are maximal at pu-
process, requiring preconditioning, and re-
berty (161, 162) and thus parallel the ontog-
eny of schizophrenia in humans. The disrup- flects a subject's ability to adjust behavior to
tion of PPI in young rats reared in isolation is changing conditions. In contrast to PPI, LI is
reversed by a broad spectrum of antipsychotic not disrupted by PCP (180).
drugs including haloperidol, risperidone, clo-
zapine, olanzapine, and quetiapine (153,163). 4.2.4 Conditioned Avoidance Response.
Perhaps the oldest animal model to predict
4.2.3 Latent Inhibition. Latent inhibition potential antipsychotic drug efficacy is the
(LI)is like PPI but includes a greater cognitive conditioned avoidance response (CAR) (108,
component. It is a retarded acquisition of a 112, 181, 182). In the conditioned reinforce-
conditioned response that occurs when a sub- ment model, experimental animals are trained
ject is exposed to the conditioning stimulus to perform a certain response to avoid a mild
before the conditioning trials (164,165). Defi- shock. Trained avoidance responses may be
cits in LI reflect the inability of the subject to active (pressing a lever, climbing a pole, or
ignore irrelevant stimuli and have been dem- jumping out of a box) or passive (remaining in
onstrated in many (164-167), but not all the darker of two compartments). Classical
(168), studies of schizophrenic patients. Like antipsychotic drugs and benzodiazepines re-
PPI, latent inhibition has been demonstrated duce avoidance responding at doses that do
Antipsychotic Agents
not impair natural (untrained) escape re- a partial agonist at M, and M, receptors and
sponding (183); antidepressants impair both an antagonist at MI, M,, and M, receptors
escape and avoidance responding (184). (201). AMPNkainate receptor antagonists
CAR is inhibited by a wide variety of struc- also cause a neuroleptic-like suppression of
turally different D, antagonists (185-188). CAR (203).
There is a positive and significant correlation
between anti-avoidance activities of various 4.2.5 Drug Discrimination. Drug discrimi-
antipsychotic drugs in rats and their D, recep- nation has been used for many years to detect
tor blockade (186)and their average daily clin- the predominant receptor activities occurring
ical dose in treating schizophrenia (189). in vivo. These inferences are based on whether
Somewhat conflicting results have been ob- behavioral responses in humans or animals
tained for clozapine in CAR tests in different that are sustained by exposure to a reference
animal species (186, 190, 191). A biphasic ef- compound are also maintained by the test
fect has been reported in mice and in squirrel compound, such as one with antipsychotic
monkeys, wherein lower doses of clozapine ac- properties (204). Drug discrimination studies
tually increase avoidance, while higher doses can identify antagonist agents that block con-
antagonize the response (190).Studies in rats ditioned responses, and agonist miomics that
have shown that clozapine blocks CAR at sustain conditioned responses. Such tests can
doses that interfere (191) or do not interfere identify antipsychotics with similar pharma-
with motor function (186, 189). Remoxipride cological properties but are not necessarily
(189), risperidone (1921, olanzapine (193), predictive for the treatment of schizophrenia.
quetiapine (1941,and ziprasidone (195) are all Systemic administration of classical anti-
active in the CAR test. psychotics or clozapine block amphetamine-
The complexity of the CAR test is evident induced discriminative stimulus responses in
from the variety of drugs that either suppress the rat (205,206). The amphetamine response
or enhance responses. Wadenberg and Hicks can also be blocked by direct injection of the
(196) conclude that CAR is a sensitive test for drug into the nucleus accumbens (207).While
potential antipsychotic drugs that act by vari- classical antipsychotics completely inhibit the
ous receptors, particularly those in the nu- response, clozapine and olanzapine are some-
cleus accumbens shell. a1-Adrenergic recep- what less active, risperidone and remoxipride
tor antagonists inhibit CAR (197). This may are weakly active, and sertindole and quetiap-
contribute to the antipsychotic effectiveness ine were inactive (208, 209). Based on these
of many neuroleptics, because almost all of findings, it has been proposed that the antag-
them bind and probably antagonize the a1 re- onism of the amphetamine discriminative
ceptor. Suppression of CAR by cholinergic po- stimulus response may be an indication of
tentiators like arecoline, pilocarpine, and phy- neuroleptic-induced deficit properties (209).
sostigmine was observed many years ago (198, Antipsychotic drugs also seem to inhibit the
199). The muscarinic cholinergic antagonist discriminative stimulus effects of hallucino-
scopolamine attenuates the impairment of genic 5-HT agonists like DOI, DOM, and LSD
CAR induced by D, antagonists, but enhances in proportion to their 5-HT,, antagonist po-
the suppression of CAR caused by the selective tencies (209-211).
Dl antagonist SCH 23390 (2001, suggesting a Early studies indicated that PCP-induced
very different mechanism for the effect of Dl discriminative stimulus responses are not an-
receptors in the CAR paradigm. Partial mus- tagonized by haloperidol (212, 213), and con-
carinic agonists including PTAC and RS86 in- flicting results have been obtained with
hibit CAR (201,202). PTAC exhibits a similar NMDA antagonists including dizocilpine
pharmacological profile to that seen with atyp- (214), whereas the metabotropic glutamate
ical neuroleptics, including a limbic-selective agonist LY 354740 is inactive (215).
inhibition of dopamine cell firing and inhibi- Drug discrimination has also been used to
tion of amphetamine induced c-fos expression compare antipsychotic drugs that act through
in the nucleus accumbens (201). Preliminary different receptor mechanisms. The close
receptor selectivity data suggest that PTAC is structural analogs olanzapine and quetiapine
4 Animal Models of Efficacy and Side Effects 61 5
generalize to the clozapine discriminative chotics (50). The induction of catalepsy re-
stimulus in rats, whereas haloperidol, remo- flects the potential of a drug to cause EPS.
xipride, chlorpromazine, and fluphenazine are Many of the newer antipsychotics, for exam- -
ineffective (216-218). Seroquel, haloperidol, ple, the benzamides (188), risperidone (192), .
i and thioridazine partially generalized, but ris- olanzapine (193), ziprasidone (2311, and aripi-
i
i
peridone, sertindole, and amisulpiride, a D,/
D,-antagonist (2191, failed to generalize (220).
In a study conducted in squirrel monkeys,
prazole (232, 233), induce catalepsy only at
high doses. Some drugs lacking neuroleptic ac-
tivity, such as opioids (234) and muscarinic
T
quetiapine fully, and olanzapine partially, agonists (2351, potently induce catalepsy. .
generalized to the clozapine stimulus, Muscarinic antagonists, on the other hand, re-
whereas risperidone, sertindole, and remo- verse the cataleptogenic actions of haloperidol
xipride failed to show generalization (221). (236).
Olanzapine itself also elicits a discriminative
stimulus in rats (222). The olanzapine re- 4.3.2 Stereotyped Behaviors Induced by
sponse generalized to clozapine, chlorproma- Amphetamine and Apomorphine. Stereotypy
zine, and thioridazine, as well as to the 5-HT,, responses in rodents consist of repetitive sniff-
antagonist ritanserin and to scopolamine. A ing, licking, biting, and gnawing (237) because
full generalization to the clozapine response of excessive activation of striatal D, receptors
was achieved with muscarinic antagonist sco- (226,228). The EPS potential of antipsychotic
polamine (2201, reflecting the prominent anti- drugs is also evaluated by their ability to an-
, muscarinic action of clozapine. A partial gen- tagonize stereotypy induced in rats by rela-
eralization occurred with the a1-adrenergic tively high doses of amphetamine (5 mg/kg) or
antagonist prazosin and the a2-adrenergic an- apomorphine (1mg/kg) (229, 238-240). Clas-
-
tagonist idazoxan (220). Given the most com- sical antipsychotics and the selective D, an-
plex receptor pharmacology of clozapine and tagonist SCH 23390 fully and potently antag-
its chemical analogue olanzapine, it is not sur- onize the stereotyped behaviors induced by
prising that many antipsychotic drugs gener- amphetamine or apomorphine (238,239,241).
alize to their discriminative stimulus cues Consistent with their atypical classification,
(220, 223). thioridazine (239, 240), clozapine (239, 240),
sertindole (242), quetiapine, and ziprasidone
4.3 Prediction of Side Effect Liability
are ineffective even at the highest doses
While EPS and catalepsy in experimental an- tested, whereas remoxipride, risperidone, and
imals are no longer considered to be necessary olanzapine antagonize amphetamine-induced
consequences of antipsychotic drug action stereotypy only at relatively high doses.
(224, 225), even the newest atypical antipsy-
chotics block D, receptors to varying degrees 4.3.3 Paw Test. In the paw test, rats are
(12,13), and with the exception of quetiapine, placed on a platform with their fore- and hind-
can induce EPS, catalepsy, and increase se- limbs lowered into four separate holes. The
rum prolactin at high doses (226-228). Thus, paw test determines the effects of drugs on the
tests for such liabilities remain in most anti- spontaneous retraction of extended forelimbs
psychotic screening batteries. and hindlimbs (243). The test was originally
developed to study the effects of GABAergic
4.3.1 Catalepsy. CPZ and reserpine were drugs on motor behavior mediated in the dor-
the first neuroleptics found to create a state of sal striatum (243). It was soon discovered that
tonic immobility, or "catalepsy," in experi- classical antipsychotics such as haloperidol
mental animals. In the catalepsy test, rats or and CPZ increase the time for retraction of the
mice are placed in an unusual position and the hindlimb (HRT) and the forelimb (FRT) with
time required for the animal to correct this equal potencies, whereas thioridazine and clo-
unusual position is determined. All typical an- zapine increase HRT at lower doses than were
tipsychotics induce catalepsy (2291, whereas required to increase FRT (244). It was pro-
clozapine does not (230), and it can even re- posed that FRT models the EPS effects of neu-
verse catalepsy induced by typical antipsy- roleptics, whereas HRT models their thera-
Antipsychotic Agents
peutic effects (244). In addition to thioridazine the use of monkeys and the long-term dura-
and clozapine, the D,-selective antagonist tion of their treatment (257).
benzamides, the Dl-selective antagonists SCH
23390 and SCH 39166, and the mixed receptor 4.3.5 Vacuous Chewing Movements. The
antagonists risperidone, olanzapine and chronic administration of classical antipsy-
sertindole, all increase HRT at significantly chotic drugs to rats induces oral dyskinesias,
lower doses than those required to increase termed vacuous (or purposeless) chewing
FRT (245,246). The predictive validity of the movements (VCMs) (258-260). This behavior
paw test is questioned (112) because SCH has been proposed as a model for orifacial TD
39166 is not an effective antipsychotic (248). in humans. Chronic treatment with clozapine
In general, however, drugs lacking neuroleptic produces far fewer VCMs than does haloperi-
do1 (261,262). Rats treated for 6 months with
activity were inactive in the paw test. These
clinically effective doses of olanzapine or
included opiates, benzodiazepines, antihista-
sertindole produced very few VCMs (263).
mines, and tricyclic antidepressants (246, Notwithstanding the limited number of new
247). Scopolamine antagonizes the increase in antipsychotic drugs that have been investi-
FRT induced by haloperidol, but not its in- gated, the VCM model seems to have use for
crease in HRT, supporting the hypothesis that the preclinical assessment of potential new
FRT is related to EPS (244). drugs for their TD-inducing liability (257),
and without limitations inherent in the sensi-
4.3.4 EPS in Primates. The ability of anti- tized primate model.
psychotic drugs to induce acute dystonia and
other neurological side effects in nonhuman 4.3.6 Prolactin Response. Classical anti-
primates closely mirrors these responses in psychotic drugs stimulate prolactin release
humans. Cebus apella monkeys are sensitized from the pituitary gland through the blockade
by long-term treatment with haloperidol to of D, receptors innervated by tuberoinfun-
produce a nonhuman primate model of dibular dopamine neurons (264).The selective
chronic EPS resulting in tardive dyskinesia Dl antagonist SCH 23390 does not stimulate
(TD) (249-252). Some of the haloperidol- prolactin release (265), consistent with the
treated monkeys develop a prolonged dyski- sole presence of D, receptors in the pituitary.
netic syndrome resembling clinical TD, and The correlation between serum prolactin in-
these are used to study the potential of anti- creases and antipsychotic potencies in hu-
psychotic and other drugs to cause or treat TD mans (266) is explained by the common D,
(251, 252). Clozapine does not elicit dystonia mechanism for both actions. Although cloza-
in sensitized monkeys and is virtually free of pine (265) and aripiprazole (267) can increase
EPS in humans. Quetiapine is also devoid of serum prolactin in rats, neither antipsychotic
EPS potential in the dystonia model, after elevates prolactin in humans (268-270). More
both oral (194) and intraperitoneal (253) ad- minor effects on human serum prolactin typ-
ministration. A large separation between pre- ify the newer antipsychotic drugs, particularly
dicted EPS and clinically effective doses was aripiprazole, risperidone, olanzapine, and zi-
found for sertindole, whereas olanzapine, prasidone (421, and these will be described in
quetiapine, and remoxipride gave an interme- greater detail in the following sections dedi-
diate separation. Haloperidol, risperidone, cated to these drugs.
and ziprasidone had little separation
(254-2561, suggestive of EPS liability. The 4.3.7 Cognitive Effects. Along with the
strength of the Cebus monkey dystonia model overt psychotic symptoms that characterize
of TD is that the symptoms closely parallel the schizophrenia, deficits in cognitive function
symptoms of TD in humans, including the in- are commonly manifested by impairments in
dividual vulnerability factor wherein some attention, information processing, and mem-
monkeys develop TD while others do not ory (14, 701-703). While antipsychotic drugs
(257).Limitations are mainly the expense and can ameliorate impaired attention and infor-
ethical considerations of the model because of mation processing, their effects on memory
i 5 Discovery of Prototype Antipsychotics
t
E
deficits are less clearly established (see Sec- by Laborit to calm patients afflicted with
tion 3.2.6). In fact, some studies in rats have schizophrenia who had been treated primarily
shown that classical antipsychotics actually by social isolation or physical restraints in
induce short-term memory deficits (271,272). mental institutions. Their unprecedented im-
In the Morris water tank model of spatial provement heralded a revolution in the treat-
memory (273), low doses of haloperidol and ment of this terrible disease. CPZ and related
risperidone decrease spatial memory in a dose- drugs enabled many institutionalized patients '
dependent fashion. These drugs also decrease to reenter society and home, something that
swimming speed, suggesting that impairment had been the exception rather than the rule.
of motor function related to D, antagonism This led to a marked decrease in the number of -
(274) may be a contributing factor. Ziprasi- hospitalized patients, from 500,000 in 1955 to
done and olanzapine impair spatial memory at 222,000 in 1983.
relatively high doses but do not affect swim- Most patients who returned to society were
ming speed (275). Clozapine slightly impairs able to function moderately well, but if they
cognitive performance initially but is without stopped their medication their condition dete-
effect in later trials. Sertindole and quetiapine -
riorated. Also, CPZ and com~ounds related to
have no effect on either spatial memory. this phenothiazine series produced motoric
The same group of antipsychotic drugs side effects, including extrapyramidal side ef-
have been evaluated for their effects on work- fects (EPS) that resemble Parkinson's dis-
ing memory (276,277) in a visual spatial ver- ease. These symptoms could be severe and de-
sion of the delayed non-matching to position veloped in up to 90% of patients on typical
paradigm (278, 279). Haloperidol and risperi- antipsychotic drugs. This condition often pro-
done exhibit marked inhibitory effects in low gressed to irreversible tardive dyskinesias, in-
doses, whereas considerably higher doses of voluntary- movements of the limbs and facial
clozapine, olanzapine, and ziprasidone are re- muscles that resemble the symptoms of Hun-
quired to produce similar effects (276).Sertin- tington's disease. In addition, such typical an-
dole and quetiapine are inactive at the doses tipsychotics, although they were effective in
tested (276). Long-term treatment reveals a treating the positive or florid symptoms of
continued memory impairment of memory by schizophrenia, did not ameliorate the negative
haloperidol, the development of tolerance to symptoms of the disease.
the effects of clozapine on memory, and con-
5.2 Discovery of Haloperidol
tinued lack of impairment by sertindole (277).
Haloperidol was discovered in the Janssen
Laboratories in 1958 (280,315,320,322). It is
5 DISCOVERY OF PROTOTYPE
a butyrophenone derivative and was pursued
ANTIPSYCHOTICS
because of its ability to block the behavioral
activating effects of amphetamine in rat mod-
5.1 Discovery of Chlorprornazine
els. The amphetamine models were used as
In the late 1940s, Laborit administered the screening tools, because symptoms observed
antihistaminic phenothiazine derivative, in paranoid schizophrenia were noted to be
promethazine, before surgery to ameliorate similar to those that developed in chronic am-
the symptoms of surgical shock. He observed phetamine abusers. The behavioral profile of
that promethazine decreased the patients' haloperidol was qualitatively similar to that of
concerns and fears of the impending surgery. CPZ, but haloperidol required up to 50-fold
In 1950, optimization of this early lead pro- lower doses to exert the same behavioral ef-
duced chlorpromazine (CPZ) a drug that dem- fects as CPZ. Around this time, the term "ma-
onstrated significant efficacy in reducing pre- jor tranquilizer" began to be replaced by the
operative fears. It is this kind of indifference term "neuroleptic." The compound was pur-
to impending stress that may form the basis sued very vigorously, and the initial clinical
for the predictive nature of the conditioned results were published only 10 months after
avoidance response (CAR) test for antipsy- the initial synthesis of the compound. Halo-
chotics (see Section 4.2.4). CPZ was also used peridol is still one of the most widely pre-
Antipsychotic Agents
scribed neuroleptics in the United States understand the relationships between chemi-
(281), partly because of its inexpensive cost, cal structure and biological effectiveness after
its potency, and availability as a decanoate the discovery by Kuhn in 1957 of the first tri-
preparation for intramuscular depot adminis- cyclic antidepressant drug imipramine. This
tration that provides long-term efficacy and sparked major interest in a number of phar-
increased compliance (282). maceutical companies to investigate variants
of such structures. Although somewhat sim-
5.3 Advent of Atypical Antipsychotics
plistic, the hypothesis developed was that in
Classical neuroleptics of the phenothiazine, such linear tricyclic systems, all of which con-
butyrophenone, and thioxanthene type cause tained a basic side-chain, if the two aromatic
undesirable side effects, which at one time rings were in the same plane, i.e., flat, as was
were considered to be an inevitable conse- the case with 6:6:6 systems, the resulting mol-
quence of their potent antagonism of D, recep- ecule was most likely to exhibit antipsychotic
tors. The search for atypical antipsychotic properties, whereas in the 6:7:6 systems
compounds focused initially on maintaining where the two aromatic rings are "twisted" or
the generally favorable efficacy while decreas- non-planar, the molecule was more likely to
ing the severity or incidence of these side ef- have antidepressant properties. Indeed, such
fects, particularly the EPS. A large number of an approach led to the discovery of the imipra-
structurally diverse compounds have been in- mine analogs, clomipramine and amitripty-
vestigated to this end. These compounds have line, and also to the discovery of amoxapine
varying degrees of receptor affinities in addi- (284). However, surprisingly, clozapine was
tion to a high D, affinity for serotonergic, his- found not to be an antidepressant but an effec-
taminergic, muscarinic, cholinergic, and tive antipsychotic agent devoid of extrapyra-
non-D, dopamine receptors. The tricyclic an- midal side effects. The drug was successfully
tipsychotic, clozapine, and the benzamide, introduced in some European countries in
amisulpiride, are associated with fewer EPS 1968, but the finding of agranulocytosis in a
and preferentially increase dopamine turn- Finnish study, where eight patients died,
over in the mesolimbic system, unlike the clas- nearly ended the further development of the
sical antipsychotics, such as haloperidol and drug. The incidence of agranulocytosis in
CPZ, that elevate dopamine turnover to the schizophrenic patients treated with clozapine
same degree in mesolimbic and striatal brain is 1-2% (285). Consequently, clozapine was
areas. This finding indicates that the limbic only used as second line therapy when at least
and prefrontal areas of the brain may be anti- two other standard antipsychotic drugs had
psychotic target sites, whereas dopamine re- failed. It was not until the 1980s in the United
ceptor blockade in the striatum is thought to States that clozapine began to find acceptance,
be more responsible for the induction of EPS. as evidence mounted that it was effective
against both positive and negative symptoms
5.4 Discovery of Clozapine
of schizophrenia. The careful blood monitor-
Classical antipsychotic agents induce EPS on ing of patients treated with clozapine has dra-
chronic administration and also do not treat matically decreased the lethality from agran-
up to 30% of psychotic patients. Clozapine, a ulocytosis in the United States. The search for
tricyclic benzodiazepine derivative, was the clozapine-like compounds (286) with compa-
first antipsychotic drug to display a dramati- rable effectiveness but without the toxicologi-
cally different pharmacological profile and did cal problems has been a quest for the past 20
so both in animals and clinically. This profile years. The success of this search has required
created the concept of a new class of "atypical" the painstaking elaboration of mechanisms by
antipsychotic drugs. which clozapine exerts its therapeutic and tox-
Clozapine has had a checkered history from icological effects (287).
its initial discovery in the late 1960s by Ger-
5.5 Discovery of Amisulpride
man and Austrian clinicians (283) to its redis-
covery in the United States in the 1980s. It With the discovery of atypical agents such as
was synthesized initially as part of an effort to clozapine and thioridazine, companies began
6 Structure-Activity Relationships
searching for other classes of compound that the diphenylbutylamine, pimozide (121, the
would be effective antipsychotics devoid of the indoline, molindone (13),and the dibenz[b,fl-
EPS problems of the classical drugs and also (1,4)oxazepine,loxapine (14). Drugs generally -
the toxicological liabilities of clozapine. A se- classified as atypical include: the benzisox-
ries of benzamides was developed starting mole, risperidone (15),the benzisothiazole, zi-
from the structure of the local anesthetic, pro- prasidone (16),the dibenzCb,fl(l,4)diazepine,
cainamide, that led progressively to the anti- clozapine (17), the thienobenz[b,fl(l,4)mepine,
emetic compound, metoclopramide, and the olanzapine (I€!),and the dibenzCb,fl(l,4)thia-
observation that it demonstrated some weak zepine, quetiapine (19) (Table 10.3).
.
antipsychotic properties. Further optimiza-
6.2 Tricyclic Neuroleptics
tion of this benzamide structure led to the de-
velopment of sulpiride, which possessed sig- After the introduction of chlorpromazine (1)
nificant antipsychotic activity and had a lower (CPZ) as a treatment for schizophrenia, thou-
incidence of EPS than classical agents (288). It sands of new agents based on its tricyclic to-
was also found somewhat later that members pology were prepared and examined pharma-
of this class compound were generally highly cologically. The neuroleptic potential of these
selective for the dopamine D, family of recep- agents was determined through a number of
tors (289, 290), although varying degrees of standardized animal tests, most notably those
selectivity across the D, receptor subtypes of motor activity and conditioned response as
(D,, D,, and D,) were found. Indeed, struc- described in Section 5.3. These assays served
tural features of the substituted benzamides to rank order the neuroleptic "potency" of new
were later incorporated into compounds de- agents relative to CPZ. This classification be-
signed to specifically block the dopamine D, came known as the "chlorpromazine index,"
receptor (see Section 8.1.1). Although exten- and the biological potencies of compounds
sive variants of sulpiride have been investi- within a selected behavioral paradigm were
gated (291), only racemic sulpiride and expressed relative to CPZ whose index was
amisulpride have been marketed widely out- unity. Although receptor binding theory and
side the United States. The active enantiomer analysis were a number of years off, these
of amisulpride, S-amisulpride, is the only an- tests were often remarkably accurate at pre-
alog still under active clinical development. A dicting the rank order of affinity for the dopa-.
highly promising analog, remoxipride (292, mine D, receptor subtype.
293), produced good efficacy against both pos- The tricyclic antipsychotics may be dis-
itive and negative symptoms of schizophrenia sected into three substructures (Fig. l O . l ) ,
and no troublesome anticholinergic effects. those being the pendent amine functionality
Unfortunately, remoxipride produced unac- (Site A), the diary1heterotricyclic (Site C),and
ceptable toxicity of aplastic anemia, and clini- the intervening alkyl chain (Site B) (295).
cal trials were discontinued in 1993. However, When considering the effect of structure on
this class of compound has provided some ex- neuroleptic activity, it is informative to exam-
cellent pharmacological "tools" and radioli- ine each of the three substructures of the tri-
gands because of their high selectivity for the cyclic~in isolation to see how individual
D, receptor (294). changes within each of these regions impacts
the pharmacological properties of the result-
ant compound.
6 STRUCTURE-ACTIVITY RELATIONSHIPS
The distance between Sites A and C is crit-
ical for neuroleptic activity, with a three car-
6.1 Drug Classes
bon chain being optimal. Shortening the chain
Antipsychotic drugs currently approved for to two carbons has the effect of amplifying the
clinical use in the United States are summa- anticholinergic and antihistaminic properties.
rized in Table 10.1. Drugs classified as typical The amino ethyl derivatives diethazine (2) has
include the following: several phenothiazine proven useful in the treatment of Parkinson's
derivatives (1-9), the thioxanthene, thiothix- disease, while promethazine (3)is effective as
ene (lo), the butyrophenone, haloperidol (111, an antihistamine.
620 Antipsychotic Agents
Chlorpromazine
Index Block of
Conditioned
No. U V W X Y Z Response in Rats
; z
Site A ; Site B I Site C
tion of the aminopropylon side-chain provides response blocking activity relative to the cor-
thioridazine (5), which is equipotent to chlor- responding alkyl (13 and 14) or alkoxy (12)
promazine (1)(297). derivatives (298.299).
A number of polysubstituted derivatives -
have been examined (16-20). In each of these
cases, a marked decrease in neuroleptic po-
tency was reported (300,301). i~
Nature of the Amino Group. The size and
nature of the basic amino group has consider-
able influence on the behavioral profile of the -
phenothiazine neuroleptics (Table 10.4). A
tertiary m i n e is optimal, both mono and des-
methylchlorpromazine are several times less
active than the parent molecule in rat condi-
tioned response (302). In many cases, larger
(4) Chlorpromazine index <0.1 N-alkyl groups decrease neuroleptic potency
(Table 10.4). The diethyl (21), pyrrolidinyl
(22), morpholinine (231, and thiomorpholine
(24) analogs of CPZ show decreased chlor-
promazine indices in the rat conditioned re-
sponse assay. In contrast, the N-meth-
ylpiperazine compound prochlorperazine (25)
has enhanced potency, as do a number of other
piperazines (26-28) (296). The azaspiranyl
analog (29) produced pronounced CNS de-
pression and sedation lasting more than 48 h
(303).
Variations Within the Tricyclic Topology.
With an eye toward maintaining the diary1na-
(5) Chlorpromazine index 1 ture of the tricyclic site C, a wide variety of
creative variations of CPZ were prepared. In-
troduction of other group VI elements in place
Effect of Aromatic Substitution Within the Tri- of the sulfur in the phenothiazines produced
cyclic System. The influence of aromatic sub- the corresponding phenoxazine and phe-
stitution on the pharmacological profile of the noselenazine derivatives. As shown in Table
tricyclic antipsychotics is fairly straightfor- 10.5, the phenoxazines are markedly less
ward. For the purposes of this discussion we active than the phenothiazines (304a,b),
will examine the effect of varying substituents whereas the phenoselenazines fall somewhere
within the 10-(3-dimethylaminopropyl) phe- in between (305). The diminished activity of
nothiazines (Table 10.3),although these influ- the phenoxazines may be a result of the short-
ences are applicable across the various classes ened oxygen-carbon bond length, which would
of tricyclic neuroleptics. tend to pull the two aromatics closer together
All other factors being equal, substitution and at a tighter angle. The carbon (acridan)
at position 2 provides compounds of enhanced derivative (35) resemble the phenosel-
potency in blocking conditioned response in enozines, although the disubstituted acridan
rats relative to both the parent compound and (36) is essentially inactive (306).
also compounds having the identical substitu- A unique series of neuroleptics results from
ent at positions 1, 3, or 4. The electronic na- the replacement of the nitrogen within the
ture of the substituent also plays a role in de- phenothiazine ring system with a methine
termining the efficacy in this model. Electron carbon. The introduction of the double bond
withdrawing groups such as chloro (1)and tri- within the propylamino chain provides for
fluoromethyl (9) show superior conditioned geometric isomers. The geometry, wherein the
Antipsychotic Agents
(CHd3R
CPZ Index Blockade
of Conditioned
No. R X Fksponse in Rats
I:1:
2-substituted aromatic ring and the amino-
ethyl substructures are on the same side of the
double bond, is referred to as "cis," and the C1
isomers having this configuration have been
shown to possess enhanced neuroleptic po-
tency relative to the "trans." For instance,
chlorprothixine (371,the direct cis thioxan-
thene analog of chlorpromazine, showed sim- N(CH3)z
ilar activity to CPZ in conditioned behavioral
tests in rats. The trans isomer (38)displayed (37)
6 Structure-Activity Relationships 623
(CH2)3N(CH3)2 P
Chlorpromazine Index Blockade
No. Y of Conditioned Response in Rats
apc1
pCl N-
(47)
ClH3 '2H3
(48) X = 0 oxyclozapine
(49) X = S clotiapine
(50) X = NH clozapine
(51) X = CH2 perlapine
Q ~ ! ~ ~ H ap
~ N-
Q b
"O)
o
(52)
(53)
Figure 10.2.
does not produce extrapyramidal side effects. of this class showed high neuroleptic potency
Indeed, clozapine has been used to suppress relative to chlorpromazine. The most effective
the motor effects of tardive dyskinesia (316). compounds of this class are of the following
The reasons for this unique profile are a mat- general structure.
ter of continuing debate and research.
Two drugs related to clozapine include the
almost identical compound, olanzapine (52),
and quetiapine (53).Both confer similar anti-
psychotic profiles and a minimal propensity to
elicit extrapyramidal side effects (317, 318).
Olanzapine has also found use in the treat- FX
<
E&
-
ment of tardive dyskinesia (319).
Comparison of the behavioral potencies
and dopamine D, receptor binding of some of
6.3 Butyrophenones
these compounds are displayed in Table 10.6.
The butyrophenone antipsychotics were dis- The first of these drugs to be introduced for
covered during studies of modified meperidine treatment of psychosis was haloperidol (54).
derivatives (320). These compounds, which The relatively low toxicity of haloperidol rela-
possess a tertiary amine at the fourth carbon tive to chlorpromazine resulted in its wide ac-
of the butyral chain, could be made to possess ceptance and it remains an important drug.
minimal analgesic activity by the addition of a Related to haloperidol are methylperidol
substituent at the 4-position of the aromatic (55), trifluperidol (56), and compound (57).
ring. Many of the clinically tested compounds Comparison of these analogs shows that po-
626 Antipsychotic Agents
Table 10.6 Behavioral and Dopamine D, Binding Data for Selected Butyrophenones
Rat
Dog Jumping Amphetamine L3H] Haloperidol
Box Testa Jumping ~ t e r e o t ~ p y 6 Binding IC,,
No. Name R (Refs. 314, 320) Box Testa (Ref. 321) (nit0 (Ref. 322)
(1) Chlorpromazine
(54) Haloperidol
(55) Methylperidol
(56) Trifluperidol
(57) Peridol
(58) Haleperidide
(59) Paraperidide
(60) Floropipamide
(61) Spiperone
6 Structure-Activity Relationships
.+"
\ /
Rat
Dog Jumping Amphetamine L3H1 Haloperid&
Box Testa Jumping Stereotypyb Binding IC,,
No. Name R (Refs. 314,320) Box Testa
(Ref. 321) (nM) (Ref. 322)
.
500 33 33 0.33
Q
;r""
..
NN
-$-J
(64) Fluoanisone 3
EN^
CH30, 5 3
tency and dopamine binding are enhanced by who were administered the antiemetic agent
substitution on the piperidino phenyl ring. Re- metoclopramide (66) exhibited behavioral
placement of the tertiary hydroxyl of haloper- properties consistent with antagonism of do-
idol by amides as in (58) and (59) produced pamine receptors (325). Metoclopramide initi-
effective compounds, as did simultaneous re-
placement of the aromatic by piperidine (60). 0
A cyclic variation of the 4-amino-4-carbox-
-
amido vattern can be seen in spiperone (61).
nl
"l'/\vilN,,,N(Et)z
A
The origin of the benzamide antipsychotics amide substructure was crucial to the elicita-
arose from the astute observation that rodents tion of behavioral effects in rodents. The 2-me-
Antipsychotic Agents
reotypies (striatal) versus agonist-induced hy- creased activity over remoxipride, although
peractivity (limbic). Based on this behavioral the latter showed a better separation in the
Table 10.7 Behavioral Properties and D, Binding Affinities of 2-Methoxybenzamides
stereotypy/hyperactivityratio. Substitution of
chlorines for bromine in both cases led to di-
minished activity and a more typical behav-
ioral profile (75 and 76).
Analysisofthehuman metabolitesofremox-
ipride identified two products resulting from
demethylation (332). The 3-bromo-6-methoxy
salicylamide (77) (FLA 797) showed a much
greater affinity for the D, receptor than did Figure 10.3. Solid state conformation of FLA 797.
either its isomer (FLA 908) (78) or the parent
compound. This result lead to speculation that amide substructure is the preferred conforma-
the neuroleptic activity of remoxipride was tion for binding of the benzamide neuroleptics
caused by this metabolite. to the receptor.
The 100-fold increase in affinity of FLA 797 Raclopride presents an exceptional separa-
tion between striatal- and limbic-mediated be-
over remoxipride was puzzling. Determina-
haviors. Although it has been suggested that
tion of the logP values for remoxipride and
raclopride is able to differentiate between re-
FLA 797 found them to be nearly identical (2.1 gional subclasses of D, receptors (3371, this
versus 2.0), indicating a similar partitioning has never been clearly substantiated in the
between octanol and water. This result is laboratory for this or any antipsychotic at the
somewhat surprising because the free hy- level of receptor binding.
droxyl group of (77) would be expected to as- Another benzamide of interest is ami-
sociate with water. A larger difference was sulpiride (83). Clinical trials have shown
found for logP where FLA 797 was found to be
more lipophilic than remoxipride (1.7 versus
0.7). However, this difference seems unlikely
to fully explain the difference in in uitro activ-
ity.
X-ray crystal structures of both compounds
were performed and indicated major differ-
ences in the conformations of the two benz-
amides (333). For remoxipride, the phenyl
group and the amide carbonyl are oriented al-
most perpendicular to each other. This orien- amisulpiride to be efficacious against the pos-
tation precludes the formation of a hydrogen itive symptoms of schizophrenia at doses that
bond between the amide hydrogen and the or- have only a low propensity to induce EPS ef-
tho methoxy group. Such hydrogen bonds fects (338). In accordance with other benz-
have been noted in other types of ortho benz- amide neuroleptics such as sulpiride and
amides (334). raclopride, amisulpiride is highly selective for
The 6-methoxysalicylamide displayed a the D, and D, receptor subtypes for which it,
planar conformation wherein a pseudo-ring is like most antipsychotics, has similar affinity
stabilized by a hydrogen bond between the (-2 nM). The high selectivity of (83)for the D,
phenol and the carbonyl oxygen. As depicted receptor family, coupled with its clinical pro-
in Fig. 10.3, this conformation also allows for file, bring into question previous theories that
an additional interaction between the amide the atypical nature of drugs such as clozapine
hydrogen and the remaining ortho methoxy. is a result of their interaction with one or a
Such additional hydrogen bonds have been number of other non-dopaminergic receptors
found in the related 2-methoxysalicylamides (339,340).
raclopride (81) (335) and eticlopride (82) Considerable evidence exists that D, re-
(336).The exceptional dopamine D, affinity of ceptors are presynaptic autoreceptors that
these compounds relative to remoxipride sug- are preferentially localized in limbic area
gests that this planar orientation of the benz- (341-343). The atypical nature of amisulpiride
6 Structure-Activity Relationships
7 PHARMACOKINETICS,
BIOTRANSFORMATION,
AND DRUG INTERACTIONS
is apparently catalyzed by CYPlA2, because mean terminal half-life of about 6 h. Oral ab-
this transformation is enhanced by cigarette sorption of the drug is rapid with peak plasma
smoke and the inducer carbamazepine and in- levels achieved within 1.5 h. Quetiapine is -
hibited by fluvoxamine. The formation of the widely distributed throughout the body with -
2-hydroxy-methyl metabolite catalyzed by an average volume of distribution of 10 L/kg.
CYP2D6 is considered to be a minor pathway The extent of plasma protein binding is 83%at,
and not likely to lead to clinically significant therapeutic blood concentrations.
drug interactions (472). A flavin nonooxygen- Quetiapine is extensively metabolized by
ase (FM03)is thought to be responsible for the cytochrome P450 oxidation. Less than 5% of _
4-N-oxide (472). unchanged drug is excreted. The major path-
Oxidative metabolism of clozapine was way involves CYP3A4 oxidation to the sulfox-
found to correlate with caffeine metabolism ide (473, 475). Other important reactions in-
(462) and is thus largely carried out by cyto- clude oxidation of the side-chain alcohol to the
chrome P4501A2 (CYPlA2). No correlation carboxylic acid, aromatic oxidation to the phe-
with CYP2D6 polymorphism was found (461, nolic 7-hydroxy-metabolite, and oxidative N-
462). Several interaction studies of clozapine dealkylation of the entire side-chain (473,
with SSRI antidepressants have been reported 475). Inducers of CYP3A4 (phenytoin and car-
(465-469). Fluvoxamine increased plasma bamazepine) significantly increased quetiap-
concentrations of clozapine in schizophrenic ine clearance, whereas ketoconazole, an inhib-
patients (463,464), presumably through inhi- itor of CYP3A4, significantly increased plasma
bition of CYPlA2 catalyzed N-demethylation levels of the drug (475).
(465). Fluoxetine was found to increase the
7.7 Butyrophenones
plasma concentrations of clozapine and its
major metabolites, suggesting that this SSRI The member of the butyrophenone class of an-
must interfere with pathways other than N- tipsychotic drugs that has been studied the
demethylation and N-dealkylation (466). Two most extensively is haloperidol (476). The
other SSRIs, paroxetine (463) and citalopram drug is rapidly and almost completely ab-
(467), had no apparent effects on clozapine sorbed after oral administration. Significant
levels. first pass metabolism in the liver largely ac-
The biotransformation (468-470) and clin- counts for the systemic bioavailability of,
ical pharmacokinetics (471) of olanzapine around 60% for haloperidol by the oral route
have been investigated extensively. The drug (477). Haloperidol is 90-92% bound to plasma
exhibits dose proportional linear pharmacoki- proteins. Peak plasma levels of haloperidol are
netics in therapeutic dose ranges (471). Olan- achieved in 1.7-3.2 h, and the drug is rapidly
zapine is bound to both albumin and a-1-acid distributed to well-perfused tissues such as
glycoproteins in the plasma. The mean elimi- the lungs, skeletal muscle, and brain, with a
nation half-life in healthy adults was 33 h and terminal half-life ranging from 6 to 33 h. Re-
the plasma clearance averaged 26 L/h. distribution of haloperidol to adipose tissue
apparently does not occur significantly (478).
7.6 Dibenz(l,4)Thiazepines
Haloperidol undergoes extensive metabo-
Quetiapine (53) is the only dibenz(l,4)thia- lism to form a myriad of primary and second-
zepine derivative currently available in the ary metabolites (479-486). The principal
United States. Very few publications have phase I reactions involve the following: oxida-
been devoted to its biotransformation and tive N-dealkylation to initially form 4-fluoro-
pharmacokinetics (473, 474). Much of the fol- phenylbenzoylpropionaldehyde (which is rap-
lowing information has been obtained from idly oxidized to the acid) and 444-
data on file from the manufacturer (475). chloropheny1)-4-hydroxypiperidine(479, 480,
Quetiapine exhibits dose proportional lin- 483-485); N-oxidation to form haloperido1-N-
ear pharmacokinetics within the clinical dose oxide (485); and reversible reduction of the
range with accumulation that is predictable carbonyl group to the alcohol, or reduced hal-
on multiple dosing. Clearance of quetiapine is operidol(481,487). A phase I1 metabolite, hal-
largely through hepatic metabolism with a operidol-0-glucuronide, accounts for as much
Antipsychotic Agents
as 60% of total haloperidol metabolites ex- The structural similarity of the pyridinium
creted in the urine (486). Reduced haloperidol metabolite (102) to 1-methyl-4-phenylpyri-
is also hydroxylated at the 2'-fluorophenyl dinium (MPPt) (103), the toxic metabolic
ring position and this phase I metabolite is
excreted in the urine as the sulfate and gluc-
uronide conjugates (486). The quaternary N-
glucuronide of haloperidol apparently does
not form (441). Formation of the dehydration
product of haloperidol, i.e., the 1,2,5,6-tetra-
hydroperidine derivative (101), is thought to
product responsible for the Parkinson's disease-
inducing agent 1-methyl-4-phenyl-1,2,3,6-tet-
rahydropyridine (MPTP) (104), prompted the
7.8 Benzisoxazoles
humans (517)through aldehyde oxidase (697). -
The pharmacokinetics of risperidone, the first Four principal routes of metabolism were
member of this structural class to be marketed identified: N-dealkylation of the arylethyl
worldwide, has been studied in experimental side-chain attached to the 4'-piperazinyl ni-
animals (510) and in humans (511). Risperi- trogen; oxidation of the sulfur, resulting in the
done (84) is well absorbed after oral adminis- sulfoxide and the sulfone; reductive cleavage
tration and rapidly distributed to the tissues of the benzoisothiazolyl ring; and hydrolytic
(70%absolute oral bioavailability). It is exten- cleavage of the C=N bond of the isothiazolyl
sively metabolized in the liver at three differ- ring.
ent sites in the molecule (510, 511). Hydroxy- Eleven days after the administration of a
lation at the 9 position is mediated by single 20-mg oral dose of 14C-labeled ziprasi-
CYP2D6, while oxidative N-dealkylation is ap- done (517) to human volunteers, 20% was re-
parently not affected by debrisoquine hy- covered in the urine and 66% in the feces. The
droxylator polymorphism (511,512). Opening absorption was rapid and the, ,C for free
of the isoxazole ring also occurs in humans, drug and metabolites occurred at 2-6 h post-
but is a less important pathway (511). In the dose. The mean peak serum concentration of
rat, some oxidation also occurs at the 7 posi- unchanged ziprasidone was 45 ng/mL with a
tion of risperidone to provide minor amounts mean area under the curve (AUC) of 336 ngkl
of 7,9-dihydroxy- and 7-keto-9-hydroxy me- mL. On the basis of AUC values, approxi-
tabolites (510). In humans, peak plasma levels mately 46% of the circulating radioactivity
of 9-hydroxyrisperidone, the major metabo- was attributed to unmetabolized ziprasidone.
lite, are reached in 3 h with extensive metabo- Less than 5% of the administered dose was
lizers and in 17 h with poor metabolizers. The excreted as unchanged drug.
terminal half-lives of risperidone averaged 3 h Metabolites apparently do not contribute
in extensive metabolizers and 20 h in poor me- to the pharmacological activity of ziprasidone
tabolizers. The average terminal half-lives of in vivo because the sulfone and sulfoxide me-
9-hydroxyrisperidone are 21 h and 30 h in ex- tabolites exhibit low affinities at 5-HT, and D,
tensive and poor metabolizers, respectively. receptors (517). Limited investigations of cy-
The fractions of risperidone and 9-hydroxyris- tochrome P450 isoforms responsible for the
peridone bound to proteins in the plasma are primary oxidative metabolic reactions for zi-
90% and 77%, respectively. Because the phar- prasidone indicate a major role for CYP3A4
macological activity of 9-hydroxyrisperidone oxidation to the sulfone, shown by Prakash et
is nearly equivalent to that of risperidone, the al. (518), and virtually no role for CYP2D6
steady-state levels of antipsychotic species are (518,519).
similar in poor and extensive metabolizers.
7.1 0 Diarylbutylamines
7.9 Benzoisothiazoles
The pharmacology and pharmacokinetics of
The atypical antipsychotic drug ziprasidone pimozide (861, the most important member of
(105) (514) was approved for the treatment of this class of neuroleptics, have been reviewed
schizophrenia in the United States in early (520). Pimozide is well absorbed after oral ad-
2001. Studies of the biotransformation of this ministration and is widely distributed into the
benzoisothiazolederivative reveal that it is ex- tissues. It has an elimination half-life of
tensively metabolized in rats (515,516) and in around 55 h, despite extensive hepatic metab-
Antipsychotic Agents
olism (521, 522). Phase I oxidative N-dealky- The Dl-like receptors include Dl and D,, while
lations occur on the bis(4-fluoropheny1)-butyl the D,-like receptors encompass D,, D,, and
side-chain of pimozide to form bis(4-fluoro- D, (531, 532). The D, receptor subtype has a
pheny1)butyric acid and N-Cpiperidinylben- short (D,,) and long (D,,) protein form that
zimidazolin-2-one, and at the 4-position of the results from alternative transcription of the
piperidine ring of pimozide to form 1-Cbis(4- D, gene. The difference between the two re-
fluropheny1)-butyllpiperidonol and benzimi- ceptors is a 29 amino acid peptide coded by an
dazolin-Pone. None of the metabolites of exon that has been spliced out of the third
pimozide that have been isolated from experi- intracellular loop of DZL.The D, receptor also
mental animals or humans seem to be phar- has several variants, and the D, receptor has
macologically active (520). multiple variants that are characterized by a
The principal cytochrome P450 isoform in- different number of repeating units also lo-
volved in pimozide oxidation is CYP3A4 cata- cated in the third cytoplasmic loop of the re-
lyzed N-dealkylation (523). CYPlA2 seems to ceptor protein (533).
also be involved. Pimozide may also interfere
with the metabolism of substrates by 8.1.1 D, Selective Compounds. In general,
CYP2D6. Potentiation of pimozide-induced the typical^ antipsychotics interact with the
cardiotoxicity (prolongation of the QT inter- D,, D,, and D, receptor subtypes but are more
val) in patients by clarithromycin-induced in- potent in their affinities at the D, receptor,
hibition by CYP3A4 N-dealkylation has been with Ki around 1-5 nM and about 10-fold less
reported (524). potency at the D, and D, receptors. Clozapine,
however, has its greatest affinity at the D, re-
7.1 1 Molindone and Related Compounds ceptor subtype, with a Kiof 21 nM, in contrast
to its 230 nM potency at the dopamine D, re-
The clinical pharmacodynamics and pharma- ceptor (534). In addition, 10-30% increases in
cokinetics of molindone (91) have been re- D, and D, receptors (535) and sixfold in-
viewed (525).The drug is reputed to be rapidly creases in the D, receptor subtype (536) have
absorbed after oral administration and rapidly been reported in schizophrenic brains, com-
metabolized. Only 2 3 % of the unchanged pared with normals, although the latter find-
drug can be recovered in the urine and feces. ing remains controversial. Autoradiographic
Molindone has a very short half-like (1.5-2 h) analyses with the D, selective ligand ,H-NGD
and is 1.5-1.7 times more bioavailable after 94-1 show D, sites to be dense in rat and hu-
intramuscular, rather than oral, administra- man hippocampus, hypothalamus, and neo-
tion (526). Molindone is less lipophilic than cortex, among other brain regions, and to be
most antipsychotic drugs and has a lower frac- absent in the striatum (537). These findings
tion (around 75%) that is bound to proteins in suggested that a D,-specific compound might
the plasma (527).Clinical studies indicate that treat schizophrenia as effectively as clozapine
the antipsychotic effectiveness of molindone but without the D, antagonist-mediated EPS
lasts more than 24 h (525,528,5291, suggest- or the clozapine constellation of side effects.
ing that one or more active metabolites may Given these initial technical hurdles, a ma-
contribute to its actions in vivo. jor effort was undertaken to discover potent,
specific D, receptor antagonists (538).
8 STRATEGIES FOR DRUG DISCOVERY Whereas the D, hypothesis may yet prove cor-
rect, it remains unclear as to whether antipsy-
chotic effects can be obtained with selective D,
8.1 Dopamine Receptor Subtype Approaches
antagonists such as NGD 94-1, L-745-870, or
Dopamine receptors were first classified into sonepiprazole, or the mixed 5-HT,,/D, antag-
Dl and D, subtypes (530). As a testament to onist fananserin. Each of these compounds
the validity of this initial classification, the have failed to show efficacy in the treatment of
five subsequently cloned dopamine receptors schizophrenia. However, the unique polymor-
fall into structural and functional groupings phism~of the D, receptor gene have lead to
-
with other CNS disorders. Associations seem to the D, and D, but not D, receptors. Isos-
to be with ADHD, substance abuse (heroin but teric replacement of the arnide functionality
not alcohol), and novelty- or risk-seeking with a pyrrole as in (107) lea to a related series
traits (539) but not with obsessive compulsive
disorders, Parkinson's disease, schizophrenia,
psychoses, mood disorders, or Tourette's
syndrome.
The quest for D,-selective ligands (540444)
began with the knowledge that the sulpiride
class of atypical antipsychotics bound specifi-
cally to members of the D, family of receptors.
One early investigation showed that confor-
mationally restricted benzamide analogs of
sulpiride-like molecules retained dopaminer-
gic selectivity after replacement of the carbox- that maintained many of the pharmacological
amide with a pyrrole ring. This was shown in characteristics of the benzamides. Extension
the case of the 2-phenyl pyrroles (545) and the of this strategy to the butyrophenones pro-
corresponding 1-phenyl pyrrole derivatives vided (108)with high affinity for D, receptors
(540). This series is closely related to NGD
94-1, the first potent and specific D, antago-
nist to be developed clinically. NGD 94-1
binds to the D, receptor with a 3.6 nM affinity
(541). Clinically, NGD 94-1 is subject to rela-
tively rapid (<1h) hydroxylation in the pyrim-
idine ring to produce the corresponding 5-hy-
droxy pyrimidine derivative. This metabolite
is also a potent D4-specificligand, but has par-
tial D, agonist properties (546). [,H]NGD
94-1 was synthesized (547) and used to local-
ize and map D, receptors in the brains of rats,
and normal and schizophrenic humans (548- (545). The analogous 2-phenylimidazole com-
550). Attempts were made to block the 5-hy- pound (109)also showed strong affinity for D,
droxylation by incorporation of a fluorine sub- as well as D, receptors (554). When the piper-
stituent at the 5-position of the pyrimidine.
While this was successful, development of the
resulting compound NGD 94 -2ISch-66712
was discontinued because of a potent inhibi-
tion of human liver cytochrome P450 2D6
(CYP2D6) (551). Some compounds like
nemonapride (106)bound equally at all three
respectively). It arose from a structural hy- than to the typical antipsychotic, haloperidol.
bridization of the selective 5-HT,, antagonist Further evidence for the value of a high D2/5-
ritanserin (130),where the fused pyrimidine HT, affinity ratio in this series of compounds
side-chain was retained, and a 1,2-benzisox- may be found with a metabolite of tiasperone-
azole-3-yl piperidine, reminiscent of the aryl (5931, where hydroxylation of the six-mem-
piperidines in the potent D, blocker, haloper- bered imide ring showed somewhat decreased
idol, was substituted. Clinical experience with D, receptor affinity relative to tiasperone buT
risperidone has confirmed its antipsychotic ef- had similar 5-HT, receptor affinity. This com-
ficacy with reduced propensity to induce EPS pound exhibited good inhibition of the condi-
(589). Similar effects are observed with ocap- tioned avoidance response in rats and did not-
eridone (1311, a compound structurally re- induce catalepsy. A series of thieno[3,2-clpyri-
lated to risperidone but with a D,/5-HT, ratio dine and furo[3,2-clpyridine compounds
of only 5 (590). structurally related to tiosperone have similar
Another member of the benzisoxazolyl pip- profiles and demonstrate that a higher 5-HT,
eridine class is HP-873 (132), which has a fa- affinity can compensate for high D, affinity
vorable D,/5-HT, ratio of 17. In contrast with (594). These compounds block conditioned
the typical antipsychotics, HP-873 also in- avoidance responses in rats despite weak D,
creases social interaction in rats, suggesting affinity (Ki 115 nM) provided that potent
an ability to ameliorate negative symptoms 5-HT, affinity was present. They also did not
(591). induce catalepsy, again suggesting an atypical
Sertindole (94) was selected from a series of profile. Using a variant of the spiro-imide side-
N-phenylindolyl-piperidines and has Ki7sof chain in tiosperone, the effect of varying the
0.39 and 4.1 nM at the 5-HT,, and D2recep- aryl group of the arylpiperazine demonstrated
tors, respectively, and a favorable ratio of 10.5. that a 1,2-benzisothiazolegroup was superior
Sertindole blocks the hyperactivity in rats in- over other aryl groups in both in vitro and in
duced by dopamine infusion into the nucleus vivo testing and retained a favorable DJ5-HT,
accumbens without causing the rebound hy- affinity ratio of 7. The structurally related
peractivity after cessation of treatment seen compound in which the 1,2-benzoisothiazole
with typical antipsychotics. Although sertin- nitrogen has been replaced by C-H to yield
dole has a low propensity to induce catalepsy, the corresponding benzothiophene, showed a
both the direct analog in which the chloro sub- similar in vivo profile with 5-HT, affinity (Ki
stituent is replaced by cyano and also the cor- = 20 nM) and a D,/5-HT, affinity ratio of 26
responding piperazine and tetrahydropyri- (595). SM-9018 (134) is another variant with
dine analogs have more classical D,/5-HT, the 1,2-benzothiazolylpiperazineimide struc-
ratios favoring D, affinity and thus potently ture and shows affinity for D, (Ki = 5 nM) and
induce catalepsy (592). 5-HT, (Ki = 0.61 nM)receptors, in vivo block-
A series related to risperidone, which was ade of dopamine-mediated behaviors, and a re-
not designed to incorporate 5-HT,, antago- duced tendency to induce catalepsy (596). Be-
nist activity, resulted in tiaspirone (133).This cause the compound also has potent 5-HT-,,
analog has a D,/5-HT, ratio of 21, similar to receptor affinity (Ki = 2.9 nM),it is possible
that found for risperidone. The behavioral that this also contributes to the in vivo profile
profile of tioperone was closer to clozapine (597, 585).
Antipsychotic Agents
cles linked by two atoms or imides linked by presumably because of sertindole's effect at
four atoms, seem to have validated the SDA voltage-gated I, ion channels that control
hypothesis and added an important role for heart rythm (607). Sertindole is contraindi-
partial agonism at the 5-HT,, receptor (602, cated in patients with a long QT interval or
603). Ziprasidone has been marketed in Swe- those receiving drugs known to prolong the
den since 2000 and in the United States since QT interval (608,609). Development of sertin-
2001 as an oral and injectable atypical antipsy- dole has been suspended in most countries,
chotic. The EPS that would otherwise be ex- and it remains unclear whether sertindole will
pected from its high D, antagonist affinity is be approved in the United States or reintro-
probably minimized by its potent antagonist duced in Europe.
affmity at 5-HT,, receptors (599,602) and its A series of 6-aminoalkyltetrahydroindol-4-
potent, partial agonist affinity at 5-HT,, re- ones related to molindone (91) show potent
ceptors (604,605). affinities for D, and 5-HT,, receptors (610).
Molindone exhibits many similarities to typi-
8.1.6 Arylpiperazines/Piperidines. Risperi- cal neuroleptics, including D, antagonism, an-
done, a potent SDA, is a highly successful tipsychotic efficacy, and EPS (611). Zotepine
atypical antipsychotic drug introduced to clin- (136) is a potent D, and 5-HT,, antagonist
ical practice in the mid-1990s. It treats the with high affinity for the 5-HT, receptor.
8 Strategies for Drug Discovery 649
mine neurons (638). Unlike other dopamine has a high affinity for 5-HT, receptors rekin- -
inactive in models of EPS liability (659). The serin, fananserin, and MDL 100907, it seems
ineffectiveness of fananserin (2) as well as the that 5-HT,, receptor antagonism by itself is
selective 5-HT,, antagonist ritaneserin as an- insufficient to produce robust antipsychotic
tipsychotics would tend to rule out 5-HT,, effects. The ability of 5-HT,, antagonism to
and D, antagonism as sufficient targets by diminish EPS induced by D, blockade and
themselves for antipsychotic drug discovery, treat anxiety associated with the disease (24,
and reinforces the critical presence of D, 580, 584, 667, 668) are likely components of
antagonism. atypical antipsychotic agents.
One sufficiently selective agent has arisen 8.2.1.2 5-HTIA Partial Agonists. Like
to test the hypothesis that 5-HT,, receptor 5-HT,, antagonism, 5-HT,, receptor agonism
antagonism alone can treat psychosis. MDL can produce anxiolytic effects and diminish
100907 (146) is an N-phenethylpiperidine de- the catalepsy induced by D, antagonists in ro-
rivative with at least 100-fold higher affinity dents (111, 597, 646) and primates (3). The
(1.5 nM)at human 5-HT,, receptors than at specific role of 5-HT,, agonism in these effects
human D, and 5-HT,, receptors (660). was confirmed by the ability of selective
MDL100907 exhibited an antipsychotic profile 5-HT,, antagonists to block the 8- OH,DPAT-
in a number of animal models for schizo- induced reversal of catalepsy (585, 597). The
phrenia. As with atypical antipsychotics, median raphe nucleus seems to be a critical
MDL100907 decreased hyperactivity induced site for this action, because infusions of
by amphetamine (661) and by PCP (662). It 8-OH,DPAT into the raphe but not into stria-
also decreased the deficits in prepulse inhibi- tal projection areas can reverse the catalepsy
tion induced by MK-801 (663) and failed to induced by the D, antagonist raclopride (645).
induce catalepsy, normally a predictor for Elevations in serum prolactin produced by
EPS. Positron emission tomography (PET) haloperidol can also be attenuated by partial
studies conducted in humans by Wong et al. 5-HT,, agonists (669). Intrinsic 5-HT,, re-
showed that low plasma levels of MDL100907 ceptor agonist activity is a property of mainly
caused by a single oral 20 mg dose produced the newest antipsychotic drugs. These include
the desired high degree of cortical 5-HT,, re- nemonapride (5971, ziprasidone (195, 670,
ceptor occupancy (664). 6711, and aripiprazole (6281, and the original
In an initial confirmation of the preclinical 5-HT,, partial agonist, clozapine (672). An-
findings, a phase I1 clinical trial using 10-40 other compound, PD 158771 (147) (673), was
mgofMDL 100907 per day for 6 weeks showed shown by Akunne et al. to be a D2D3 partial
improved Parkinson rating scale (BPRS) agonist and 5-HT,, agonist with preclinical
scores relative to placebo and no EPS liability properties that are very similar to those of
(665). A larger phase I11 trial showed some aripipirazole. This profile is consistent with
minor improvement in positive symptoms at the lack of elevations in serum prolactin seen
the 10-mg/day dose, but this effect disap- in clinical trials with the mixed DJBHT,,
peared at the 20-mgJday dose, compared with partial agonist aripiprazole (269,270) and the
placebo. Functional PET scans of selected pa- D, antagonistl5-HT,, partial agonist ziprasi-
tients in the trial showed minor metabolic done (195, 670, 671, 674).
changes in the frontal cortex at the 10 but not Among these drugs, clozapine, aripipra-
20 mg dose. Clinical trials of MDL100907 have zole, and PD 158771 can decrease dopamine
been halted, presumably because of limited ef- and serotonin synthesis in the neostriatum,
ficacy (666). Between the findings with ritan- and most of these drugs share a similar behav-
8 Strategies for Drug Discovery
ioral profile suggestive of low EPS liability be constructed from either doublets ( N R l d
(645, 673, 675). Again consistent with partial NR2y) or triplets (NRlx/NRZy/NRZz or N R l d
.
-
5-HT,, agonism (and 5-HT,, antagonism), NR2y/NR3) of these subunits (678). A glycine
some of these compounds are anxiolytic in pre- modulatory site resides on the NR1 subunit,
clinical tests including the Vogel conflict test, and its occupation is required for receptor ac-
social contact test, and the elevated plus maze. tivation (679). A inhibitory site, identified by
As with aripiprazole (232), most of these com- the binding of ifenprodil, is present on recep-
pounds are also weaker at inducing catalepsy tors containing the NR2 subunit (680).
or blocking apomorphine-induced stereotypy The noncompetitive NMDA antagonist ion
than in preventing apomorphine-induced channel blockers such as PCP (148) and the
climbing. It may be that the potent, partial competitive antagonists such as CGS 19755
5-HT,, agonism of these compounds miti- (149) are both psychotomimetic in humans
gates against D, antagonist effects, including
the propensity to produce EPS (597,647,649,
675) and elevation of serum prolactin (669).
Partial 5-HT,, agonism may also lessen the
depression and anxiety that frequently accom-
pany psychosis (662, 668, 672). The effective-
ness of ziprasidone or clozapine against the
negative symptoms of schizophrenia may also
derive from their intrinsic partial 5-HT,, ago-
nism, which mediates the selective increases
in frontal cortex dopamine release produced
by either drug (647).
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CHAPTER ELEVEN
Neurodegenerative Conditions
JAMES DAVID ADAMS
JR.
Department of Molecular Pharmacology and Toxicology
USC School of Pharmacy
Los Angeles, California
THOMAS F. WOOLF
HyBar BioScience, LLC
Ann Arbor, Michigan
Contents
1 Introduction, 674
1.1 Alzheimer's Disease, Parkinson's Disease,
Amyotrophic Lateral Sclerosis, Multiple
Sclerosis, and Other Neurodegenerative
Conditions, 674
1.2 Stroke and Other Ischemic Conditions, 675
2 Necrosis and Apoptosis, 676
3 DNA Damage and Repair, 677
3.1 DNA Damage, 677
3.2 DNA Repair, 677
3.3 PARP and DNA Repair, 677
4 Cellular Energetics, 679
4.1 PARP Inhibitors, 680
4.2 NAD Glycohydrolase Inhibitors, 686
4.3 NAD Precursors and Cellular Energetics,
687
5 NMDA Receptor Antagonists, 689
6 Nitric Oxide Synthase Inhibitors, 695
Recent Citations for Drugs Used in Alzheimer's Disease, Amyotrophic Lateral Sclerosis (ALS),
and Multiple Sclerosis (MS).Ache Refers to Acetylcholinesterase.
Agent First Author Citation
- --
why not use a drug that induces hypothermia? damage, and other conditions and may be
Hypothermia is routinely used in open chest caused by temporary interruption of blood
surgery, where patients are mechanically flow to areas of the brain. Infarction produces
cooled to enhance survival. Intracranial pres- a large area of dead tissue that eventually may
sure may increase in patients after stroke. It is form an extensive glial scar. In stroke, infarc-
known that increased intracranial pressure is tion involves necrotic and apoptotic cell death
dangerous in patients suffering from trau- (2,3).The majority of the cell death in the core
matic brain injury. It is routine to monitor of the infarction is necrotic. However, apopto-
intracranial pressure in traumatic brain dam- tic cell death also occurs in the core and is
age patients and to decrease intracranial pres- more prominent in the limit area surrounding
sure by withdrawing cerebrospinal fluids. Yet, the core, sometimes called the penumbra. The
it is not routine to monitor or correct intracra- term limit will be used in this chapter. Penum-
nial pressure in stroke patients. There are bra is a term originally used to refer to an area
drugs available that are known to decrease in- that is not as ischemic as the core and that
tracranial pressure. Yet, they are not used in reperfuses more readily than does the core.
the treatment of stroke. The point here is that The penumbra disappears during reperfusion.
it is the patient that counts. Too often thera- Eventually, the inner part of the penumbra
peutic strategies are neglected because it is may become part of the core of the infarction.
decided that the drug therapy induces an arti- The outer penumbra becomes the limit area
factual effect that is not an intended effect. where apoptosis is prominent. Necrosis is also
For instance, if a drug inhibits excitotoxic seen in the limit area. The point here is that
mechanisms and also induces hypothermia, necrosis must be prevented in order to prevent
the drug may be neglected because the hypo- extensive infarction. However, apoptosis pre-
thermic effect is considered artifactual. Yet, vention may also be important.
such a drug could be a real benefit to patients. Cells die in the brain by two main pro-
It is important to realize that it is the survival cesses, necrotic and apoptotic. However, some
of the patient that is important. authors find other forms of cell death in the
The overall goal of therapeutics with these brain, such as oxytosis. Necrosis is a rapid pro-
drugs is to prevent the death of cells in the cess in which the defense mechanisms of the
brain. Of course it is important to protect neu- cell are overwhelmed. The cell nucleus swells
rons because they do not normally regenerate and ruptures. Cellular mitochondria may
well. However, stem cells in the brain may al- swell. The cytoplasm swells and forms small
low some neuronal regeneration. Endothelial vacuoles. The cell membrane may eventually
cells maintain the blood-brain barrier and rupture. Necrosis may be maximal in the
must be protected if the brain is to survive. brain at about 6-12 h after an insult.
Astrocytes support neuronal activity by deac- Apoptosis is delayed and may be maximal
tivating neurotransmitters, providing trophic at 24-48 h after an insult. However, apoptotic
factors and other activities. Therefore, astro- cells can be seen even within 6 h of an insult,
cytes must be protected if the brain is to sur- but are not common. Apoptosis involves con-
vive. Oligodendrocytes make myelin that is densation of the nucleus. Condensation of the
vital for axonal survival. Therefore, oligoden- cytoplasm occurs with large vacuole forma-
drocytes must be protected. It is obvious that tion. Some mitochondria may condense. Very
all of the cells of the brain are vital to brain uncommonly, some mitochondria swell in ap-
survival and should be protected if possible. optosis. The cell splits up into membrane-
bound apoptotic bodies that can be seen in the
limit areas surrounding the core of an infarc-
2 NECROSIS A N D APOPTOSIS tion. Apoptosis is a programmed form of cell
death that occurs following a small but suffi-
Infarction of the brain is involved in stroke, cient amount of damage to a cell. This initiates
ruptured berry aneurysm, traumatic brain a program that eventually kills the cell.
3 DNA Damage and Repair
and the nuclear location signal. The middle very similar to those found in the bacterial
section contains the automodification area. toxins that are mono(ADP-ribosyl) trsms-
The C-terminal contains the active site that ferases (6).
binds NAD (Fig. 11.1). The active site has a Several amino acids are involved in the ac-
nicotinamide-binding site and an adenine- tive site of PAW: Glu988, Tyr896, Ala8198,
binding site. Lys903, His862, Ser904, Gly863, and Tyr'907
The crystal structure of the active site of (6). A crystal structure for NAD bound to
PARP has been described (5). It is made up of PARP is not available because the complex is
a five-stranded antiparallel beta sheet and a unstable. However, a model system of N'AD
four-stranded mixed beta sheet (Fig. 11.2). bound to diphtheria toxin, a mono(ADP-riibo-
The two beta sheets are connected by two hy- syl) transferase, has yielded a useful cry;jtal
drogen bonds. The beta sheet structure is sup- structure (7). NAD binds with the ribose in a
ported by a surrounding protein structure 3'-endo conformation and the nicotinamidce in
made up of five alpha helices, three 3,, helices, a syn position (Fig. 11.3). Several hydro;gen
and beta sheet excursions. The active site is bonds and hydrophobic interactions occur be-
Figure 11.2. Ribbon representation of the PARP active site. A strongly conserved motif that binds
NAD (black) is shown in gray. This figure is from Ref. 5 and is used with permission.
dlular Energetics
Figure 11.3. Stereo view of atomic interactions of NAD and a model of the PARP site. NAD is shown
in black. The PARP site amino acids are shown in gray. Hydrogen bonds are shown as dashed lines.
This figure is from Ref. 7 and is used with permission.
twe~ en the enzyme and the substrate. The hy- proteins, making them anionic. This estab-
drof;en bonds are as follows: Gly876, Asp770, lishes an electrostatic repulsion from nega-
and Arg878 bond to the adenine; Ser864 or tively charged DNA.
His:562 bonds to the ribose connected to the PARP is a family of enzymes including
adeinine; Asp766, Gln763, and Tyr896 bond to PARP-1, PARP-2, PARP-3, tankyrase, and V-
the phosphates; Gly863 or Ser904 bond to the PARP (8).PAW-1 is perhaps the most impor-
niccjtinarnide; and Tyr907 and Glu988 bond to tant enzyme involved in DNA repair in stroke.
the ribose connected to the nicotinamide. Hy- However, if PARP-1 is deactivated, perhaps
dro]~hobicbonds are as follows: Leu877 and the other enzymes can take its place. The
Ile872 bond to adenine; Tyr907 bonds to nico- PARP inhibitors described so far in the litera-
tina.mide;and T~I-896, which may bond to the ture are PARP-l inhibitors. It is not known
riboise connected to the nicotinamide. Glu988 whether these compounds also inhibit the.
ma) be involved in the catalytic mechanism by other forms of PARP.
polarizing the NAD and the ADP-ribose accep-
tor 1through hydrogen bonding. This polariza-
tion would stabilize the NAD transition state 4 CELLULAR ENERGETICS
and increase the nucleophilicity of the accep-
tor. The NAD transition state involves a ri- During ischemia, oxygen levels diminish, thus
bosf:oxocarbenium ion that leads to cleavage shutting down some mitochondrial functions
in tin S,2 mechanism. and greatly decreasing adenosine 5'-triphos-
I)NA can be stabilized under potentially phate (ATP) levels. When oxygen reperfuses
dam aging conditions by PARP inhibition. into the brain, ATP levels rise to normal levels
Whf?nDNA damage occurs, PARP is activated within about 5 min. ATP is required for cellu-
and binds to DNA, thus stabilizing it. PARP lar energetics, for oxidized glutathione
inhibitors cause PARP to remain bound to (GSSG) reduction, for DNA repair, and for
DNI4, thereby providing long-term stabiliza- many other functions. Therefore, without
tion, even when oxygen radical generation oc- ATP, DNA repair cannot proceed. The cell
C U EI. After the generation of oxygen radicals must turn to other ways to make ATP until
has ceased, the PARP inhibitor can dissociate oxygen returns and mitochondrial function re-
fronI PARP, allowing the DNA to be repaired. turns.
ASter enough DNA repair has occurred, PARP depletes cellular energy resources. It
PAF1P causes the detachment of the repair does this by using NAD as a substrate. When
machinery and of PARP itself from the DNA. DNA damage is severe, such as during isch-
PAF1P does this by poly(ADP)-ribosylating emia and reperfusion, NAD levels can become
680 Investigative Agents for Use in Neurodegenerative Conditions
Several inhibitors are known that bind to the biochemical pathways (22). Recent attempts
adenosine site of PARP (19). These inhibitors to make more specific and more potent inhib-
include 5-bromo-2'-deoxyuridine,caffeine, 5- itors of PARP have centered around making
bromouracil, diadenosine-tetraphosphate, 1- conformationally restricted analogs. For in-
methyladenine, 5-nitrouracil, theophylline, stance, the 5-substituted dihydroisoquinolino-
theobromine, thymidine, and other compounds. nes below are restricted to anti conformations
These compounds are not as well studied as the and are PARP inhibitors. However, the 7-sub-
nicotinamide analogs. In addition, it is not stituted dihydroisoquinolinonesare restricted
known whether these compounds can interact to syn conformations and are inactive. 5-Meth-
with the adenosine (A,) receptors that are in- yl-3,4-dihydroisoquinolinone (PD128763) is
volved in modulation of synaptic transmission being investigated for use in stroke and other
and neuroprotective effects. conditions. Several isoquinolinones appear to
Inhibitors based on the structure of nico- be good inhibitors of PARP (21), with signifi-
tinamide or benzamide have been reported to cant inhibition at concentrations of 10 pM or
have toxicity problems arising from the fact less (Fig. 11.7).
that some of them may be antimetabolites for Benzoxazoles have become of interest be-
NAD synthesis or interact with other NAD cause they may have intramolecular hydrogen
Investigative Agents for Use in Neurodegenerative Conditions
Figure
- ,5. PARP inhibitors: 1,5-
dihydroxyisoquinoline, 5-aminoiso-
quinolin-l(2H)-one, 0-(2-hydroxy-3-
piperidinepropy1)pyridine carbonic
acid amidoxime, 6(5H)-phenanthridi- 0
none, 3,4-dihydro-5-[4-(1-piperidinyl)
butoxyl-l(2H)-isoquinoline,2,B-dimeth-
yl-3-hydroquinazolin-4-one,8-methyl-2-
(p-nitrophenyl)-3-hydroquinazolin-4-
one, 5-iodo-6-aminocoumarin. These I
inhibitors are from Ref. 11 and have CH3
been redrawn with permission.
palmitic acid, palmitoleic acid, stearic acid, ment of pellagra. Before nicotinamide, the
thymidine, theophylline, linoleic acid, vitamin mortality from pellagra was 30% in the United
K2, and vitamin K1 (25, 27,28,29). States. About 10,000 deaths occurred every
year, mostly caused by neurodegeneration. Af-
4.3 NAD Precursors and Cellular Energetics ter the introduction of nicotinamide in 1938
The most potent neuroprotective agent used (301, the mortality from pellagra decreased to
clinically is nicotinamide, used in the treat- nearly 0%. Of course, pellagra is caused by nic-
otinamide, or niacin, deficiency. Both nicotin-
amide and niacin are called vitamin B3. Pella-
gra is a disease of NAD deficiency.
Nicotinamide, but not niacin, is taken up
into the brain by an active uptake process (31)
and is converted into NAD (Fig. 11.9). Niacin
released in the brain from catabolic processes
is converted into nicotinamide as shown. Nic-
otinamide can increase brain levels of NAD by
50% or more (32). By increasing brain NAD
levels, nicotinamide prevents ATP depletion
(9) and protects cellular DNA (32). Therefore,
nicotinamide maintains cellular energetics in
the presence of oxidative stress.
Many compounds have been investigated
that may maintain cellular energetics, espe-
cially in pellagra (Fig. 11.10). All but one of the
compounds found to be active can be con-
Figure 11.7. Isoquinolinone inhibitors of PARP verted into NAD in the body (33). This is im-
from Ref. 21. portant because compounds that make NAD
688 Investigative Agents for Use in Neurodegenerative Conditions
Compounda Structure R X
OH
Me
Orne
Me
Orne
Me
Orne
Me
Orne
Me
Orne
Me
Orne
Orne
Me
Orne
Me
OH
OH
OH
Me
OH
"Data are used with permission from Ref. 23 (0 1998 American Chemical Society).
*3AB is 3-aminobenzamide; 3HB is 3-hydroxybenzamide; QN is quinazolin-4(3H)-one;PD128763 is 5-methyl-3,4dihy-
droisoquinolinone.
analogs in the body may be toxic and interfere zymes. The other active compounds can be
with normal biochemical processes. However, converted in the body into nicotinamide by es-
compound (21) may make NAD analogs in the terases, amidases, decarboxylation, and
body and has been reported to be active in methyl oxidation to form carboxyl, N-dealky-
NAD deficiency. Perhaps the NAD analogs lation, and other processes. Tryptophan can
from (21) are not toxic and can substitute for be converted into nicotinamide in the body.
NAD in normal biochemical processes. This The pathway is tryptophan, kynurenine, 3-hy-
seems unlikely, given that the Cposition of the droxykynurenine, 3-hydroxyanthranilic acid,
NAD pyridine ring is involved in hydride l-amino-4-formyl-l,3-butadiene-1,2-dicar-
transfer reactions catalyzed by several en- boxylic acid, quinolinic acid, niacin, and nico-
1 5 NMDA Receptor Antagonists
k
i
I
I
114 0 N H 2 x
11 < ~& C O N ~H ~ ~ ~ ~
,YZ NO2 'Y' NH2
tinamide. Oral NAD is active because it is two subunits, NR1 and NR2, and multiple
cleaved in the gut to make nicotinamide. binding sites for ligands and inhibitors. The
receptors are glutamate receptors. Glutamate
5 NMDA RECEPTOR ANTAGONISTS is released during ischemia such that activa-
tion of NMDA and other excitatory amino acid
NMDA receptors are ligand-gated ion &an- receptors may be involved in damage caused
nels that allow the influx of calcium upon li- by ischemia and reperfusion. The binding sites
gand binding. The receptors contain at least include sites for AMPA, glycine, phencyclid-
CONH2 COOH
1
N Nicotinamide N Niacin
1
CONH2
0 N
I
Nicotinamide
mononucleotide
..
I mononucleotide
Figure 11.9. NAD synthesis pathway
Ri-P Ri-P from nicotinamide or niacin: (1) nicoti-
namidase (E.C.3.5.1.19); (2) nicotinamide
phosphoribosyl transferase, which re-
quires ATP; (3) NMN adenyl transferase,
which requires ATP; (4) nicotinic acid
phosphoribosyl transferase, which re-
quires ATP; (5) NMN adenyl transferase,
which requires ATP; (6)NAD synthetase,
which requires ATP. Ri, ribose; P, phos-
phate; Ad, adenine; NMN, nicotinarnide
Ri-P-P-Ri
mononucleotide. This figure is from Ref.
NAD 33 and is used with permission.
Investigative Agents for Use in Neurodegenerative Conditions
COOH
COOH
aCooH
N
(7)
COOH
n C
N O N H " '
I
glucosiodide
(20)
Figure 11.10. Nicotinamide analogs tested in pellagra or animal models of pellagra. The active
compounds include (1,2,6, 11, 12,13,15,18,and 20). Compounds active in some models or in
pellagra include (7,17,21),pyrazine-2,3-dicarboxylicacid, NAD, pyridyl-3-aldehyde, pyridyl-3-carbi-
nol, tryptophan, and 3-hydroxyanthranilic acid. The inactive compounds are (3,4,5,8,9,10,14,16,
19),3-arninopyridine, thiazole-5-carboxylic acid, 2-methylpyridine, 3-methylpyridine, 2,6-dimethyl-
pyridine-3,5-dicarboxylic acid, pyridine-3,5-dicarboxylicacid, kynurenine, 3-hydroxykynurenine,
and formylkynurenine. This figure is from Ref. 33 and is used with permission.
5 NMDA Receptor Antagonists
COOH
H2N
c COOH
COOH
F
ine, kainic acid, polyamines, and other binding 11.11). These compounds were synthesized
sites. AMPA is ~~-c~-amino-3-hydroxy-5-meth- because it was noticed that several competi-
ylisoxazole-4-propionic acid. Compounds that tive inhibitors of the NMDA receptor had al-
alter glutamate release and thereby alter pha-amino carboxylic acid and phosphonic '
NMDA channel activation are known. Activa- acid functionalities separated by 3-5 carbons.
tion of NMDA receptors is associated with ox- All of the inhibitors shown in Fig. 11.11 have
ygen radical formation and activation of nitric Ki values for inhibition of the NMDA receptor
oxide synthase. of 10 p M or less. In general, nitrogen substitu-
Several NMDA receptor antagonists have ents larger than those shown decrease recep-
been synthesized and tested in stroke models, tor affinity. In addition, substitution on the
epilepsy models, and in clinical trials. Many of second nitrogen decreases affinity.
the antagonists are limited by side effects such A series of N-phosphonoalkyl and N-(phos-
as hemodynamic abnormalities, hypotension, phonoalky1)phenyl-spaced alpha-amino acids
neuronal vacuolation, memory disturbances, have been explored as NMDA receptor antag-
cognitive disturbances, motor dysfunction, onists (35). The compounds are competitive
seizures, hallucinations, unpleasant dreams, inhibitors and are shown in Table 11.3. A
psychotomimetic episodes, and other effects. folded conformation of the inhibitors seems to
This has made the search difficult for NMDA be favored that places the phosphonic acid and
receptor antagonists that can be used as neu- the alpha-carboxylic acid moieties in close
roprotective agents. proximity in the receptor. The phosphonoal-
Many NMDA receptor antagonists have kyl group must be long and flexible enough to
been synthesized that are based on the struc- allow the phosphonic acid to stretch across the
ture of glutamate, NMDA, and AMPA. Several molecule and fit into the acidic binding site of
3,4-diamino-3-cyclobutene-1,2-dionederiva- the receptor. Addition of too much length cre-
tives (34) have been synthesized and found to ates excessive bulk that hinders the fit of the
be good inhibitors of the NMDA receptor (Fig. molecule into the receptor.
692 Investigative Agents for Use in Neurodegenerative Conditions
I
COOH
HN\ /
H
HOOC, ,NH I
C
I
H
C
A
Structureb Position, Xb n m R % Inhibc Ic,o (rUM)
(A) o 0 0 (C0OH)l H 4
(A) o 0 0 H 27
(A) o 0 1 H 48
(A) 0 0 2 H 0
(A) 0 0 2 (ElZ H 9
(A) 0 1 1 H 21
(A) 0 1 1 CH3 4
(A) m 0 2 H 0
(A) m 0 3 (E)' H 13
(A) m 0 3 H 5
(A) m 1 0 H 21
(A) m 1 1 H 2.4
(A) m 1 1 CH3 31
(A) m 1 1 CH(CH& 0
(A) m 2 1 H 13.7
(A) P 1 1 H 2.3
(B) - 2 0 H 14
(B) - 3 0 H 3
(B) - 4 0 H 17
(B) CH=CH(E)' 1 1 H 1.0
(B) CH=CH(E)' 1 1 CH3 42.4
(B) CH=CH(ZE)' 2 0 H 2.3
(B) S 2 1 H 60
(B) S(0) 2 1 H 4
"Data are adapted from Ref. 35 with permission (0 1992 American Chemical Society).
*Position,Xrefers to, for structure A, the position of the phosphate substituent on the phenyl ring, or for structure B, the
nature of X.
'% Inhib refers to the percentage inhibition at 100 a. (1) The phosphate is replaced by COOH; (2) the geometry of the
double bond is entgegen or a combination of entgegen and zuzammen.
may be of interest in clinical trails against tion between activity and peptide tertiary
stroke. These compounds are based on the structure. The ability of these peptides to
structures of benzoCblquinolizinium cations modulate the NMDA receptor suggests there-
and dizocilpine, which have been clinically may be endogenous peptides that perform the-
tested against stroke. same function.
A family of peptides has been found in ma- Bis(pheny1)guanidines have been explored
rine snails of the Conus genus that are NMDA as NMDA receptor antagonists (39). These
receptor antagonists by virtue of their interac-compounds are similar to N-l-naphthyl-N1-(3-
tions at the polyamine site (38). These pep- ethylpheny1)-N'-methylguanidine(aptiganel,
tides apparently can cross the blood-brain bar- below) that has been tested in clinical trails for
rier and cause ataxia, respiratory paralysis, traumatic brain damage and stroke. Many of
and death. They are noncompetitive inhibi- these compounds are potent NMDA receptor
tors of polyamine responses at NMDA recep- antagonists, but are also sigma receptor an-
tors. However, the peptides do not produce the tagonists. The sigma receptor is not well un-
same effects as those of other polyarnine inhib-derstood. Some sigma receptor antagonists
itors, such as arcaine, ifenprodil, and 1,lO-dia-
may block calcium entry into cells and prevent
minodecane, which implies that the peptides apoptosis (40). These receptors may modulate
act at a novel polyamine site on the NMDA NO synthase activity and appear to bind
receptor. Conantokin-G is the model peptide neurosteroids. However, the effects of sigma
in this series and is Gly-Glu-Gla-Gla-Leu-Gln- receptor antagonists may be dose related,
Gla-Am-Gln-Gla-Leu-Ile-Arg-Gla-Lys-Ser-An- causing potentiation of NMDA receptor activ-
NH,. The peptide contains several gamma- ity at low doses and inhibition of NMDA recep-
carboxyglutamate (Gla) residues that make it tor activity at high doses (41). Some sigma re-
resistant to peptidases. Conantokin-G has an ceptor antagonists have been found to be
IC,, of 0.2 pM in NMDA receptor assays. neuroprotective. However, several of the bis-
Many peptides were synthesized with one (pheny1)guanidines were found to have higher
amino acid difference compared to that of co- affinity for the NMDA receptor than for the
nantokin-G. Only the peptide with an Ala at sigma receptor (Table 11.5). For R1 substitu-
position 7 instead of Gla had enhanced activity ents, iPr and F do not produce NMDA recep-
(IC,, = 0.05 pM). All other amino acid substi- tor-selective agents. In general, for R2 and R3,
tutions either did not enhance or decreased one or both of the two substituents should be
activity. Although conantokin-G possesses al- SMe or CF, to retain NMDA receptor selectiv-
pha-helicity, there was no apparent correla- ity.
694 Investigative Agents for Use in Neurodegenerative Conditions
A\/B
OEt Oet
OEt Oet
OEt Oet
OPr OP~
OEt Oet
OEt Oet
OEt Oet
OEt Oet
OEt Oet
OEt Oet
3-C4H30 3-C4H30
3-C4H30 3-C4H30
3-C4H30 3-C4H30
3-C4H30 3-C4H30
3-C4H30 3-C4H30
3-C4H30 3-C4H30
3-C4H30 3-C4H30
3-C4H30 3-C4H30
C6H5 C6H5
-
"Data are adapted from Refs. 36 and 37 with permission (01994, 1995 American Chemical Society).
SMe H
Et Et
Et Srne
Et Srne
Et Srne
CF3 Srne
CF3 Srne
SMe Et
SMe Et
SMe Srne
SMe Srne
SMe Br
0CF3 Et "Adapted from Ref. 42 with permission (0 1999 Amer-
Br Srne ican Chemical Society).
"Adapted from Ref. 39 with permission (63 1997 Amer-
ican Chemical Society).
*These compounds have Kivalues for inhibition of the placed with several other side-chains with re-
NNDA receptor that are at most 10 times lower than their tention of activity, such as methyl, ethyl, pro-
ICbOvalues for inhibition of the sigma receptor. pyl, butyl, pentyl, and hexyl. These side- .
chains must be unbranched, given that
site antagonist. However, more potent glycine branched side-chains may have no activity.
site antagonists are the brominated and the This implies steric restrictions exist in the re-
trifluoromethylated phosphoalanine deriva- ceptor for these side-chains. A variety of fluor-
tives. In general, increasing the size of the R inated hydrocarbon side-chains can be added
substituent tends to decrease activity at the in place of trifluoromethoxy, with retention of
glycine site. activity. These side-chains must be at the
6-position to be active. Perhaps a lipophilic re-
ceptor site exists that can interact only with
6-substituents. Many different substituents
can be placed on the ring nitrogen with reten-
tion of antiseizure activity. It is very possible
Riluzole that many of these side-chains are metaboli-
cally removed upon injection into animals.
Riluzole is an NMDA antagonist with ac-
tions at the pre- and postsynaptic levels. It is 6 NITRIC OXIDE SYNTHASE INHIBITORS
currently being used in amyotrophic lateral
sclerosis patients. Riluzole also inhibits gluta- Nitric oxide synthase (NOS) is a family of en-
mate-induced convulsions. Several riluzole zymes consisting of at least three isoforms, in-
analogs have been synthesized and tested for ducible (iNOS) found in macrophages and
inhibition of glutamate-induced seizures (44). other cells, neuronal (nNOS) found in neu-
The trifluoromethoxy side-chain can be re- rons, and endothelial (eNOS) found in endo-
Found to be Active at AMPA (ICs, in p M ) and Glycine (MDL, ICso in p M l Sites
Table 11.7 5-Amino-quinoxaline-2,3-diones
and Found to Have Anticondant Activity (ESM in mglkg)"
R
HOOcQN
(lb) NO,
H O O C ~ N H
HOOC ( 1 ~ ) NO,
CNH
HOOC- NH (Id) NO,
(le) NO,
H O O c NH
~
HOOC-NH (10 NO,
---
HOOc+
~NH (lg) NO,
Oh) NO,
.OOYNH
I
H O O c ~ N H
CH300
YNH +
%
HOOC- NH
HOOC-NH
.
--
H O O c ~ ""
HOOC
CNH
HOOC-S
HOOCVO
OI
rD
4'
(18a) NO, HOOCVO
(18b) NO,
H O O c ~ O
thelial cells throughout the body. Induction of are important in neurodegeneration. Inhibi-
iNOS depends on exposure to cytokines and tors based on the structure of arginine include
other stimuli and is important in the immune p-methyl-L-arginine,p-nitro-L-arginine and
response and other defense mechanisms. its methyl ester, p-amino-L-arginine, N-6-
Blood flow to inflamed tissues may be con- (iminoethy1)-L-ornithine, and others.
trolled by iNOS. Blood pressure regulation N-Phenylamidines are known to reversibly
and antithrombosis responses involve eNOS. inhibit nNOS (46). Slight structural varia-
Neuromodulation involves nNOS. Both eNOS tions can switch isoform specificity with these
and nNOS are constitutively expressed iso- compounds as seen with the two acetamidine
forms. structures shown below. N-(3-(Aminomethyl)-
NOS is a homodimer and requires dimer- phenyllacetamidine, below, was modified by
ization involving calmodulin for activation. changing the aminomethyl side-chain (Table
However, iNOS contains tightly bound cal- 11.8). Ethylamino, methylaminomethyl, and
modulin, such that exogenous calmodulin dimethylaminomethyl side-chains retain nNOS
binding is not required for activation. The en- specificity. A methanol side-chain produces
zyme contains a cytochrome P450 reductase a n nNOS-specific inhibitor. However, acetyl
domain and an oxygenase, heme protein do- and modified acetyl side-chains abolish
main. There are binding sites for NADPH, activity.
FAD, FMN, and tetrahydrobiopterin. The ox- Modification of the acetamidine functional-
ygenase domain oxidizes arginine with the for- ity also produces nNOS-specific inhibitors
mation of citrulline and nitric oxide. Activation (Table 11.9). Several alkyl side-chains were
of the enzyme requiresdimerization because the found to be effective, including aminomethyl,
flow of electrons from the redudase occurs to fluoromethyl, 2-pyridyl, 2-furanyl, and 2-thie-
the tram oxygenase. Tetrahydrobiopterin facil- nyl. This may imply that the acetamidine side-
itates dimerization, stabilizes the enzyme, shifts chain is involved in hydrophobic interactions
the iron to a high spin state, and facilitates elec- with the enzyme that can be enhanced by ali-
tron transfer (45).NADPH supplies electrons to phatic and aromatic groups. The 2-furanyl
the reductase. Electron transfer in the reduc- and 2-thienyl groups may be able to bind in the
tase involves FAD and FMN. NOS makes NO hydrophobic pocket created by Pro336,
that is a reactive radical, especially after inter- Val338, and Phe355 in the active site of nNOS.
action with oxygen when peroxynitrite is Thiocitrulline and other citrulline analogs
formed. Peroxynitrite can damage DNA and have been found to be good inhibitors of NOS
other macromolecules. (47,48). The inhibitors reported so far do not
Many inhibitors have been synthesized seem to have isoform specificity. Thiocitrul-
based on the structure of L-arginine. These in- line is the most potent inhibitor in the series.
hibitors are not isoform specific. Specificity However, its N-hydroxy analog and other an-
may be important in the treatment of neuro- alogs, below, are nearly as potent. The inhibi-
degenerative disorders. It is possible that inhi- tors shown inhibit NOS with IC,, values in
bition of nNOS could be very important in the the range of 10 pM. It has been proposed (48)
inhibition of neurodegeneration. However, that the sulfur or imidazole nitrogen binds to
NO can diffuse long distances in the brain. For the oxygenase heme iron. Nearby on the en-
this reason, it is possible that eNOS and iNOS zyme is a cationic site that binds the amino
6 Nitric Oxide Synthase Inhibitors
H2N
ACOOH H2N
ACOOH
H2N
COOH
H2N
hCOOH
ACOOH ACOOH
H2N H2N
acid nitrogen. An anionic binding site may equally well. However, none of the dipeptides
bind the carboxylic acid. An H-bonding site inhibits NOS as potently as S-L-isothiocitrulline
may bind the amino hydrogen. (IC,, = 0.06 pM for nNOS). The ability of these
Several dipeptides have been synthesized dipeptides to inhibit NOS suggests that there
that incorporate isothiocitruline into the may be endogenous peptide regulators of NOS.
peptide (49). Some of these dipeptides are Imidazole-containing amino acids can
selective for inhibition of nNOS and iNOS make good NOS inhibitors, given that imida-
but not eNOS. These peptides include S-methyl- zole ligates heme (50). The inhibitors display
L-isothiocitrullinyl-L-phenylalanine,S-meth- some isoform specificity (Table 11.10) in that
yl-L-isothiocitrullinyl-L-leucine, S-meth- at least two of them inhibit nNOS with affini-
yl-L-isothiocitrullinyl-L-tryptophan(-CHO), ties at least 10 times greater than their affin-
S-methyl-isothiocitrullinyl-L-phenylglycine, ities for eNOS. The inhibitors with odd num-
S-methyl-L-isothiocitrullinyl-L-glycine, S- bers of carbons in the spacer between the
methyl-L-isothiocitrullinyl-glycine, S-methyl- imidazole and amino acid are the most selec-
isothiocitrullinyl-L-tyrosine, and S-methyl-L- tive inhibitors. However, when a phenyl sub-
isothiocitrullinyl-4-nitro-L-phenylalanine. All stituent is added to the imidazole, all isoform
of these dipeptides inhibit nNOS with IC,, specificity is lost. Perhaps hydrophobic inter-
values of about 10 pM,and they inhibit iNOS actions exist between the enzyme and the hy-
Table 11.8 N-PhenylacetamidineInhibitors of NOSa
Ki (cJM) Selectivity
R Isomer Salt
CH,NH, 3 2HC1
CH,NH, 4 2HBr
NH, 3 2HBr
CH,CH,NH, 3 2HC1
CH,NHMe 3 2HBr
CH,NMe, 3 2HBr
2HBr
CH(0H)Me HBr
CH,CO,H HBr
CH,CO,Me HC1
C0,tBu HC1
CH,C(O)-N-morpholinyl HBr
CH,NHC(O)-N-morpholinyl HBr
H HBr
F HCl
Br HCl
NO, ' HCl
CN HCl
NMe, 2HBr
SMe HC1
OMe HC1
OH HBr
C(0)Me HC1
C0,H HC1
C(O)NH, HC1
SO,NH, HBr
J 2HBr
HBr >50 27
NHNH,
C(NH)NH,
NHC(NH)Me
"Data are reprinted from Ref. 46 (O 1998 American Chemical Society).
702 Investigative Agents for Use in Neurodegenerative Conditions
Ki (CLM) Selectivity
eNOS/ iNOS/
Compound R R' R" Salt iNOS eNOS nNOS nNOS nNOS
drocarbon spacer. An odd number of carbons amine. The m i n e may be able to bind in the
in the spacer may provide the optimal hydro- nNOS active site better than in the eNOS ac-
phobic interactions, while keeping the amino tive sites.
acid in the proper position for binding to the Phenyl-2-aminopyridines have been ex-
anionic and cationic sites. The phenyl analogs plored as potential nNOS inhibitors (52).
may be too bulky for binding to the site with These compounds have some selectivity for
the best orientation of the amino acid portion nNOS over eNOS. They point out that the
of the inhibitor. nNOS active site appears to be less sterically
Several nitroarginine dipeptides have been hindered than the eNOS active site. When the
found to be very selective inhibitors of nNOS side-chain in the compound below is quino-
(51).These peptidomimetic agents, below, ap- line, the IC,, values (@ I)inhibition
for NOS
pear to fit much better into the nNOS active are 0.21 nNOS and 0.83 eNOS; for the
site than into the iNOS or eNOS sites. The PhCOCH, side-chain, the values are 0.14
amide side-chain containing compound below nNOS and 0.89 eNOS; for the PhCH,CO
has Ki(a) values as follows: 0.13 nNOS, 25 side-chain the values are 0.14 nNOS and
iNOS, and 200 eNOS; the compound without 0.69 eNOS; for the PhCH,CH2 side-chain
the amide side-chain has Kivalues of 0.54 the values are 0.26 nNOS and 0.45 eNOS,
nNOS, 100 iNOS, and 199 eNOS; the com- demonstrating almost complete loss of iso-
pound without the amide or the carbonyl has form selectivity. For the iBu side-chain the
Ki values of 0.12 nNOS, 39 iNOS, and 314 values are 0.35 nNOS and 0.37 eNOS, dem-
eNOS. Elongation of the m i n e side-chain onstrating no isoform selectivity. For the
tends to decrease potency and isoform selec-
tivity. All of these compounds are very selec-
tive for nNOS. The fact that the amide and
carbonyl can be eliminated with retention of
nNOS inhibitory activity and isoform selectiv-
ity shows that these functionalities, although
not critical to nNOS binding, may be more im-
portant for binding to iNOS and eNOS. In ad-
dition, elimination of the carbonyl converts
the amide nitrogen to a basic, secondary
i
I
p 6 Nitric Oxide Synthase Inhibitors
K,(rn
Compound iNOS nNOS ENOS
(la) 950 170 500
(lb) 10 2 33
(lc) 35 65 150
(Id) 8 2 50
(le) 40 150 250
(2a) 100 80 50
(2b) 50 100 17
(2c) 120 70 350
"Data are from Ref. 50, used with permission from Elsevier Science.
704 Investigative Agents for Use in Neurodegenerative Conditions
A B
Compound R2 R4 R5 R6 R7 G o (P.M)
aDataare from Ref. 53, used with permission (63 1999 American Chemical Society).
iPrNHCOCH, side-chain the values are 0.33 tion, perhaps by increasing affinity for the hy-
nNOS and 0.82 eNOS. Apparently, an aryl or drophobic binding site. Substituents other
carbonyl functionality with sp2 characteris- than hydrogen at R7 may not be beneficial.
tics should be adjacent to the terminal piper- Several imidazoles have been found to
azine to retain isoform selectivity. inhibit NOS, including 1-phenylimidazole,
4-Amino-5,6,7,8-tetrahydropteridinesand 2-phenylimidazole, and 4-phenylimidazole.
2,4-diamino-pteridineshave been synthesized These imidazoles bind heme in NOS and other
and tested as nNOS inhibitors (53). These in- enzymes. A search for isoform-specific inhibi-
hibitors are designed to fitinto the tetrahydro- tors based on an imidazole structure has led to
biopterin site of nNOS (Table 11.11). A bind- the discovery of 1-(trifluoromethylphenyl)im-
ing site model has been proposed for these idazole, N-(4-nitrophenacyl) imidazole, and
compounds. The nitrogen in the 3-position, N-(4-nitrophenylacy1)-2-methyl-imidazole,
the exocyclic nitrogen at the 4-position, and below (54). The nitrophenylacylimidazoles are
M may bind to the heme side-chain. Arg375 selective for nNOS rather than eNOS inhibi-
may bind to the exocyclic nitrogen, R4 and N5. tion. They appear to bind to the tetrahydro-
R6 and R7 may bind to a hydrophobic environ- biopterin site and are competitive inhibitors of
ment made up of Phe470 and Serll2. The tetrahydrobiopterin binding. They are non-
pteridine ring may be involved in 7~ electron competitive inhibitors of arginine binding. It
stacking with Trp457. In general, R2 substitu- appears that electron-withdrawing N-1 sub-
ents other than hydrogen do not enhance ac- stituents enhance activity and nNOS selectiv-
tivity. Perhaps bulky groups in this position ity.
inhibit binding to the heme. Similarly, R5 sub- Some imidazoles are selective inhibitors of
stituents other than hydrogen are not benefi- iNOS and could be useful in the treatment of
cial in nNOS binding, perhaps because of pre- stroke. Polymorphonuclear leukocytes and
vention of binding to Arg375. R6 substituents other inflammatory cells are prominent in the
that are hydrophobic enhance nNOS inhibi- limit area within 24 h or less of ischemia and
6 Nitric Oxide Synthase Inhibitors
COOH
H
K NH
I
-
NADPH, 02 -NKm
I I
- -'I
citrulline
OH R
heme
--.
heme
6 Nitric Oxide Synthase Inhibitors
f
H
H H
SEt N+H2
CH3S
SCH3
HN A NH-HX
-
- - - - - -
"Data adapted from Ref. 60 with permission (63 1997 American Chemical Society).
also inhibit cytochrome P450 reductase and that bind to heme might inhibit cytochrome
similar reductases. This would be detrimental P450 and other heme enzymes, leading to in-
to patients, in that drug metabolism might be hibition of drug metabolism. NOS inhibitors
inhibited by these compounds. NOS inhibitors that inhibit tetrahydrobiopterin binding
708 Investigative Agents for Use in Neurodegenerative Conditions
might inhibit other tetrahydrobiopterin en- 18. A. Y. Sun and J. S. Cheng, Acta Pharmacol.
zymes such as tyrosine hydroxylase. This Sinica, 19, 104-108 (1998).
might lead to drug-induced parkinsonism. 19. M. Banasik and K. Ueda, Mol. Cell. Biochem.,
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search. 20. P. W. Rankin, E. L. Jacobson, R. C. Benjamin, J.
Moss, and M. K. Jacobson, J. Biol. Chem., 264,
4312-4317 (1989).
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ams, Biochim. Biophys. Acta, 1525, 136-148
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Moran, B. Schiavoni, J. R. Hsu, and S. K.
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109-112 (2000). Haven-Hudkins, B. Ault, J. L. Herrman, J. S.
References 709
JOHN L. NEUMEYER
Ross J. BALDESSARINI
Harvard Medical School
McLean Hospital
Belmont, Massachusetts
School of Pharmacy
The University of North Carolina
1 Chapel Hill, North Carolina
i Contents
1 Introduction, 712
2 Parkinson's Disease. 712
2.1 Pathophysiology, 713
2.2 Etiology, 715
2.2.1 Genetic Factors, 715
2.2.2 Developmental Factors and Oxidation
of Dopamine, 716
2.2.3 Environmental Toxins, 717
2.2.4 Parkinsonism and MPTP, 717
3 Treatments for Parkinson's Disease, 721
3.1 Dopaminergic Treatments, 721
.
3.1.1 L-Dowa Metabolism and Therawv. 721
A " ,
tors), and inhibitory D,-type (D, with splice rect descending influences, the internal (me-
variants, and less abundant D, and D,) sub- dial) globus pallidus and pars reticulata send
families of membrane proteins. These pep- inhibitory GABAergic projections to modulate
tides, composed of 387-477 amino acids, are activity of ascending thalamic neurons, partic-
typical of the superfamily of GTP-binding pro- ularly in the ventral (mainly anterior and lat-
tein (G-protein)-associated membrane pro- eral) thalamic nuclei. The thalamic nuclei
teins that include most monoaminergic recep- project ascending glutamatergic excitatory ef-
tors and other physiologically important f e r e n t ~to motor cerebral cortex, thus exerting
membrane proteins. Their structure is charac- a major regulatory influence over the descend-
terized by seven relatively hydrophobic, puta- ing corticospinal motor output pathway that
tive transmembrane regions linked by four controls the cholinergic spinal ventral motor
extracellular and four intracellular loop seg- horn cells innervating skeletal muscle (18).
ments, starting from an extracellular amino DA modulates the activity of local and ef-
terminus, and extending to an intracytoplasmic ferent inhibitory GABAergic neurons as well
carboxy end of the receptor polypeptide chain as acetylcholinergic interneurons of caudate-
(15, 16). The third intracellular loop and car- putamen (12, 15, 17). Excitatory DA Dl-type
boxy-terminus segment vary most among Dl receptors, together with the neuropeptides sub-
and D, receptor subtypes, and probably inter- stance P and dynorphin, are mainly expressed
act critically with excitatory or inhibitory G- by the striatonigral GABAergic neurons in the
Therapeutic and Diagnostic Agents for Parkinson's Diseasc
Glu
(+)
Glu
(+I
GABA GABA
Figure 12.1. Circuitry of the basal ganglia. Shown are major relationships to the neostriatum
(caudate nucleus-putamen complex) with its prominent dopaminergic innervation (particularly of
putamen in man) from the midbrain substantia nigra zona compacta (SNc), as well as descending
control by corticostriatal glutamatergic projections. Dopamine exerts excitatory effects through D,
receptors on efferent medium spiny neurons that are GABAergic and inhibitory to the substantia
zona reticulata (SNr) and the internal portion of the globus pallidus (GPi), thus limiting a secondary
inhibitory influence of nigrothalamic and pallidothalamic GABA neurons (that also express sub-
stance P [SP] and dynorphin [DYN] peptides) to facilitate the ascending excitatory glutamatergic
(Glu) thalamocortical circuits. This short outflow loop from the neostriatum is paralleled by a long
loop that involves D, dopamine receptors inhibitory to generally excitatory acetylcholinergic (ACh)
interneurons (which are inhibited by GABA neurons) as well as to GABA efferents to the external
portion of globus pallidus (GPe) and co-express enkephalins (ENK). The globus projects GABAergic
neurons that tonically inhibit the excitatory glutamatergic neurons of the subthalamic nucleus
(STN)that stimulate an inhibitory influence of nigra, internal globus pallidus, and pedunculopontine
nucleus on the thalamus (including its ventral lateral nucleus) to yield a net reduction of thalamo-
cortical activation. In Parkinson's disease (PD) the nigrostriatal dopamine projections degenerate
and dopaminergic influences in the neostriatum are initially compromised and eventually lost. Loss
of dopamine in PD leads to reduced influences through both the direct and indirect pathways to result
in a net decrease in thalamocortical stimulation, with clinical bradykinesia. Excessive dopaminergic
stimulation encountered in the treatment of PD increases thalamocortical activation, with clinical
dyskinesias. This traditional model remains tentative and incomplete, and does not include simulta-
neous Dl and D, influences that may occur on some GABAergic neurons in neostriatum. More
inclusive models have been proposed (9).
2 Parkinson's Disease
direct output pathway. In contrast, inhibitory nal development of effective therapeutic and
DA D,-type DA receptors, along with the neu- prophylactic pharmacotherapies. Several po-
ropeptide enkephalin, are predominantly local- tentially convergent hypotheses have been -
ized to striatopallidal GABAergic efferents of proposed regarding the cause of PD. They con-
the indirect output pathway. These relation- sider: (1) genetic factors; (2) neurodevelop-
ships lead to a complex role for DA in the basal mental factors associated with advancing age,
ganglia. By stimulating excitatory Dl recep- including oxidation of DA and dysfunction of '
tors, DA appears to have a net facilitatory ef- oxidative metabolism; and (3) effects of envi-
fect on the direct GABAergic pathway to inter- ronmental or endogenous neurotoxins.
nal (medial) globus pallidus and midbrain, Several neurodegenerative disorders af- -
which can diminish their inhibitory connec- fecting motility are genetically determined.
tions to thalamus, to increase ascending exci- Striking progress in defining the genetic basis
tatory thalamocortical activity. In contrast, of Huntington's chorea greatly increased in-
activation of inhibitory D, receptors in the in- terest in genetic contributions to other neuro-
direct pathway inhibits GABAergic neurons degenerative disorders, including PD (21). Ep-
projecting to external (lateral) globus pallidus, idemiological studies have found that, apart
and reduces its inhibitory influence on the from advancing age, a family history of PD is a
subthalamic nucleus. These effects result in a predictor of increased risk of the disorder, sug-
net disinhibition of an excitatory glutamater- gesting a genetic contribution (22). Recent
gic link from the subthalamic nucleus to mid- studies strongly indicate a genetic contribu-
brain that increases nigral GABAergic inhibi- tion in the disorder, although the majority of
tion of thalamus. The outcome is to decrease cases of PD are considered sporadic, given that
thalamocortical stimulation, opposite to the genetic factors account for only a minority of
effect initiated by DA through the direct path- cases, predominantly those of early onset (23,
way (Fig. 12.1). 24).
In sum, the overall effect of DA is to facili- A specific genetic hypothesis has consid-
tate cortical excitation by thalamocortical ered mutation of the gene for the protein
glutamatergic projections through the direct a-synuclein in an autosomal dominant form of
pathway, but to decrease thalamocortical familial PD. This molecule is a highly con-
stimulation through the indirect pathway. Ac- served, relatively abundant, 140-amino acid.
cordingly, in PD striatal DA deficiency alters polypeptide of unknown function that is ex-
the modulation of excitatory outflow from pressed mainly in presynaptic nerve terminals
ventral thalamus to motor cortex (9, 12, 16). in the brain. It is controlled by a gene in the
Presumably, a balance of Dl- and D,-mediated long-arm (4q) of human chromosome 4 (25).
dopaminergic function would be optimal in re- The gene for a-synuclein can be expressed as
storing the functional losses that follow de- distinct structural variants that have been as-
generation of the DA-producingneurons. Neuro- sociated with some pedigrees involving PD
chemically, striatal DA deficiency seems to (26, 27), but not in a larger number of other
8ccountfor the major motor symptomsof PD, par- families (28, 29) or in sporadic cases (27).
ticularly bradykinesia. The mainstay of phar- Other genetic findings have implicated muta-
macological treatment (8) continues to be re- tions in the gene coding for ubiquitin carboxy-
placement therapy with the a-amino acid, L-3,4- terminal hydrolase in another autosomal
dihydroxyphenylalanjne (L-dopa), the immediate dominant form of PD, and other genetic fac-
biochemical precursor of DA, discussed below, tors associated with autosomal recessive
which should produce nearly physiological ago- forms of the disorder (30). However, these spe-
nism of both D, and D, DA receptors. cific genetically based disorders, collectively,
do not account for the great majority of evi-
2.2 Etiology dently sporadic cases of PD.
Nevertheless, a large proportion of the un-
2.2.1 Genetic Factors. Although the neu- common cases of early-onset PD appear to be
ropathology of PD is well defined, the primary inherited by a mechanism involving abnormal
cause remains unknown, thus limiting ratio- molecular processing of intracellular neuronal
716 Therapeutic and Diagnostic Agents for Parkinson's Disease
proteins by the process of conjugation with the 2.2.2 Developmental Factors and Oxidation
protein ubiquitin required for the orderly deg- of Dopamine. DA itself has also been impli-
radation of intracellular proteins by proteases cated in the disease process of PD through pro-
(23, 24). A specific autosomal dominant ge- duction of chemically reactive products of
netic defect produces a mutant form of the oxidation, including reactive quinones, perox-
protein parkin. Normally, parkin enables ides, and free radicals (33). Toxic effects of
ubiquitin ligase-mediated polyubiquitination these compounds may contribute to the appar-
of an 0-glycosylatedform (aSP22) of the pro- ently normal progressive loss of DA neurons
tein a-synuclein. Because polyubiquitin conju- with maturation and aging, at the rate of
gation is required for normal degradation of about 13%/decade(36,37). Clinical symptoms
a-synuclein by proteasomes, ubiquitin-uncon-
of PD emerge as losses of DA neurons exceed
jugated aSP22 accumulates in neurons, possi-
60-70% (38). However, the significance of this
bly contributing to their degeneration (31).It
trend toward spontaneous, possibly geneti-
remains unclear why such mechanisms might
occur selectively in DA neurons, or whether cally programmed (apoptosis) or degenerative,
they pertain to the more prevalent, nonfarnil- but evidently normal loss of DA neurons with
ial forms of PD. The neuropathology of spon- aging for the pathoetiology of PD is not clear
taneous PD, in contrast to the less common (39,40).
familial forms, is characterized by prominent Among products of the metabolism of DA,
accumulation of Lewy bodies rich in polyubiq- the monoamine oxidase (MA0)-catalyzedoxida-
uitnated aSP22 and other proteins, presum- tion of this and other monoamine neurotrans-
ably through insufficient degradation by pro- mitters generates hydrogen peroxide (Equation
teases that remains unexplained. 1 of Fig. 12.2). The peroxide can undergo a redox
The proposal that an increase of a-synuclein reaction with superoxide in the Haber-Weiss re-
may be responsible for PD has inspired new action (41) to form the extremely cytotoxic free
laboratory models of PD involving transgenic hydroxy ( H a ) radical (Equation 2 of Fig. 12.2).
mice (32) or the fruit fly Drosophila (33) made Moreover, auto-oxidation of DA can yield the
to express the human wild-type a-synuclein correspondingcytotoxic quinone (Fig. 12.2) (42).
and recreate certain features of PD. In both Manganese ion can catalyze oxidation of DA,
models, cytoplasmic inclusions of a-synuclein and the resulting quinones have been implicated
resembling Lewy bodies were detected, but in manganese neurotoxicity, an environmen-
these were fibrillar in structure, as in clinical tally determined form of parkinsonism (43). Ad-
PD, only in the fruit fly model. Mechanisms by ditional evidence for a role of oxidative stress in
which abnormal a-synuclein oligomers might PD includes increased levels of oxidized by-prod-
selectively cause dysfunction and death of DA ucts of nucleic acids such as 8-hydroxy-
neurons remain unclear (34). Possibilities guanosine in cerebrospinal fluid or plasma (44).
considered include abnormal cellular distribu- Auto-oxidation of catecholamines also
tion of DA, with an accumulation of toxic by- leads to formation of the polymeric pigment
products, and an effect of DA on a-synuclein neuromelanin that increases with age and is
oligomerization (31). Support for the second responsible for the dark coloration of DA-pro-
possibility arose from recent screening of a ducing cells in the substantia nigra and the
large number of compounds for effects on norepinephrine neurons of the locus caeruleus
a-synuclein fibrilization; 151169 compounds (42). A physiological role for this polymer is
inhibited the process, and 14 of these were not established, and there is no evidence to
catechols, including L-dopa and DA (35). DA support its involvement either in normal loss
underwent oxidative ligation to a-synuclein of DA neurons with aging or in the pathophys-
and selectively inhibited the conversion of pro- iology of PD.
tofibrils to fibrils, causing accumulation of the Conceivably, the symptoms of PD may result
a-synuclein protofibril. These findings suggest from two processes: a specific disease-related in-
a basis for DA-neuron selectivity and may sult combined with changes attributed to nor-
open a novel line for developing novel thera- mal aging that may include accumulation of
peutic approaches to PD (35). endogenous neurotoxins. Such a two-factor
2 Parkinson's Disease 717
\ /
H o ~ c H 2 c H 2 N H +2O2 + H20 - HO CH2CH0 + NH3 + H202 Equation 1
HO HO
Dopamine 'x,
Quinone -
Figure 12.2. Toxic derivatives of dopamine. These include hydrogen peroxide, the free hydroxy
radical, and the quinone.
ical findings in PD is its lower incidence in and monkeys that is similar in neuropathology
cigarette smokers than in nonsmokers (51, and motor abnormalities to idiopathic PD (55-
52). Chemical components of cigarette smoke, 58). Its significance emerged from a serendip-
including nicotine, may protect against an en- itous series of events. In 1977, a young college
vironmental or endogenous toxin relevant to student developed acute parkinsonian symp-
the neuropathology of PD. For example, car- toms with severe rigidity, bradykinesia, and
bon monoxide in cigarette smoke may detoxify mutism (58). The abrupt and early onset of
potentially neurotoxic free radicals from envi- symptoms was so atypical that the patient ini-
718 Therapeutic and Diagnostic Agents for Parkinson's Disease
tially was thought to have catatonic schizo- electron oxidation of MPTP at the allylic
phrenia. Subsequent diagnosis of parkinson- a-carbon, to give the intermediate, l-methyl-
ism was substantiated by his therapeutic 4-phenyl-2,3-dihydropyridinium species
response to L-dopa. When he admitted having (MPDPt, 7; see Fig. 12.3). This unstable
synthesized and used several illicit drugs, his charged substance undergoes further two-
psychiatrist visited his home and collected electron oxidation to the more stable, reactive
glassware that had been used for chemical species 1-methyl-4-phenylpyridinium(MPP+,
syntheses. 8 ) by auto-oxidation, disproportionation, and
Chemical analysis of the glassware re- enzyme-catalyzed mechanisms (59- 61). In-
vealed several pyridines, including MPTP (3), hibitors of MAO-B can prevent MPTP-in-
formed as by-products in synthesizing MPPP duced parkinsonism in primates (62), further
(N-methyl-4-propionoxy-4-phenylpiperidine), supporting the currently accepted view that
(4) and also known as "designer heroin" or MPP (8)is probably the major toxic metabo-
f
"synthetic heroin." MPPP is also the N-des- lite of MPTP (3)responsible for destruction of
methyl analog of a structurally similar nar- DA neurons, although a role for the unstable
cotic analgesic, alphaprodine (5), and is the dihydropyridinium species MPDP+ (7) has
reverse ester of the analgesic meperidine (De- not been ruled out.
merol, 6). It was unclear initially whether The relationship of MA0 and MPTP has
MPTP or other constituents of the injected neurobiological relevance beyond MPTP neu-
mixture accounted for the neurotoxicity. The rotoxicity. Types A and B MA0 catalyze the
patient continued to abuse drugs, died of an a-carbon oxidative deamination of mono-
overdose, and autopsy revealed degeneration m i n e neurotransmitters and other aromatic
of his substantia nigra, but without the Lewy amines. These genetically dissimilar isozymes
bodies typical of the neuropathology of idio- show differential selectivity for specific sub-
pathic PD (58). strates and inhibitors (63). Most intraneuro-
Other persons exposed to preparations of nal MA0 is mitochondrial and type A, and ox-
MPTP (3) or MPPP (4) were later identified idizes primary amines to aldehydes, which are
after presenting with similar acute parkinso- then converted by aldehyde reductases to al-
nian symptoms. In several patients, MPTP cohols or carboxylic acids, including dihy-
was the principal or sole compound impli- droxyphenylatic acid (DOPAC) from DA. Ad-
cated, supporting the hypothesis that MPTP ditional 3-0-methylation yields the major final
is a parkinsonism-producing neurotoxin. Al- human metabolite of DA, homovanillic acid
though more than 400 people are known to (HVA; see Fig. 12.4).
have self-administered MPTP, only a few have In contrast, MAO-B is found in other cell
developed parkinsonian symptoms, though types, including glia in the nervous system.
the incidence may increase as they age. Both Oxidation of the heterocyclic tertiary-amine
the clinical and neuropathological features of MPTP by MAO-B is an unprecedented reac-
MPTP-induced parkinsonism resemble idio- tion that suggests a novel physiological role
pathic PD more closely than any other model for this enzyme. For example, MAO-B may
elicited by toxins, metals, viruses, genetic ma- regulate the oxidation state of pyridine sys-
nipulation, or other means. Accordingly, un- tems, such as those involvingnucleic acids and
derstanding the molecular pathophysiology of NADH (63).In turn, oxidative systems are im-
MPTP neurotoxicity was aggressively pur- plicated in MPTP neurotoxicity (see below).
sued to clarify neurodegenerative mecha- Interesting, too, is evidence that cigarette
nisms that might also occur in idiopathic PD. smokers have been found to have 40% less
The chemical structure of MPTP suggests postmortem brain MAO-B activity than non-
that the compound should be relatively inert smokers or former smokers (64), as well as a
chemically, given that no reactive functional lower incidence of PD (64, 65). That cigarette
group is present, and that MPTP might un- smokers reportedly have depressed MAO-B
dergo metabolic activation to a more reactive activity (64) may be a potential link to their
derivative. Researchers discovered that brain decreased risk of PD (65). Nicotine is not a
MAO-B (mainly in glial cells) catalyzes a two- potent inhibitor of MAO, and may even in-
2 Parkinson's Disease
I
CH3
MPTP MPPP Alphaprodine Meperidine
(designer heroin) (Nisentil) (Demerol)
(5) (6)
MAO-B
II
CH3
MPPP MPTP
deprenyl
(4) inhibit
Nigrostriatal
cell death *
metabolic changes - uptake
catecholamine
neurons
I
0 N
I
CH3
A I MPP'
\ inhibits )
Figure 12.3. Phenylpiperidine analgesics and metabolic activation of MPTP. In efforts to synthe-
size the meperidine-like analgesic agent MPPP ("designer heroin,") (4), MPTP (3)can be formed. It
is converted selectively by monoamine oxidase type B (MAO-B, inhibited by agents including depre-
nyl (selegiline)and pargyline to MPDP (7),and thence to MPP+ (8)the proposed toxic species that
f
accumulated in dopamine neurons to result in disruption of their cellular respiration and death.
nydroxylase
I T
OH
Me0
OH
NH2 L-Tyrosine 3-Methoxy-4-hydroxy
COOH
OH
HO 3-Methoxy-4-hydroxy
phenylpyruvic acid
OH HO
111
L .l
L-3,4-dihydroxyphenylananine OH
3,4-Dihydroxyphenylpyruvicacid
(L-dopa) ,CHO
OH
3,4-dihydroxyphenylacetaldehyde HO
NH2
3,4-Dihydroxy \
phenylacetic acid I
PI
Dopamine
(3,4-Dihydroxyphenethylamine) OH
3-Methoxytyramine (MTA) OH
NHCH3 Homovanillic acid (HVA)
I
(3-Methoxy-
4-hydroxyphenylacetic acid)
OH
8
Epinephrine
NH2
I
/
HO
OH
Figure 12.4. Pathways in the metabolism o f L-dopa (1)and i t s major decarboxylated product dopa-
mine (2). Major (heavy arrows) and minor (light arrows) reactions are indicated. AD, aldehyde
dehydrogenase; AAD, aromatic L-amino acid decarboxylase; COMT, catechol-0-methyltransferase;
DH, dopamine P-hydroxylase; MAO, monoamine oxidase; PNMT, phenylethanolamine-N-methyl-
transferase.
3 Treatments for Parkinson's Disease
crease MPTP neurotoxicity (66). Other com- critical components of most theories of nigral
pounds in cigarette smoke, however, do in- cell degeneration in PD (8). Moreover, discov-
hibit MA0 and cigarette smoke protects ery of the selective ability of MPTP to induce -
against MPTP-induced depletion of striatal nigral cell death has stimulated broad interest .
DA in mice (67). in identifying potential environmental or en-
Extensive investigation indicates that ni- dogenous compounds as potential causative
grostriatal degenerative properties of MPTP agents in PD, even though there is no evidence '
are mediated by the MAO-B-derived metabo- that MPTP itself is involved in idiopathic PD.
lite MPP+ (8). First, MPTP (3) binds selec- Another example of a relevant toxin effect
tively to MAO-B, which is highly concentrated is evidence that chronic, systemic inhibition of -
in glial cells in human substantia nigra and complex I by the lipophilic pesticide rotenone
corpus striatum (681, and is oxidized to MPTP causes highly selective degeneration of nigro-
(3)to MPDP+ (7).Several factors may account striatal DA neurons that can produce hypo-
for selective damage of nigrostriatal DA neu- kinesia and rigidity, as well as accumulation of
rons by MPTP (69; Fig. 12.3). Notably the fibrillar cytoplasmic inclusions that contain
MPP+ (3) produced from MPTP (3) is selec- ubiquitin and a-synuclein (75). These findings
tively accumulated by DA transporters into ni- lend additional support to the view that envi-
gral DA cells and striatal DA nerve terminals ronmental toxic factors may contribute to risk
(69). Within the DA nigral cells, MPP+ binds of PD.
to neuromelanin and may be gradually re-
leased in a depot-like fashion, maintaining
3 TREATMENTS FOR PARKINSON'S
toxic intracellular concentrations that inhibit
DISEASE
mitochondrial respiration in DA neurons.
MPP can also displace DA from presynaptic
f
Pramipexole (Mirapex)
(17 )
Piribedil
(18)
Despite controversy regarding long-term effi- example of a rationally predicted and logically
cacy, adverse effects, and even potential neu- pursued clinical treatment in a neurological
rotoxicity of this amino acid precursor of DA, disorder, based on neurochemical pathology
most PD patients derive a substantial benefit and basic pharmacological theory (13,141.The
from L-dopa throughout their illness. More- effectiveness of L-dopa treatment requires its
over, L-dopa increases life expectancy among penetration into the central nervous system
patients with PD, particularly if instituted (CNS) and local decarboxylation to DA. DA
early in the course of PD (82). does not cross the blood-brain diffusion bar-
In 1960 Ehringer and Hornykiewicz as- rier because its amino moiety is protonated
sayed DA in the brains of patients dying with under physiological conditions (pK, 10.61,
PD and found that DA levels in the corpora making it excessively hydrophilic (86). How-
striata of many of these patients averaged only ever, its precursor amino acid L-dopa is less
20% of normal (83). The signs of illness in PD basic (pK, 8.72) and polar at physiological pH,
patients resembled behavioral changes in rats and more able to penetrate the CNS, in part
treated with reserpine or other amine-deplet- facilitated by transport into brain with other
ing agents. These findings led Birkmeyer and aromatic and neutral aliphatic amino acids
Hornykiewicz to administer high oral doses of (86-88).
racemic dopa to PD patients in Vienna in 1960 L-Dopa is normally a trace intermediary
(84).Subsequent clinical trials led by Barbeau metabolite in the biosynthesis of catechol-
in Montreal in the early 1960s and by Cotzias amines, formed from L-tyrosinein a rate-lim-
in New York in the late 1960s confirmed this iting hydroxylation step by tyrosine hydroxy-
effect of racemic dopa (85). Barbeau in Mon- lase, a phosphorylation-activated cytoplasmic
treal, and later Cotzias, also demonstrated the mono-oxygenase. L-Dopa is readily decar-
greater potency and safety of the physiological boxyated by the cytoplasmic enzyme L-aro-
levo enantiomer (80,81). matic amino acid decarboxylase ("dopa decar-
Development of L-dopa (1;Larodopa; Fig. boxylase") to form DA (2). The effects
12.4) as a therapeutic agent in PD is a rare observed after systemic administration of L-
724 Therapeutic and Diagnostic Agents for Parkinson's Disease
dopa have been attributed to its peripheral creasing decarboxylase activity, with more
and cerebral metabolites, mainly DA, with peripheral conversion of the amino acid to DA
much less conversion to norepinephrine by
0-hydroxylation, or epinephrine formed by N-
-
to make less L - ~ O available
D ~ to the CNS. How-
ever, blockade of peripheral decarboxylation
methylation of norepinephrine by phenyleth- with carbidopa minimizes this effect of
anolamine-N-methyltransferase(86, 87) (Fig. pyridoxine.
12.4). A small amount of L-dopa is O-methyl- DA itself is relatively rapidly metabolized
ated to L-3-0-methyldopa (L-3-methoxyty- to its principal inactive excretion products of
rosine), which accumulates in the CNS be- MA0 (largely by MAO-A in mitochondria of
cause of its long half-life. However, most aminergic nerve terminals) and by extraneu-
exogenous L-dopa is rapidly decarboxyated to ronal catechol-0-methyltransferase (COMT).
DA in peripheral tissues, including liver, Tissue concentrations of the methyl-donor co-
heart, lung, and kidney. Because only about factor of methylpherases, S-adenosyl-L-methi-
1%of an administered dose reaches the brain, onine (SAMe), can be depleted with large
L-dopa, by itself, has very limited dose effec- doses of L-dopa (90). The main by-products of
tiveness (89). In humans, appreciable quanti- DA are deaminated 3,4-dihydroxyphenylac-
ties of L-dopa enter the brain only when ad- etic acid (DOPAC)and deaminated, 3-0-meth-
ministered by itself in doses (3-6 g daily) high ylated homovanillic acid (HVA, 3-methoxy-4-
enough to overcome losses caused by periph- hydroxyphenylacetic acid; see Fig. 12.4).
eral metabolism. A common adverse effect of L-dopa therapy
Inhibition of peripheral decarboxylase ac- is nausea and vomiting, possibly because of a
tivity by coadministration of L-dopa with a hy- combination of gastrointestinal irritation as
drophilic, peripheral decarboxylase inhibitor well as stimulation by DA (and perhaps
(see Fig. 12.8 below) such as carbidopa (9; Lo- L-dopa) of the chemoreceptor trigger zone
dosyn; combined with levodopa in Sinemet (CTZ) in the area postrema of the brainstem,
and Sinemet-SR) or benserazide (10; com- an emesis-inducing center. The blood-brain
bined with levodopa in Prolopa) markedly in- barrier is poorly developed in area postrema,
creases the proportion of L-dopa that reaches making the CTZ accessible to circulating
the brain, where it can be converted to DA by emetics. An important advantage of combin-
widely available aromatic amino acid decar- ing L-dopa with a peripheral decarboxylase in-
boxylase and replace its deficiency associated hibitor. in association with the 75-80% reduc-
with PD. Doses of L-dopa required are corre- tion of the required doses of L-dopa, is less risk
spondingly much lower (typically only 0.2-1.2 of emesis or other adverse effects associated
glday), and most commonly attained with 251 with peripheral formation of excess DA. These
100 mg doses of standard preparations of can include activation of peripheral adrener-
carbidopa/levodopa, or 15/200 mg of SR prod- gic and DA receptors, in part by releasing en-
ucts, although preparations with other dosage dogenous adrenergic catecholamines (88),
ratios are available (20). Patients with PD are with a variety of cardiovascular effects. Theo-
typically started on one of the combination retically, vasoconstriction and hypertension
products, either alone or with other adjunctive might occur by stimulation of peripheral a-ad-
agents discussed below. The extended-release renoceptors, tachycardia by stimulation of
preparations should theoretically provide cardiac P-adrenoceptors, and direct renal and
more sustained benefits with less "wearing mesenteric vasodilatation by DA. However,
off' of benefit after several hours, but the bio- such effects are rarely encountered clinically
availability of these products is variable. In with the use of a peripheral decarboxylase in-
general, tissue uptake of L-dopa is highly de- hibitor with L-dopa (20).
pendent on competition with other aromatic After about 5 years, at least half of L-dopa-
and neutral aliphatic amino acids, and can be treated PD patients develop fluctuating motor
decreased substantially by a protein meal. responses, and nearly three-quarters do so by
Pyridoxine (vitamin B,) is the cofactor for 15 years (90). These fluctuations include "off'
aromatic amino acid decarboxylase. It can re- periods of immobility, and "on" periods with
verse the therapeutic effects of L-dopa by in- abnormal involuntary movements or dyskine-
sias (see Fig. 12.1). This "on-off' phenomenon been developed for clinical application, though
may reflect progression of the disease with apomorphine has some Dl as well as potent D,
more severe striatal nerve terminal degenera- agonist effects (110).
tion and further loss of DA, along with in- Bromocriptine (Parlodel, 11; Fig. 12.5) is
creased sensitivity of its receptors. an ergot alkaloid-peptide that acts as a weak
Psychiatric disturbances such as hypersex- partial agonist (or antagonist) at Dl-type and
uality, mania, visual hallucinations, and para- a partial agonist at D,-type DA receptors, with f
noid psychosis also are quite common adverse moderate intrinsic activitv." It was the first di-
responses to treatment with L-dopa or direct rect DA agonist to be employed in the treat-
DA agonists. These psychiatric disturbances ment of PD, after its development as a prolac- -
tin inhibitor (101, 111, 112). Bromocriptine
are widely proposed to reflect excessive stimu-
inhibits prolactin release from anterior pitu-
lation of DA receptors in mesolimbic or meso-
itary mammotrophic cells that express D, DA
cortical DA systems. They can greatly com-
receptors selectively. These receptors respond
plicate clinical management of depression com-
to DA produced in the arcuate nucleus of the
monly associated with PD, and of dementia
hypothalamus and released at the median em-
that sometimes arises in late stages of the dis-
inence into the hypophysioportal blood ves-
ease. Modern antidepressants usually are well
sels, and carried to the pituitary to act as a
tolerated, with inconsistent and probably mi- prolactin-inhibitory hormone. Bromocriptine
nor risk of worsening bradykinesia with sero-
is an effective prolactin inhibitor at low doses
tonin-enhancing antidepressants (91). Use of (typically 1-5 mgtday), for which it is used to
antipsychotic drugs, however, is limited to
treat hyperprolactinemia associated with pi-
those with minimal risk of worsening bradyki-
tuitary adenomas, or to suppress prolactin
nesia and other aspects of extrapyramidal mo-
output in prolactin-sensitive metastatic carci-
tor dysfunction (7). Clozapine is best toler-
noma of the breast. The partial-agonist acts as
ated; quetiapine and low doses of olanzapine
an agonist at pituitary D, receptors that are
are sometimes tolerated; and the new atypical
normally in a high sensitivity state. At higher
antipsychotic agent ziprasidone has not been
doses (typically 10-20 mglday), bromocriptine
evaluated in PD patients with psychotic reac-
and other D, partial-agonist ergolines act as
tions, although the chemically related risperi-
D, agonists with antiparkinson, and perhaps
done and older neuroleptic D, antagonists are
mood-elevating, effects. This agonism evi-
not tolerated by PD patients (7,92-95). dently reflects the supersensitized status of
denervated DA receptors in PD (94, 113).
3.1.2 Clinically Used Dopamine D, Recep- The peptide component of bromocriptine
tor Agonists. The nigrostriatal neurodegen- evidently is not necessary for its dopaminergic
eration underlying PD reduces the number of activity, and pergolide (Permax, 12; see Fig.
striatal nerve terminals available to decarbox- 12.5) was the first nonpeptide ergoline used
ylate L-dopa to DA, but also increases sensitiv- successfully in the treatment of PD, as well to
ity of DA receptors as well as loss of the inac- inhibit release of prolactin from the pituitary
tivation of DA by neuronal reuptake at DA (96, 103, 107, 108, 111). Pergolide shows
transporters virtually expressed only by DA greater agonist effects at both D,- and Dl-type
neurons and their terminals. Drugs that act DA receptors than does bromocriptine. Sev-
directly to stimulate DA receptors do not re- eral additional experimental ergolines have
quire functioning DA nerve terminals or en- been developed with a range of D, agonist or
dogenous synthesis of DA, and can be particu- partial-agonist actions, including cabergoline
larly useful in managing late-stage PD. (13),lergotrile (14a), pergolide (14b), and lis-
Several agents with direct DA-agonist activity uride (15) (11,95,96, 101, 104, 105,109).
have been used in the treatment of PD (20, Other small molecules are direct D, ago-
96-109). All are primarily agonists or partial nists or partial agonists, and are effective in
agonists of the D, family of DA receptors. No the treatment of PD. They include ropinirole
agents with selective Dl agonist activity, or hydrochloride (Requip 16), and pramipexole
well-balanced Dl and D, agonist actions have dihydrochloride (Mirapex, 17) (11, 102, 103,
726 Therapeutic and Diagnostic Agents for Parkinson's Disease
107, 108),as well as piribedil(18)(98)and the peripheral and central dopaminergic effects,
experimental agent R-PNU-95666-E (19) (99, including nausea, hypotension, and agitation
100) (Fig. 12.5). In contrast to the ergolines, also are encountered with ropinirole and
these compounds are more selective in their pramipexole. They also can produce paradoxi-
interactions with DA receptors, and have cal somnolence as well as edema, and have
much less effect at nondopaminergic sites. been associated with uncommon narcolepsy-
Currently, ropinirole (16) and pramipexole like sleep attacks during the daytime, with po-
(17)are among the most commonly prescribed tential risk during driving (114).
direct DA agonists for PD in the United States R-(-)-apomorphine (20; Fig. 12.5) is an
(20). They were introduced primarily for use acid-rearrangement product of morphine em-
in advanced stages of PD to limit fluctuations ployed in experimental neuropharmacology
in response to L-dopa therapy and as a "res- since the late nineteenth century (110, 115).
cue" therapy when L-dopa became less effec- Apomorphine is an agonist for both D, and Dl
tive. However, their relative tolerability, cou- DA receptors. With a pK, of about 9, it is
pled with clinically unresolved concern that mostly protonated at physiological pH, but
L-dopa therapy might contribute to additional sufficiently lipophilic to cross the blood-brain
toxic damage to DA neurons through forma- barrier readily. Apomorphine hydrochloride
tion of reactive oxidized by-products, has led was resurrected as a useful adjunct in the
to their growing use as a first-line treatment therapy of PD a decade ago (1161, following
option, sometimes before L-dopa is added. An years of neglect after promising early observa-
additional advantage of these agents is that tions (85, 117). Lack of oral bioavailability,
they have a relatively prolonged dopaminergic short duration of action, and potent central
action, to provide more sustained clinical ben- emetic action discouraged its clinical use. Nev-
efit with less risk of fluctuation than with L- ertheless, in 1993, apomorphine received reg-
dopa, even as modified by cotreatment with ulatory approval in the UK for control of
inhibitors of its peripheral metabolism by de- refractory motor dysfunction and wide fluctu-
carboxylase and COMT. This impression is ations in responses ("on-off' syndrome) to L-
supported by controlled comparisons of the dopa or DA agonists (118-120). Improved mo-
two forms of treatment (101-103). A recent tility in response to an acute challenge dose of
study in patients initially treated with apomorphine can also predict responsiveness
pramipexole (17) demonstrated a reduction in to L-dopa treatment (121-123). Apomorphine
loss of striatal (1231)-/3-CIT(54, see Fig. 12.11) can be administered subcutaneously by inter-
a marker of DA neuron degeneration, com- mittent self-administration with a small self-
pared with those initially treated with L-dopa injector (Penject), or continuous infusion with
during a 46-month period (77). a portable minipump (115, 120). Catechol-di-
The direct DA agonists generally produce ester and methylenedioxy aporphine prodrugs
similar adverse effects, including initial nau- limit first-pass metabolic inactivation, while
sea and vomiting, postural hypotension, and retaining much of the activity of catecholapor-
fatigue. These effects are more likely with the phines (124). The 11-monohydroxy congener
ergolines, which are started in low doses and of the potent DA agonist R-(-)-N-n-propyl-
increased slowly, as tolerated, whereas ropini- norapomorphine (2 I), R-(-)-N-n-propyl-1 l-
role and pramipexole can usually be dosed hydroxynoraporphine (22; Fig. 12.5), retains
more rapidly to clinically effective levels. Ad- the critical free hydroxyl group analogous to
ditional risks include psychotic reactions the meta-3-hydroxy substituent of DA, has po-
when the DA agonists are given alone or with tent dopaminergic activity, and is orally bio-
L-dopa. These reactions include hallucina- available (124-127).
tions, delusions, and confusion, suggesting de-
lirium, which is most likely to occur in elderly 3.1.3 Experimental Dopamine D, Receptor
PD patients with mild dementia. Treatment is Agonists. The opposing actions of the direct
similar to psychotic reactions to L-dopa ther- and indirect pathways in the basal ganglia (see
apy, and usually includes use of low doses of Fig. 12.1) suggest that coordinated movement
clozapine or quetiapine (6, 7, 91-94). Adverse requires neurotransmission to be activated in
3 Treatments for Parkinson's Disease
(24) X = R = H: SKF-38393
(25) X = CI, R = H: SKF-81297
(26) X = Br, R = CH2CH=CH2: Br-APB
(27) X = CI, R = CH2CH=CH2: SKF-82958, CI-APB
Dihydrexidine Dinapsoline
(28) (29)
-7
Figure 12.6. Representative experimental Dl agonists. These agents have unproved or untested
clinical utility in the treatment of Parkinson's disease.
the direct pathway and attenuated in the indi- first selective Dl partial agonists such as
rect pathway. DA in the striatum may achieve the benzergoline CY-208-243 (23; Fig. 12.6)
such neuromodulation by stirnulatory actions and the phenylbenzazepine SKF-38393 (24)
at Dl-type receptors and inhibitory actions at showed that these drugs were either short-act-
D,-type receptors. Consistent with a role for ing or lacking in efficacy (11,128, 129). Other
the Dl receptor as a regulator of the direct phenylbenzazepines are also short-acting Dl
output pathway, stimulation of Dl receptors partial agonists (25-27). It was later hypothe-
represents a plausible pharmacotherapeutic sized that adequate testing of D, agonist util-
approach in PD. Initial clinical trials with the ity in PD required a full, not a partial, agonist.
728 Therapeutic and ~iagnosticAgents for Parkinson's Disease
The benzophenanthridine dihydrexidine (28) zures, but an analog, A-77636 (32), showed
was the first full-efficacy Dl agonist to be de- antiparkinson effects without inducing sei-
veloped, though it also has some D,-type activ- zures in MPTP-treated marmosets (144).
ity (130, 131). In MPTP-lesioned monkeys, However, A-77636 showed rapid desensitiza-
dihydrexidine essentially eliminates all par- tion to its beneficial effects (1451, possibly re-
kinsonian signs, and this effect was fully lated to its prolonged action (>20 h) (142).
blocked by the Dl antagonist SCH-23390 but
not by the D, antagonist remoxipride, consis-
tent with a Dl mechanism of action (132). 3.1.4 Structural Modeling for Dopamine
Moreover, continuous administration of dihy- Agonists. Rational molecular design of DA
drexidine to rats for 2 weeks produced mini- agonists for treating PD is limited by a lack of
mal or no changes in either Dl receptor den- validated three-dimensional models of inter-
sity or Dl receptor-mediated DA-stimulated actions of DA with critical amino acid residues
adenylyl cyclase activity, suggesting that tol- at DA binding sites within DA receptor pep-
erance should not develop to its antiparkinson tide chains. Moreover, structural models that
effects. In PD patients, however, dihydrexi- would differentiate the known DA Dl-type
dine has a narrow therapeutic index and (Dl, D,) or D,-type (D,, D,, D,) receptors are
dose-limiting adverse effects including flush- also lacking, and the physiology of the low-
ing, hypotension, and tachycardia after single abundance subtypes, including D,, D,, and D,
intravenous doses (133). receptors, remains highly tentative, particu-
The Dl receptor pharmacophore model de- larly at the level of motor control (15). Devel-
veloped for dihydrexidine subsequently was opment of selective DA receptor agents con-
used to design novel molecular structures as tinues to be guided by quantitative structure-
full-efficacy Dl agonists (134). One such com- activity relationships (QSAFt) based on lead
pound is dinapsoline (29) (135). Numerous molecules, discovery of which in turn remains
other analogs have since been produced with highly empirical, based largely on affhities to
structural elements of both dihydrexidine and receptor subtypes in brain tissue or expressed
dinapsoline (29). For example, the dihydrexi- in genetically transfected cultured cells, all la-
dine isostere A-86929 (30a) and its diacetyl beled with receptor-selective radioligands (15,
prodrug ABT-431 (30b) are full Dl agonists 146).
with sustained antiparkinson effects in Because the side-chain of DA possesses un-
MPTP-lesioned monkeys (136). In patients limited flexibility and unrestricted rotation
with PD, ABT-431 was highly effective against about the p-carbon-phenyl bond, little infor-
bradykinesia, but produced dyskinesias (137). mation can be obtained concerning conforma-
Several full-efficacy R-(+)-phenylbenzaz- tional requirements for activation of DA re-
epine Dl agonists have been developed based ceptors using the endogenous ligand itself.
on SKF-38393 (24), including SKF-81297 (25; Accordingly, compounds in which the catechol
Fig. 12.6) and its 6-halo derivatives, 6-Br-APB ring and the aminoethyl side-chain of DA are
(26) and 6-CI-APB (SKF-82958, 27) (138, held in rigid conformation have been synthe-
139). In MPTP-lesioned monkeys, 6-Cl-APB sized. Several groups of such heterocyclic
(27) produced antiparkinson effects (140),but agents have contributed important insights
its utility was limited by very brief action (<1 into SAFt requirements of DA agonists. Nota-
h; 141) and severe adverse effects (142).More- bly, aporphines, aminotetrahydronaphtha-
over, SKI?-81297 (25) and dihydrexidine (28) lenes (aminotetralins),and aminobenzoquino-
(141) showed beneficial results only in mon- lines have yielded D,-like agonists, and the
keys with severe parkinsonism, supporting phenylbenzazepines and isochromans have
the suggestion that Dl agonists may be most provided Dl-like agents (146, 147).
useful in late stages of PD, if tolerable agents Structural models of the DA-agonist R-(-)-
with prolonged action can be developed (143). enantiomers of apomorphine (20) and its more
Several isochromans are also full Dl ago- potent congener, N-n-propylnorapomorphine
nists. The first compound in this series to be (21; Fig. 12.5) have been proposed, based on
tested, A-68930 (31; Fig. 12.61, produced sei- their X-ray crystal structure (148). Such anal-
3 Treatments for Parkinson's Disease
HO
/
NH2
1
:
I HO
/
NH2
1
:
H
HO
0
/
r N H 2
'i
OH 1- /
trans, a-rotamer trans, p-rotamer cis, a-rotamer . -
yses show that these tetracyclics contain mo- 3.1.5 Dopamine-Potentiating Agents. Sev-
lecular features in common with DA in its eral agents have been used in the treatment of
trans-a-rotamer conformation (Fig. 12.7). PD that limit the metabolism of cat-
Experimentation with rigid dopaminomi- echolamines. They include the MA0 inhibitor
metic agents strongly suggests that the pre- selegiline (37) and several inhibitors of
ferred conformation of D2-like DA agonists COMT. Because active neuronal transport
models DA in its extended trans-a-conforma- ("reuptake") mediated by the DA transporter
tion. The trans-a-rotameric conformation of is the principal means of inactivating DA in
DA is also likely to be an important determi- the synaptic region (151),it is also plausible to
nant of agonist activation of Dl-type recep- expect that DA transport-inhibiting agents
tors, based largely on consideration of a series might also potentiate DA available in remain-
of phenylbenzazepines that also contain the ing dopaminergic neurons in early PD, and in
elements of DA within a rigid heterocyclic sys- potentiating DA produced from L-dopa. Al-
tem (149) (Fig. 12.6). though stimulant agents have some beneficial
Computational chemistry methods such as effects in mild PD, they are rarely used cur-
comparative molecular field analysis (CoMFA) rently and evidently have not been evaluated
also have been applied to elucidate quantita- as a means of potentiating L-dopa (114).
tive three-dimensional structure-activity re- 3.1.5.1 Monoamine Oxidase Inhibitors.
quirements (3D-QSAR) for activation of DA Among MA0 inhibitors, those selective for the
receptors. One such analysis considered 16 MAO-A in monoaminergic nerve terminals
structurally diverse, prototypical template Dl might be expected to potentiate DA. These in-
and D2 DA agonists (150). Interactions of ago- clude the long-acting, irreversible, nonselec-
nists with DA receptors were best described by tive MAO-IVB inhibitors phenelzine (Nardil;
a pharmacophore consisting of one protonated 33; Fig. 12.8) and tranylcypromine (Parnate;
nitrogen (at physiological pH) and at least one 34) (136,152). There are also short-acting, re-
electronegative center able to participate in versible inhibitors selective for MAO-A, which
hydrogen bonding (e.g., catechol, critically po- include the antidepressant moclobemide
sitioned hydroxy, or equivalent electronega- (Manerix, 35 and the experimental agent bro-
tive moieties). The pharmacophore maps for faromine 36; Fig. 12.8) (153). Presumably be-
Dl- and D2-type receptors differed primarily cause of their short and reversible actions,
in requiring a higher position of the amino- they have less risk of inducing hypertensive
nitrogen atom above the plane of the electro- crises when combined with indirect sympatho-
negative hydrogen bonding group Dl receptor mimetic amines that release endogenous cat-
activity. These analyses indicate that the cat- echolamines, though they have some risk of
ionic nitrogen moiety is more important than inducing cerebral intoxication when given
the rotameric conformation of the electroneg- with serotonin-potentiating agents including
ative hydroxy or other hydrogen-bonding serotonin (5-hyroxytryptamine) reuptake in-
groups in determining pharmacophore selec- hibitor antidepressants, meperidine, and
tivity for Dl and D2 receptors. other agents (136,152). Combinations of long-
730 Therapeutic and Diagnostic Agents for Parkinson's Disease
M A 0 inhibitors
L(-)-Carbidopa(Lodosyn) Benserazide
(9) (10)
COMT inhibitors
NO2
Entacapone (Comtan) Rolcapone (Tasmar)
(38) (39)
Figure 12.8. Dopamine potentiating or protective agents. These include MA0 inhibitors (only
selegiline is used clinically to treat PD),inhibitors of the peripheral decarboxylation of L-dopa, and
inhibitors of the 0-methylation of dopamine and L-dopa.
acting MAO-A/B inhibitors like phenelzine to neurotoxic by-products (see Fig. 12.3).
and tranylcypromine with L-dopa are contra- Probably by protecting DA, selegiline can in-
indicated because of the risk of inducing hy- crease the potency and duration of action of
pertensive crises and delirium (20). L-dopa, and do so safely provided that doses
Selegiline hydrochloride (Eldepryl; L-de- are kept low (153, 154). However, at doses
prenyl; R-(-1-N,2-dimethyl-N-2-propynyl- above 20 mg/day, selegiline has an inhibitory
phenethylamine, 37; Fig. 12.81, at low doses effect on both MAO-A and MAO-B. In addi-
(a10 mglday), is a selective inhibitor of mainly tion, it can be converted to methamphetamine
nonneuronal MAO-B, which oxidizes MPTP or similar metabolic by-products, inhibit neu-
3 Treatments for Parkinson's Disease
ronal transport of monoamines, and release fective, and with clinical monitoring of hepatic
DA (153). In this way, selegiline resembles tra- functioning (156-159). Other adverse effects
ditional stimulants that release DA and other of these agents include severe diarrhea and
monoamines (114). It had also been hoped risk of dyskinesias and psychosis in PD p a
that use of an MAO-B inhibitor like selegiline tients when combined with L-dopa (160).
might prevent formation of neurotoxic oxida-
r
tion products of DA or perhaps MPTP-like
compounds and slow the progressive neurode- 3.2 Agents Acting on Nondopaminergic
generation in PD, but evidence for such a neu- Systems
-
roprotective effect of this agent or of antioxi-
dants including vitamin E is lacking, and 3.2.1 Anticholinergic Agents. Cholinergic
selegiline has only limited clinical benefits in interneurons in the striatum exert mainly ex-
early or mild PD (75, 76, 155). citatory effects on GABAergic output from the
3.1.5.2 Catechol-0-Methyltransferase In- striatum (see Fig. 12.1). Drugs that increase
hibitors. The peripheral metabolism of L-dopa cholinergic neurotransmission (e.g., the cho-
given alone leads to very limited access of the linesterase inhibitor physostigmine and the
amino acid to the CNS. It is rapidly decarboxy- direct agonist carbachol) have long been
lated by L-aromatic amino acid decarboxylase known to aggravate parkinsonism in humans,
and 3-0-methylated by COMT. In addition to whereas centrally active muscarinic antago-
potentiating L-dopa with peripheral decarbox- nists (such as the belladonna alkaloids, includ-
ylase inhibitors including carbidopa and ing atropine), have moderately beneficial ef-
benserazide, there are also inhibitors of fects (20, 161-164). Accordingly, before the
COMT. This methyl transferase, with its discovery of L-dopa, drug therapy for parkin-
methyl-donor cofactor S-adenosyl-L-methio- sonism depended primarily on the limited ef-
nine ( W e ) ,converts dopa and catecholamines ficacy of the natural belladonna alkaloids and
preferentially to their m-methoxy derivatives newer synthetic antimuscarinic alkaloids, as
(see Fig, 12.4). These include 3-0-methyl-dopa well as antihistamines that also exert central
and 3-0-methyldopamine (3-methoxytyra- antimuscarinic actions (Fig. 12.9). Synthetic
mine, 3-methoxy-4-hydroxyphenethylamine), central anticholinergic agents include benz-
as well as the 3-0-methylated, deaminated tropine mesylate (40; Cogentin and others),
compound homovanillic acid (HVA),the major biperidin (41; Akineton), diphenhydramine
metabolite of DA in humans. Treatment with (42; Benadryl and others), ethopropazine (43;
L-dopa can reduce tissue concentrations of Parsidol), orphenadrine (44; Disipal and oth-
SAMe (87), with uncertain consequences that ers), procyclidine (45; Kemadrin), and tri-
should be limited by cotreatment with a hexylphenidyl (46; Artane and others). Such
COMT-inhibitor. COMT acts in both periph- drugs continue to be used to control parkin-
ery and CNS, though the use of COMT inhib- sonism and other adverse extrapyramidal
itors to potentiate L-dopa is most effective in neurological effects of the potent D,-receptor
peripheral tissues (20, 140, 156-158). antagonist neuroleptic antipsychotic agents,
COMT inhibitors in current clinical use in- for which they are quite effective (20,91).
clude entacapone (Corntan; 38) and tolcapone In contrast, central antimuscarinic agents
(Tasmar; 39; Fig. 12.8). Although these drugs have limited therapeutic benefit in PD. They
lack beneficial effects on PD themselves, they also exert a range of undesirable adverse ef-
potentiate L-dopa and prolong its actions by fects because of their blockade of peripheral
inhibiting the metabolic inactivation of L-dopa parasympathetic function. These include dry
and DA. Entacapone is more widely used, acts mouth, impaired visual accommodation, con-
only in the periphery, and is relatively short- stipation, urinary retention, and tachycardia.
acting (-2 h). Tolcapone is longer acting, but Adverse CNS effects include delirium of vary-
has about 2% risk of elevating hepatic ing severity, marked by confusion, memory
transaminases, and has been associated with impairment, and psychotic symptoms. Despite
several cases of hepatic failure, leading to its their relatively unfavorable benefitlrisk ratio,
preferred use when other options are not ef- these agents are still sometimes employed in
Therapeutic and Diagnostic Agents for Parkinson's Disease
CHO- CH2CH2- N
,
, CH3
S N-CH2-CH-N
/ CH3
\
I CH3
CH3
C N-CH2CH2-C- OH
C
0
I
Trihexyphenidyl (Artane)
(46)
12.9. Agents with central antimuscarinic activity sometimes used to treat idiopathic or
leptic drug-induced parkinsonism.
3 Treatments for Parkinson's Disease
the treatment of PD in combination with L- ger systems (173) and reduce the binding af-
dopa, particularly to help control tremor (163, finity of DA for D, receptors (173). An A.,,
164). antagonist presumably would block these A.,,
receptor-mediated inhibitory effects on DA
3.2.2 Adenosine Antagonists. KW 6002 neurotransmission and perhaps provide bene-
(46; Fig. 12.10) is a potent and selective antag- fit in PD. Activation of A.,, receptors also stim-
onist at adenosine AZAreceptors (165, 166) ulates release of acetylcholine in striatum
currently in clinical trials for treatment of PD (174). Because muscarinic acetylcholine re-
after improving motor disability in primate ceptor antagonists can ameliorate some signs
models of PD (167, 168). The adenosine A, of PD, A,. receptor antagonists may exert ad-
receptor is one of four cloned adenosine recep- ditional benefits in PD by reducing striatal
tors that are members of the seven-transmem- cholinergic neurotransmission (171).
brane, G-protein-coupled receptor superfam-
ily. AZAreceptor mRNA is highly concentrated 3.2.3 Glutamate Antagonists. Amantadine
in the striatum, nucleus accumbens, and olfac- (Symadine, Symmetrel, 48; Fig. 12.10) is an
tory tubercle, and colocalizes with D, receptor adamantane with an unusual cagelike struc-
mRNA in these brain regions (169).Activation ture, originally developed as an antiviral
of A, receptors inhibits GABA release in stri- agent. Amantadine, as a primary amine with a
atum and reduces GABA-mediated inhibition pK, of 10.8, is mainly in the protonated form
of striatal medium spiny output neurons at physiological pH, but it can enter the brain
(170). Thus, antagonism of A.,, receptors is because of its unusual structure that not only
expected to increase GABA-mediated inhibi- increases its lipophilicity, but also prevents its
tion of the medium spiny output neurons to catabolism by oxidative enzymes so that most
help compensate for the loss of DA Dl recep- of it is excreted in the urine unchanged.
tor-stimulated GABA release and D, receptor- Amantadine has some ability to release DA
mediated inhibition of these neurons in PD and norepinephrine from intraneuronal stor-
(171) (see Fig. 12.1). Adenosine A, receptors age sites and block reuptake of DA, and was
also oppose the actions of Dl and D, receptors initially considered a DA-potentiating agent
on gene expression (172) and second-messen- for use in mild PD (172, 173, 175-177). How-
734 Therapeutic and Diagnostic Agents for Parkinson's Disease
ever, in addition, amantadine and its conge- kinsonism-like catalepsy induced by haloperi-
ner, memantine (1-amino-3,5-dimethylada- do1 (189), 5-HT,, receptor activation might
mantane, 49; Fig. 12.10) have some activity as also counteract losses of nigrostriatal DA neu-
NMDA (and possibly AMPA) glutamate recep- rotransmission in PD (188). Moreover, in pa-
tor antagonists that might also provide neuro- tients with advanced PD, intact striatal5-HT
protective effects (178). Both have been used terminals are an important site of decarboxyl-
to treat PD (20,178). Amantadine has moder- ation of exogenous L-dopa to DA (190). A
ately beneficial effects early in PD, can en- 5-HT,, agonist might act at striatal seroto-
hance the effects of L-dopa, and perhaps limit nergic terminals to limit release of DA pro-
the severity of dyskinesias induced by L-dopa duced by L-dopa treatment and released from
therapy (179). Also, memantine has been used 5-HT terminals as a "false transmitter" (85).
as a spasmolytic agent in the treatment of Sarizotan (EMD-128130, 51; Fig. 12.10) is
both PD and dementia (180). The potential an arninomethylchroman derivative with po-
that such agents might afford long-term neu- tent central 5-HT,, agonist activity. Given by
roprotective effects that might include slow- itself to MPTP-lesioned monkeys, sarizotan
ing the progression of PD remains speculative. had no effect on the severity of motor deficits
Riluzole is a benzothiazolamine (50) with or on beneficial responses to L-dopa, but it re-
sodium channel blocking activity that inter- duced L-dopa-induced choreiform dyskinesias
feres with glutamatergic neurotransmission by more than 90% (187). It is in clinical trials
by blocking glutamate release in the subtha- for the treatment of PD and of dyskinesias
larnic nucleus, an area of increased neuronal resulting from L-dopa therapy. The lack of in-
activity in PD (181). It is currently given to teraction of sarizotan with L-dopa in MPTP-
patients with amyotrophic lateral sclerosis lesioned monkeys (187) is unsupportive of
(ALS) in an attempt to slow the progression of concern that it might limit release of DA from
this degenerative disorder of motor neurons of 5-HT terminals. Although sarizotan also has
the spinal anterior horn by inhibiting gluta- weak DA D, receptor affinity (10 times less
mate neurotoxicity (182). In the MPTP-le- than its 5-HT,, affinity), its beneficial effects
sioned monkey model of PD, riluzole delayed in parkinsonian monkeys seem specific to
appearance of parkinsonian motor abnormal- 5-HT,, agonism, in that they were reversed by
ities by a mechanism not involving decreased a selective 5-HT,, antagonist (187).
formation of MPP', the neurotoxic metabolite
ofMPTP (183,184). Recently, riluzole was as-
sessed for safety, tolerability, and efficacy in 4 DIAGNOSTIC AGENTS FOR
treatment of PD patients who had L-dopa-in- PARKINSON'S DISEASE
duced dyskinesias (185). Riluzole was effective
in lessening dyskinesias in these patients, was Even for an experienced clinical neurologist,
well tolerated, did not interfere with L-dopa, diagnosis of PD can be difficult to confirm,
and is undergoing further trials in PD. especially in the early stages of this disease.
Signs of PD vary markedly among patients
3.2.4 Serotonin Agonists. Dysfunction of and in the same person over time; disability
neurotransmission mediated by 5-hydroxy- can fluctuate dramatically, and progression
tryptamine (5-HT; serotonin) occurs in the of the disorder is unpredictable. In addition,
basal ganglia of patients with PD, and exces- a number of conditions mimic PD disease,
sive serotonergic transmission may contribute and vary in their responses to antiparkinson
to dyskinesias associated with dopaminergic drugs (191). Given these difficulties, brain
treatments (186). 5-HT,, receptors are ex- imaging techniques are increasingly applied
pressed presynaptically on 5-HT terminals, to both diagnostic and neuropharmacologi-
where they limit serotonin release as autore- cal studies of brain function in PD patients.
ceptors (187). Their activation should de- Positron emission tomography (PET) and
crease 5-HT release and perhaps alleviate do- single photon emission computed tomogra-
paminergic dyskinesias in PD (188). Because phy (SPECT) are sensitive methods em-
5-HT,, receptor stimulation can reverse par- ployed in such studies.
4 Diagnostic Agents for Parkinson's Disease
Cocaine
(53)
(1231)-p-~~~ (Iz3!)-FP-CIT(loflupane)
DOPAScan DATScan
Figure 12.11. Radioligands for dopamine neurons. These include [lsF]-labeled dopa and nonhydro-
lyzable, long-acting phenyltropane analogs of cocaine, which bind selectively to the dopamine trans-
porter proteins and are highly specific markers of dopamine neur,ons. These agents are useful for
imaging with [lsF] for PET and [lZ3I]for SPECT.
Spatial resolution is greater with PET, but is rapidly hydrolyzed at its benzoyl ester func-
SPECT technology is less expensive and more tion, making it an impractical candidate for
widely accessible in many clinical settings. In use in imaging (193). However, linking the
addition, positron-emitting nuclides used in phenyl ring of cocaine directly to the tropane
PET imaging have very short half-lives (llC, system yields nonhydrolyzable, long-acting
20 min; 18F,109 min) and usually require an phenyltropanes. Many such compounds have
on-site cyclotron for their production. SPECT proved to be potent stimulants and some have
nuclides have longer half-lives (1231,13 h; high affmity and varying selectivity for DA
""Tc, 6 h), often can be supplied commer- transporters in the brain. Some compounds of
cially, and [99mTc]-labeledradioligands can be this type have been prepared as clinically use-
f prepared locally as needed. Specifically, quan- ful radiopharmaceuticals (Fig. 12.11).
titative assessment of nigrostriatal presynap- The first such agent was p-[1231]phenyl-
tic DA nerve terminal function by PET using labeled 2-P-carbomethoxy-3P-(4-iodophenyl)-
[18F]-labeled L-6-fluorodopa ([l8F1dopa, 52; tropane (54 [1231]p-CITor RTI-55; Dopa-
Fig. 12.11) has proved useful for the early di- Scan), although this agent requires about 8 h
agnosis of PD (192). for peak uptake before imaging, thus limiting
Additional radioligands have recently been its practicability (77, 191, 194). However, the
developed for probing the DA transporter pro- radioiodinated N-3-fluoropropyl analog of
teins that are highly characteristic gene prod- P-CIT [N-3-fluoropropyl-2-P-carbomethoxy-
ucts of DA neurons and nerve terminals in the 3P-(4-iodopheny1)-tropane,(55),[12311FP-CIT
basal ganglia. Cocaine (53; Fig. 12.11) binds to or [1231]ioflupane(DATScan)] has the advan-
the DA transporter (DAT) and other mono- tage of more favorable kinetics for clinical use
amine transporters, but radiolabeled cocaine because patients can be imaged 1-2 h after
736 Therapeutic and Diagnostic Agents for Parkinson's Disease
injection of the radioligand (195). Moreover, a leads discussed above, and by gradual progress
radioligand suitable for PET imaging is ob- toward a better molecular understanding of
tained by replacing the fluorine atom with the primary pathoetiology of the disease. In all
[lsF] in FP-CIT (196). [1231]-FP-CITis also of these efforts, medicinal chemists working in
being studied in patients with PD and other collaboration with basic and clinical biomedi-
neuropsychiatric disorders, and is commer- cal research colleagues will continue to play a
cially available for clinical application. central role.
Research related to PD has been directed to- We thank the Branfman Family Foundation,
ward developing more effective and better-tol- the Bruce J. Anderson Foundation, and the
erated new treatments, largely guided by the McLean Private Donors Neuropharmacology
central role of DA in the pathophysiology of Research Fund for financial support, as well as
the disorder. However, recent research has in- Susan Trofinow and Drs. Xiao-Hui Gu and
creasingly included efforts to clarify the Wennan Xiong for assistance in preparing the
pathophysiology of PD and to define its pri- manuscript.
mary causes, which remain obscure. Research
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References
Alzheimer's Disease:
Search for Therapeutics
RZESZOTARSKI
W. JANUSZ
Food and Drug Administration
Rockville, Maryland
Contents
1 Introduction, 744
2 Epidemiology, 745
3 Etiology, 745
3.1 Genetic Determinants, 745
3.1.1 Presenilins 1 and 2, 745
3.1.2 Amyloid Precursor Protein (APP),746
3.2 Genetic Risk Factors, 746
3.2.1 Apolipoprotein E W O E ) , 746
3.2.2 Susceptibility Locus on Chromosome
10, 747
3.2.3 Susceptibility Locus on Chromosome
12,747
3.2.4 Tau Protein, 748
3.3 Nongenetic Risk Factors, 748
3.3.1 Traumatic Head Injury (THI), 748
3.3.2 Hypercholesterolemia and High
Density Lipoprotein (HDL)
Cholesterol, 749
4 Prevalent Hypothesis: Amyloid Cascadecum-
Cholesterol Et Inflammation, 749
4.1 Inhibitors of Ap Production, 752
4.1.1 a-Secretase Inhibitors, 752
4.1.2 0-Secretase Inhibitors, 753
4.1.3 y-Secretase Inhibitors, 754
4.2 Statins, 756
4.3 Compounds Affecting Fibril Formation, 758
4.4 Inhibitors of Ap Aggregation, 758
4.5 Accelerators of Ap Fibril Disaggregation, 760
4.6 Ap Catabolism and Removal, 760
4.7 Modulators of Ap Neurotoxicity, 760
4.8 APP Gene-Knockdown Agents, 761
4.9 a-Secretase Shift, 761
5 Attenuation of Cholinergic Transmission, 761
5.1 Acetylcholine Esterase Inhibitors, 761
Burger's Medicinal Chemistry and Drug Discovery 5.2 Muscarinic Acetylcholine Receptor Agonists
Sixth Edition, Volume 6: Nervous System Agents and Antagonists, 762
Edited by Donald J. Abraham 5.3 Nicotinic Acetylcholine Receptor Agonists,
ISBN 0-471-27401-1 O 2003John Wiley & Sons, Inc. 764
Alzheimer's Disease: Search for Therapeutics
average duration of AD is 6-10 years, and in- genetic etiology, reflecting perhaps the effects
tercurrent infection is the most common cause of several genes, is classified as sporadic or
of death (6). late-onset AD (SAD or LOAD). When the age-
at clinical onset is considered, over 90% of AD
develops after the age of 65 (21).By definition,
2 EPIDEMIOLOGY
in all FAD, LOAD, and SAD groups the patho-
logical changes observed postmortem are assoy
Given the available demographics, AD should
ciated with the formation of SP and NFT in
be one of the most thoroughly researched dis-
the affected neurons (22). That association
eases. Regretfully, this is not the case. An au-
lends credence to Kdizuka,s (23) hypothesis-
thor may be tempted to paraphrase Charles
of protein precipitation as the common etiol-
Maurice de Talleyrand-PBrigord (1754-1838):
ogy of neurodegenerative disorders.
"You will kindly observe, ladies and gentle-
men, that I neither condemn nor defend, I 3.1 Genetic Determinants
merely narrate." According to Brookmeyer et
At present it has been postulated that between
al. (18, 19) the estimated prevalence of AD in
30-50 and 95% of the population risk for de-
the United States in 1999 was 2.44 million
velopment of AD may be attributed to genetic
with the range of 1.15 to 4.78 million. Over the
factors (21,24).However, when the autosomal
next 50 years the incidence of AD is expected
dominant mutation cases of high penetrance
to triple from about 420,000 new cases in 1999
are considered, that percentage drops to less
to 1.32 million per year. Assuming no success-
than 5% (21). The most comprehensive and
ful intervention, the prevalence of AD could be
current lists of all mutations that may be con-
expected to rise over the next 50 years by a
sidered as AD genetic determinants or risk
factor of about 3.7 to 8.94 million with the
factors are maintained by Online Mendelian
range of 4.55 to 15.81 million in the United
Inheritance in Man (OMIM) (25) and Alzhei-
States alone. With the exception of early-onset
mer Disease Mutation Database (ADMB) (26).
familial AD (FAD) the disease is age.+ correlated
and its incidence grows exponentially with
3.1 .I Presenilins 1 and 2. Mutations in the
age. In part, because of their increased longev-
genes coding for the membrane proteins pre-
ity (294), women represent and will continue
senilin 1 (PSI) and presenilin 2 (PS2) are as-
to represent the majority (68% in 1997) of the
sociated with the early-onset FAD (27).Muta-
affected population. If one adds the year 2000
tions in the PSI gene on chromosome 14 are
U.S. census figures to the population of the
responsible for 30-50% of early-onset FAD
European Union (EU-15)and Japan, the prev-
and AD with the onset before the age of 55
alence of AD in "the major pharmaceutical
years. Some mutations in the PS2 gene on
markets" for that year could be estimated as
chromosome1 have also been observed but are
close to 8.7 million (20).
not as highly penetrant as those of PSI. It
appears that intracellular binding of the amy-
3 ETIOLOGY loid precursor protein (APP) to either PSI or
PS2 leading to cell-cell adhesion (28) repre-
Although a few genetic determinants and a sents a part of their normal function. The pre-
number of genetic and nongenetic (called in- senilins and their mutated forms participate
terchangeably medical, metabolic, lifestyle, in APP processing leading to production,
environmental, etc.) risk factors are recog- among others, of 4-kDa P-amyloid peptide
nized, or suspected as causative, the etiology (AP) of varied, predominantly 40-42 (39-43
of AD remains unclear. Only a small percent- overall) amino acid (aa) lengths (29). It was
age of AD cases can be segregated within fam- postulated that the PSI mutations represent a
ilies as early-onset (<60 years of age) or late- "gain-in-function" effect and contribute to or
onset (>60 years of age) FAD representing the are responsible for an aberrant increase in
autosomal dominant mutation cases of high production of amyloidogenic (having greater
penetrance and therefore considered a genetic tendency to misfold into a p-sheet and to form
disease. The prevailing rest, lacking the clear amyloid fibrils aggregating into amyloid) AP of
Alzheimer's Disease: Search for Therapeutics
42-aa length (30). PSI and PS2 may also affect cells appears to involve the static cell-sub-
other intracellular pathways linked to cell strate adhesion and/or neurite outgrowth
death (27). At present at least 35 to 50 differ- (381, synaptogenesis, synaptic plasticity, and
ent, mostly missense, and two splicing defect promotion of neuronal cell survival (39). APP
mutations in PSI and two in PS2 have been and an APP-binding protein FE65 are in-
identified in AD patients (21, 31). The PSI, volved in the regulation of cell movement (40).
PS2, and APP mutations linked to FAD were Exposure of cortical neurons to monoclonal
reported to increase secretion of AP (299). antibody, which binds to the extracellular do-
main of APP (human, rat, or mouse), leads to
3.1.2 Amyloid Precursor Protein (APP). neurite degeneration followed by caspase-de-
Mutations in localized on the chromosome 21 pendent apoptosis (41). One wonders whether
gene for APP have also been connected to a the AD-associated neuronal cell death is at-
limited number of early-onset FAD cases (32). tributable to aberrant processing of APP over-
At least 16 homologous amyloid-like proteins producing neurotoxic AP aggregates or to APP
(APLP) and APP species have been isolated deprivation.
and characterized (32). From the identified
major APP isoforms of 695, 751, and 770 aa, 3.2 Genetic Risk Factors
the 695-aa form is expressed preferentially in
the neuronal tissue. Alternative splicing of 3.2.1 Apolipoprotein E (APOE). Although
APP provides a total of eight isoforms, with the APP and PSI mutations represent a clear
lengths of 677, 695, 696, 714, 733, 751, 752, autosomal-dominant Mandelian trait of high
and 770 amino acids (33).Seventeen single-aa penetrance, their participation in all AD cases
and one two-aa (Swedish APP,7,Nm67,,) is minuscule (<5%). The case of PS2 remains
mutations of APP have been identified so far far from being clarified but its participation is
(25).APP, a type 1 cell surface glycoprotein, is negligible. Nevertheless, the determined mu-
produced in many cells and processed through tations provide, after expression in cell and
the secretory or endosomal-lysosomal path- animals, useful tools for in vitro and in vivo
ways (34). Processing of APP is carried out by studies.
the proteolytic enzymes named secretases. The polymorphism of the ApoE gene is con-
When the cleavage takes place in the presence sidered a major, best-documented, genetic
of distinct PSI or PS2 mutations regulating susceptibility risk factor for the late-onset AD
the y-secretases cleavage sites, it may lead to (21). The last decade observed the emergence
an increase in the production of amyloidogenic of ApoE as a dominating factor in aging-re-
AD42 (Apl-42). Under "normal" conditions, lated diseases like cardiovascular disease
in the brains of nondemented elderly individ- (0) or dementia and longevity in general
uals, Ap42 is actually the ubiquitous form (42). Besides being synthesized in liver (90%of
(37). Although AP42 is considered "amyloido- ApoE in circulation), lung, ovary, muscle,
genic," one should keep in mind that it is AP42 spleen, and kidneys, ApoE is also synthesized
that is a major component of the SPs that are in the central nervous system (CNS) by glia,
of diffused "preamyloid" nature and present macrophages, and neurons (43). ApoE Ad its
in the old but otherwise AD-free individuals low density lipoprotein (LDL) receptors are
(296). It is the AP40 that dominates in the employed in transport and metabolism of lip-
more compact, dense, "mature" SP, consid- ids, and in neural tissue repair after injury.
ered by some to be of greater inflammatory Located on chromosome 19, the ApoE gene
potential at a 1 : l O ratio of AP42IAp40 (35-37). occurs in three natural allelic variants ( ~ 2E,3,
The diffuse, "preamyloid" SPs that can be ob- 84).The mature protein of 34.2 kDa is secreted
served throughout the brains of normal, aged in mono- or disialylated form but most of the
individuals are therefore considered an age- sialyl
- is removed in circulation. The most
related phenomenon (6).The question of why, ubiquitous allele, ~ 3 is, found in approxi-
when, and how the diffuse SPs mature into the mately 78% of the Caucasian population (44).
dense AD-characteristic SPs remains unan- The presence of the ~4allele (allelic frequency
swered. The normal function of intact APP in ~4 = 0.15) is considered a major genetic risk
3 Etiology
factor for the late-onset FAD, SAD (45),CVD, Ap deposition. The model thus confirmed a
and longevity. The e4 heterozygous individu- critical and isoform-specific role for ApoE in
als have a three- to fourfold increased risk of (1)AP trafficking and (2) SP formation. Con-
AD, which doubles for the e4 homozygotes versely or additionally the binding of ApoE
(19). The 82 allele (allelicfrequency e2 = 0.08) isoforms to tau may affect phosphorylation of
seems to indicate the opposite (46). that protein and lead to NFT formation (52, ,
The allelic frequency varies in different 55). The connection between ApoE isoforms,
populations, with race being only one factor their serum concentration, high intake of di-
(47). The evident North-South allelic gradient etary cholesterol, and/or high cholesterol .
-
in Europe (30-35% of Scandinavians are e4 blood levels and an increased risk of AD, car-
carriers) might have a greater impact on lon- diovascular disease, and longevity is visible
gevity than the Mediterranean diet of red and rational but remains to be proved (47).
wine, olive oil, and feta cheese (47-49). Pa- The reports of observably lower incidence
tients carrying at least one e4 allele were re- of SAD among the patients prescribed statins
ported to have an increased density of SP and (56),the differentiated cholesterol response to
AP deposition. The exact role of polymorphism statin treatment dependent on the allele
of this 299-aa, 34-kDa protein is unknown and present (571, and the surprising response of e4
probably very complex. The structural differ- carriers with CVD to statin in a myocardial
ences are relatively small. The most ubiqui- infarction survival study (58) emphasize the
tous isoform ApoE3 has cysteine (Cys) and ar- need for large enrollment studies of these
ginine (Arg) in positions 112 and 158, links. An additional link between the ApoE
respectively. In the most common variant of allele and AD comes from the increased CSF
ApoE2, the amino acid Arg15' is replaced by cortisol as a function of ApoE genotype (59).
Cys. InApoE4 Argreplaces the Cys112residue. The highest levels of cortisol that may contrib-
ApoE binds to the usual AD suspects, AP and ute to neuronal degeneration were observed in
the microtubule-associated protein tau, and e4/e4 individuals with AD.
has been localized in the SP. The purified,
nonlipidated e4 isoform was reported to have 3.2.2 Susceptibility Locus on Chromosome
higher affinity than e3 to AP (50), suggesting 10. As mentioned above 95% of AD cases show
it may act as a pathological chaperone (51,52) no clear pattern of inheritance and out of that
stabilizing the P-sheet structure of AP fibrils, group only 50% carry ApoE e4 allele(s).A two-
and impairing AP clearance. The more recent stage genomewide screen, used to locate addi-
data on binding of ApoE isoforms and AP sug- tional genetic susceptibility risk factors (60,
gest that the native lipid-associated 83 isoform 61), located additional AD locus on chromo-
binds to Ap with two- to threefold higher af- some 10. From the presented evidence the
finity than that of lipid-associated e4 (53). chromosome 10 locus modifies risk of AD not
These findings may indicate that perhaps linked to the presence of ApoE genotype (60).
ApoE is involved in the clearance or routing It appears that the locus increases the risk of
out of Ap from the CNS and that the presence AD by elevating AP42 in carriers, as might be
of e4 impairs the process, thus leading to its evidenced by its high blood levels (61).
accumulation. Either alone or in complex with
Ap, ApoE seems to have no effect on APP pro- 3.2.3 Susceptibility Locus on Chromosome
cessing (52). A critical and isoform-specific 12. Another genomic screen of families with
role of ApoE in SP formation has been demon- LOAD (62) revealed AD susceptibility locus on
strated in a mouse model (54). In that model of chromosome 12 also not linked to the presence
FAD, APPV717Ftransgenic mice expressing of ApoE e4 allele. The a2-macroglobulin and
mouse, human, or no ApoE, the neuritic de- lipoprotein receptor related protein (LRP)
generation was virtually absent in ApoEP'- genes present on this chromosome were ini-
mice, although significant AP deposition was tially considered as suspects (63).
observed. When the mice expressing ApoE4 It has been proposed that a genetic varia-
were compared with those expressing ApoE3, tion in a transcriptional factor LBP-lc/CP2/
the former showed a 10-fold greater fibrillar LSF gene and not the LRP gene is the suscep-
Alzheimer's Disease: Search for Therapeutics
tibility factor for LOAD (63). The search is far ceptibility factor, the ApoE genotype, has been
from over and one can expect additional loci to implicated as a modulator of the environmen-
be discovered as the result of genomewide tdmetabolic risk factors, such as head injury,
screening. stroke, hypertension, arteriosclerosis, and
thrombosis, serum cholesterol, or estrogen re-
3.2.4 Tau Protein. Intracellular NFTs, con- placement therapy to mention a few (68). The
sisting mostly of paired helical filaments putative environmental risk factors, consid-
(PHFs), occurring in the selected populations ered among the others, are diet (high choles-
of nerve cells before degeneration carry the terol andlor high caloric intake), smoking, al-
subunit protein: the microtubule-associated cohol consumption, depression, chronic or
protein tau in the hyperphosphorylated, insol- oxidative stress, and the like, affecting the
uble form (64). A number of familial neurode- vascular or endocrine equilibrium (69). What
generatory diseases grouped into the so-called is hoped for is to establish a link between a
"frontotemporal dementia and parkinsonism genetic susceptibility factor, or the lack of it,
linked to chromosome 17" (FTDP-17) show and an environmental trigger (e.g., head in-
tau protein pathology indistinguishable from jury) (66). The overwhelming number of fac-
that of AD (64). Although no pathogenic mu- tors, which appear to contribute to AD, en-
tations in tau protein have yet been associated couraged attempts to formulate a unified
with the AD, the abundance of NFTs corre- hypothesis including or explaining all of them.
lates with the degree of neurodegeneration Particularly of interest is the unifymg hypoth-
(65). The prevailing opinion at present is that esis of Heininger (70-73), which advances the
the presence of NFTs in AD manifests a concept of AD as a metabolic disease. The ge-
"downstream" response to the pathological netic and environmental/metabolic factors,
events initiated by that disease. In transgenic according to the hypothesis, could contribute
mice expressing the Swedish mutation of APP to the deterioration of homeostasis of the cal-
(APP695,,5N,N,,,,), the Ap amyloidosis in- cium ion-energy-redox triangle and disrupt
duces the initial stage of tau accumulation the cerebral reserve capacity under metabolic
(66). Nevertheless, given the large number of stress. The brain in AD would then attempt to
neurodegenerative disorders with tau pathol- adapt to energetic stress by releasing soluble
ogy, the mechanism of NFT formation is sub- Ap as an agent mediating the metabolic switch
ject to detailed studies (67). that preserves glucose for anabolic needs and
promoting the oxidative use of ketone bodies.
3.3 Nongenetic Risk Factors
From that point of view, AD should be consid-
Although a much higher percentage (30-50 to ered as a progeroid systemic disease and all
95%) of genetic risk factors has been postu- the factors leading to it as progeroid factors
lated, the genetic determinants of high pen- (73).
etrance listed above and linked to FAD repre- Factors for which the anecdotal threshold
sent only a small (<5%) percentage of total has been crossed are briefly discussed below.
clinical manifestations of AD. FAD, LOAD, Unsurprisingly, they link to the ApoE genotype.
SAD, and the process of aging show many sim-
ilarities, whereas the differences appear to be 3.3.1 Traumatic Head Injury (THI). A num-
of a quantitative, chronological, and kinetic ber of studies suggest a likely link between a
nature. It is highly unlikely that AD pathology risk of developing AD and THI acting as an
can originate in response to environmental environmental trigger (74). It is evident in the
challenge alone. The elucidation of interde- pig model of a brain injury that even a mild
pendency between genetic and nongenetic risk trauma with full recovery leads to the accumu-
factors implicated in AD should benefit from lation of Ap and formation of neurofilament
the completion of long-term, multicenter, inclusions (75). Similarly, an overexpression
large-enrollment clinical studies, planned or of APP was observed in the brains of gunshot
under way at the present, and from exhaustive survivors (76) and in head-injured sheep (77).
epidemiological investigations. For example, Regretfully, studies involving large groups of
the already mentioned notorious genetic sus- participants, like the MIRAGE (78) and the
4 Prevalent Hypothesis: Amyloid Cascadecum-Cholesterol Et Inflammation 749
Rotterdam (79) studies, provide contradictory provide an atlas of therapeutic targets of op-
results. The presence of ApoE s4 allele(s) is portunity a la arachidonic acid cascade. The
associated with worse neurological impair- amyloid cascade hypothesis (29, 87) is so
ment in head injury or stroke patients, sug- widely circulated and accepted that it leads
gesting that ApoE4 might actually impair neu- one to recall a classic Russian proverb: "Ha
ral tissue repair (80). 6e3n~nsen mona conosefi" ("In birdless field, ,
even . . ."). It has also awakened a vocal oppo-
3.3.2 Hypercholesterolemia and High Den- sition rejecting /3-amyloidocentrism and the
sity Lipoprotein (HDL) Cholesterol. In the be- Church of Holy Amyloid (CHA) (88).Proposed _ -
ginning, the relationship between ApoE geno- by Hardy and Allsop (89), and modified ever
type, total serum cholesterol, its high and low since, it may be coarsely summarized (Figs.
density lipoprotein fractions, and AD was the 13.1 and 13.2) as follows.
subject of narrow epidemiological studies in APP is expressed in neurons and glia,
old and very old. A small Finnish study in- where it is synthesized and cotranslationally
ferred that, independent of genetic status, inserted into the endoplasmic reticulum (ER)
high serum cholesterol represents an indepen- (90). Of all the APP mutations identified, most
dent risk factor (81). The conclusion of the of them take place in the immediate vicinity of
equally modest in size PAQUID study sug- the N- and C-terminal of the AP fragment or in
gested that, independent of genetic and other the middle of its @-turnarea (Fig. 13.1).
risk factors, elevated high density lipoprotein Two distinct paths carry out the proteolysis
cholesterol (HDLC) significantly reduced the ofAPP. Both appear to be regulated by numer-
risk of dementia of AD and vascular type (82). ous factors including stimulation of acetylcho-
The same study reported that age, low HDLC, line, serotonin, glutamate, and neuropeptide
the presence of apoE4 allele, low education, receptors (91, 92). The dominant, nonamyloi-
and low wine consumption were significantly dogenic a-pathway involving a-secretase,
associated with AD. Studies of patients taking cleaves APP between Lysl6 and Leu 17 of the
cholesterol level-lowering 3-hydroxy-3-meth- Ap domain in the extracellular space, and pro-
ylglutaryl coenzyme A reductase inhibitors duces a soluble APPa fragment (sAPPa) and
(statins)confirmed its, perhaps pivotal, role in aC-terminal fragment (aCTF). aCTF (also
the pathophysiology of AD (56, 83, 84). The known as C88 for 88 aa) is in turn cleaved by
reports indicated that the patients on statins y-secretases to produce fragments p3 (from its
did show a reduction in AD incidence (56,831. 3-kDa molecular weight, synonymous with
Epidemiological observations are begin- some Apx-40 and Apx-42). Most (90%) of the
ning to be confirmed in vitro. For example, in APP is processed that way. One should keep in
APP751-transfected HEK 293 cells the ele- mind that the activity of y-secretase indicates
vated, by various means, level of cholesterol a relatively distance-specific [from the trans-
caused a dramatic reduction in secretion of the membrane domain (TM)], not aa-specific, (93)
soluble APP fragment sAPPa (84). At the cel- enzyme.
lular level it is by now proved that cholesterol The second amyloidogenic p-pathway em-
depletion (by 70%) in cultured neurons by ploys p-secretase [beta-site APP cleaving en-
combination of lovastatin treatment and zyme (BACEl), Asp2, memapsin21, which is
methyl-p-cyclodextrinextraction reduces pro- largely present in the distal Golgi membrane
duction of Ap below detectable levels (85) by (94). p-Secretase is responsible for cleaving
shifting the APP processing to a-secretase APP at the N-terminus of AP between Asp1
(86). and Met-1. Thus, the generated soluble
APPP fragment (sAPPp) leaves the scene,
4 PREVALENT HYPOTHESIS: AMYLOID whereas the membrane-bound PC-terminal
CASCADECUM-CHOLESTEROL ET fragment (PCTF or C99) is in turn cleaved by
INFLAMMATION y-secretases to produce Ap of 40- or 42-aa
length. The y-secretase cleavage of C99 leaves
The delineation of the AD pathogenic cascade, an unstable CTFy of 57- to 59-aa residues (95).
leading to neuronal death by apoptosis, should Both AP40 and AP42 are produced in normal
75 0 Alzheimer's Disease: Search for Therapeutics
Extracellular I transmembrane
p-secretase a-secretase
I y-secretase(s?)
and SAD individuals but in different propor- ported to be significantly lower in the CSF and
tions (37). The ratios differ, with AP401AP42 plasma of AD patients (99). The sites of pro-
ratio of 10 in SAD. In nondemented geriatric duction of AD of various lengths and its
controls Ap42 was dominant. The soluble Ap N-truncated fragments were the subject of nu-
are present in cerebrospinal fluid (CSF) of nor- merous investigations. According to Green-
mal and AD individuals at similar levels field et al. (go), APP translocated into the lu-
(10-8-10p10 M ) (96). Their serum and CSF men of ER forms N-truncated Apx-42, which
levels were suggested (971, but do not appear stays in the ER in an insoluble form. The un-
to be indicative of the presence of AD (98). processed APP moves through the Golgi appa-
Naturally occurring antibodies to AP were re- ratus (GA) to the trans-Golgi Network (TGN)
I psecretase
I a-secretase
I Y-secretase
Y-secretase path
CTFB / c&xI
where AP40, AP42, and their N-truncated teristic of being processed in the transmem-
APx-40 and Apx-42 peptides are generated. brane domain (108, 110-113). That discovery
The unprocessed APP, PCTF (C99),and solu- presents the seekers of y-secretase inhibitors
ble AP are packaged for release into post-TGN with a predicament of selectivity, how to pre- -
secretory vesicles. When released they can vent processing of APP without affecting the
travel to the surface of neuronal cells, where fate of Notch. -F
a-secretase generates the bulk of sAPPa One of the remaining questions is how the
(loo),secreted together with AP40, AP42, and Aps, generated inside the cell, pass through or
the N-truncated fragments. The unprocessed detach from the cell membrane and leave for
APP and the a- and P-CTFs can be internal- extracellular space where the SPs are formed.
ized to the endosome or lysosome and re- To that effect, an ATP-binding cassette (ABC)
turned to TGN for additional processing. That transporter, a p-glycoprotein (p-gp), was pro-
pathway was confirmed when production of posed as an Ap efflux pump (114). Equally im-
Ap40 and AP42 was abolished by preventing portant is the question of degradation of the
C99 from leaving ER (101). generated AP. One of the candidate enzymes
The M P a fragment was reported to have was reported to be neprilysin, one of the major
neurotrophic and neuroprotective (against endopeptidases in the brain (115) and the fast-
glucose deprivation and glutamate toxicity) est to degrade both Ap40 and AP42 (116). In
properties, to stimulate neurite outgrowth, the brains of neprilysin-deficient mice the ele-
and to regulate synaptogenesis (39, 102). The vated Ap located in the same order and regions
memory-enhancing properties of sAPPa were as reported for humans (hippocampus > cor-
reported in normal and amnestic mice (103). tex > thalamus/striatum > cerebellum).
Recently, the sAF'Pa fragments originating Once secreted, the Ap may aggregate sponta-
from APP751 and APP695 were identified as neously, with more hydrophobic AP42 showing
ligands for the class A scavenger receptor (SR- greater rate of assembly (96,117).The length of
A), suggesting a possible way for their clear- AP is not the only determinant of the rate of
ance (104). Evidently, the Kunitz-type pro- aggregation. The studies of a pathogenic muta-
tease inhibitor (KPI) domain present on tion of APP, the "Arctic" mutation (E693G1,
sAPPa originating from APP751 is not re- was reported to produce AP, which forms proto-
quired for binding. fibrils at a much greater rate than does the wild-
The biological activity, if any, of sAF'PP is so type (118).To form the core of SP after being
far unknown. The role or contribution of generated through proteolytic cleavage of APP,
N-truncated AP (p3, for example) to AD is the AP have to undergo a continuous process of
subject of a hotly contested debate, entitled the structural transformation (117, 119). The re-
shorter amyloid cascade hypothesis (87,105). leased AP initially stay in solution as a random
A mutation in either APP or PSI or even coil or a-helix (120, 121). The transition from
PS2 (29) alters the way APP is processed, ei- random coil or a-helix to p-sheet conformation
ther in favor of increasing the ratio of amyloi- begins the process of oligomerization and fibril
dogenic AP42 (which predominates in the im- formation, with AP42 assembling more readily
mature, diffused SPs) or simply increasing the than AH0 (122). A stable AP dimer appears to
production of AP4O. Indeed, some FAD-linked be the building block of the AP filament. The
mutations in PSl(A9) lead to formation of growth of the fibrils proceeds by the addition of
higher levels of AP42 in TGN-to-plasma mem- Ap dimers and tetramers (123). The amyloid
brane transport vesicles (106). Presenilin is fibrils thus formed are straight, unbranching fi-
necessary for production of AP as it complexes bers of 70-120 A in diameter and of indetermi-
with the CTFs (C99 and C83) (107). The evi- nate length (124).
dence exists of a physical interaction between The in vitro studies of that process using
PSI and Notch receptors, Notch being a pro- synthetic Ap40 at low pH suggested a kinetic
teinlike APP, a large single-transmembrane model in which (1) a nucleation step is re-
protein important in development and differ- quired, (2)nuclei are produced from seeds and
entiation of adult self-renewing cells (108, from AP micelles, and (3) fibril elongation is
109). Both proteins have an atypical charac- achieved by irreversible binding of AP mono-
Alzheimer's Disease: Search for Therapeutics
mers to the fibril ends (125). Concentration of rectly in contact with Ap, the astrocytosis was
Ap appears as a critical parameter for micelle evidently doubling the number of apoptotic
and fibril formation. Either AB monomer or cells (132).
dimer polymerization models could be used to The presence of reactive microglia, macro-
determine the rate of fibril formation under phage-like phagocytic cells, associated with
given
- conditions (126). The maximum rate of the mature, neuritic SP is one of the charac-
formation was given as t , , for AP42 of approx- teristics of AD. The presence of these main
imately 18 min and for AP40 as approximately inflammatory response cells in AD represents
6 h. ApoE and antioxidants decreased the final the foundation of the inflammation hypothe-
-
amount of fibril formed in a dose-dependent sis. The question whether reactive microglia
way, although only ApoE extended the time to are there to help or to harm is still open. An-
other question is whether they appear at the
proceed to equilibrium (126). These findings
beginning or at the end of development of AD
are in agreement with the observation that
(133). It is the fibrillar AP that induce strong
free radicals, some perhaps generated by the cellular activation in primary cell culture
A0 fragments (127), appear to promote AP (134).Small aggregates of fibrillar AP and sol-
fibril formation (126). As always, there are uble AP, despite minor differences in interac-
also reports that Ap does not form spontane- tion with microglia, do not show a great degree
ously free radicals (128). The other problem of degradation after internalization (96).
with the fibrillogenesis models is that a num- Some of the fibrillar AP might be initially de-
ber of Apx-40142 N-terminal fragments have graded by microglia but the degradation stops
been reported in the SPs and they appear to after 3 days. The soluble AP is released rapidly
aggregate much faster than do the full-length after internalization and very little is de-
Ap (129). The identified pyroglutamyl N-ter- graded. Almost accepted is the hypothesis that
mind fragments AP3pE and APllpE, by ag- the neurotoxicity of Ap is not linked to its mo-
gregating faster and being more resistant to nomeric forms or insoluble amyloid precipi-
proteolysis than the full-length AP, would tate but to its soluble intermediate oligomers
make perfect seeds for amyloid formation of full-length AP and its fragments (131, 135,
(130). From in vitro assays a number of com- 136).
pounds have been identified to affect that con- All the above-described events happen in
tinuous process of assembly. A conclusion was the environment sensitive to cholesterol levels
also drawn that the processes of fibril assem- linked to or influenced by the genetic risk fac-
bly and disassembly appear to be distinct tor ApoE. The presence of the ApoE4 allele,
(119). The process of AP fibrillogenesis is still ~ 4 1 in
~ 4particular, indeed correlates with the
waiting for its Melvin Calvin to unravel it. degree of Ap deposition (119,137-139).
Secreted AP and their fragments in various
oligomeric forms are considered to be, to a dif- 4.1 lnhibitors of AP Production
ferent degree, cytotoxic and to induce apopto-
sis in neuronal cells (131). 4.1 .I a-Secretase Inhibitors. Proteolysis of
The transformation of immature, diffuse APP by a-secretase takes place at the cell sur-
SPs composed almost exclusively from amyloi- face, manifesting an end to its processing by
dogenic AP42 into the mature, neuritic SPs is the cell (100). Inhibition of a-secretase would
poorly understood. It appears to be a continu- serve no purpose in AD but, as one of three
ous process, with numerous intermediates ob- enzymes or groups of enzymes processing
servable in the postmortem brains. The con- APP, it deserves attention. Although not iso-
struction and composition of mature, neuritic lated as yet, it appears to belong to a group of
SPs are fairly complex processes, in which the zinc metalloproteases and to be closely re-
core consists mainly of AP40/42 and Apx-40142 lated, by inhibition profile, to angiotensin-con-
fragments and is surrounded by dystrophic verting enzyme (ACE) secretase and to colla-
neurites, activated microglia, and reactive as- genase (100, 140). Some of the investigated
trocytes (96). In neuronal cultures condi- (140) a-secretase inhibitors, such as batima-
tioned by AP-treated astrocytes, but not di- stat (1)and marimastat (21, initially devel-
4 Prevalent Hypothesis: Amyloid Cascadecum-Cholesterol Et Inflammation
(2)
0
fH3k
H
CH3
H cHT
CONHOH CONHOH
(4)
NHCH3
CONHOH
HO
(5)
oped as antineoplastic agents, and their ana- produced no profound phenotypic defects with
logs can be seen in Fig. 13.3. simultaneous reduction of Ap (142). The fact
that the knockout mice developed normally
4.1.2 p-Secretase inhibitors. p-Secretase, and showed no consistent differences with
also known as memapsin 2 or p-site APP their wild-type littermates makes p-secretase
cleaving enzyme (BACE1) [BACE2 was a perfect target of opportunity for the develop-
mapped to Down syndrome (14111, the pri- ment of AD therapeutics. The observed ab-
mary membrane-anchored aspartic protease sence of astrocytic BACE immunoreactivity in
in mammalian brain, does not appear to be of young transgenic Tg2576 mice (143) and the
importance in mammals (142). The loss of consecutive expression at later stages as a pos-
p-secretase activity in BACE knockout mice sible result of astrocytes activation should en-
Alzheimer's Disease: Search for Therapeutics
I phr.r HNfi
HOOC COOH
R = H (OM99-1) (6)
courage vigorous pursuit of that enzyme. The 13.111 was found to be a perfect substrate for
accumulated knowledge and experience with recombinant memapsin 2 (144). X-ray crystal-
the approved peptidomimetic HIV protease in- lography studies of (7) bound to memapsin 2
hibitor drugs is available. Yet, despite the en- permitted the design of inhibitors of lower mo-
couraging experiments with the knockout lecular weight (145).The most potent of them
mice, the trepidation persists that there might (8-11,Fig. 13.4) have Ki values between 2 and
be a role for p-secretase in normal physiology 10 nM.
and that the selectivity against other pro-
teases might be difficult to attain. 4.1.3 y-Secretase Inhibitors. PSI-associ-
The first reported p-secretase inhibitors, ated y-secretases participating in processing
OM991 and OM992 (144) (6 and 7, Fig. 13.4), of APP are also needed for proteolysis of an-
were designed by use of the template of the other transmembrane protein, Notchl (146,
Swedish mutant APP. The Swedish mutation 147). Both proteins appear to be competitive
site of p-secretase action [SEVNLDAEFR in- substrates: APP, or rather its C99 fragment,
stead of SEVKMDAEFR wild-type (see Fig. for APs and Notchl for a signaling molecule
4 Prevalent Hypothesis: Amyloid Cascadecum-Cholesterol Et Inflammation
Notch C-terminal domain (NICD) (148). The clear translocation but to preserve Notchl
dipeptide aldehyde (12, Fig. 13.5) (149) served signaling (156).This was further confirmed in
as a lead to a group of inhibitors, with one fetal thymus organ cultures, where they inter-
compound (13)showing 10-fold improvement fered in T-cell development, indicating loss or
in IC,, values (150).A study of difluoro ketone reduction of Notchl function (109). An aspar-
peptidomimetics developed from a lead (14) tyl protease transition state mimic, L-685,458
provided a valuable insight into the mecha- (17) was reported to be a potent (IC,, = 17
nism of the action of y-secretases (151-153). nM) inhibitor of y-secretase (157). One of the
At a micromolar level these compounds calpain inhibitors, MDL28170 (18), was re-
blocked the formation of AP40 and AP42 and ported to block production of AP40 but not
increased formation of AP42 at a subinhibi- AP42 (158). The search has been recently en-
tory level (152,153).A similar group of dipep- hanced by introduction of a y-secretase assay
tide aldehydes (15) confirmed the findings based on detection of the elusive and unstable
(154). A group of fenchylamine sulfonamides CTFy (95).
(16) was reported to inhibit y-secretases at mi- The first group of y-secretase inhibitors
cromolar concentrations in HEK293 cells free of interfering with Notch processing (19-
transfected with the double-mutant form of 21, Fig. 13.6) or endoproteolysis of presenilins
human APP (155). reduced AP40 and AP42 in favor of C99 and
The first peptidomimetic y-secretase inhib- C83 CTFs arising, respectively, from P- and
itors and aspartate mutations in PSI were re- a-secretase action (159). Their design was
ported to impair Notchl proteolysis and nu- based on observations that (1)disruption of
Alzheimer's Disease: Search for Therapeutics
the chymotrypsin-like activity of the protea- CoA) reductase] are prescribed chronically to
some correlates with the inhibition of AP se- the elderly (162). The initial success of statins
cretion (160) and (2) that the serine-protease was impressive and the FDA approved six of
inhibitor AEBSF modulates AP recovery them (22-27, Fig. 13.7) (163).Although all ap-
(161). At 100 pit4 concentration, compounds proved statins have been associated with a
(19),(20), and (21) achieved 70-80% inhibi- very low incidence of rhabdomyolysis, only
tion of total Ap40142 recovered. one, cerivastatin (271, was withdrawn from
the U.S. market because of it (164, 165). The
4.2 Statins ability of statins to reduce the levels of Aps in
Developed to control elevated low density li- uitro and in uiuo was reported (85, 86, 166,
poprotein cholesterol, statins [the inhibitors 167) but at doses exceeding the approved hu-
of 3-hydroxy-3-methylglutaryl-CoA (HMG- man dosage. A cyclohexylalanine-based statin
4 Prevalent Hypothesis: Amyloid Cascadecum-Cholesterol Et Inflammation
NaOOC
HO
(22) Lovastatin [mevacor] (1 987) (23) Pravastatin sodium [pravachol] (1991)
COONa
CH3
(28, Fig. 13.8) was trawled out from the patent justice, the cited study did not show any differ-
literature along a lipophilic tetraline (29) as ence in total cholesterol and HDL cholesterol
an inhibitor of AP formation in cell culture either.
(155). A small study of age- and gender- The actual mechanism of how the statins
matched asymptomatic Japanese patients, ei- affect AD remains unknown. For amyloid cas-
ther on pravastatin or simvastatin or na'ive for cade enthusiasts, it is possible that the re-
an unknown length of time, did not show any ported reduction in the AD prevalence in pa-
observable difference in serum A@ levels tients on statins (56, 83) reflects the
(168). One should remember that the AP lev- cumulative effect of small but effective de-
els in serum or CSF are not considered the creases in Ap production over a long period of
best measure of therapeutic progress. To do chronic administration. The explanation of
Alzheimer's Disease: Search for Therapeutics
experimentally observed shift, favoring ecule will self-assemble or complex with simi-
y-secretase processing of APP, attributed to larly attractive molecules. Aggregation (self-
lowering of membrane cholesterol, might be assembly) of APs and their neurotoxicity are
the peculiar nature of y-secretase (84). The intimately linked but may be affected by the
y-secretase site of APP processing belongs to a different groups of compounds. If the SPs are
very limited number of intrarnembrane pro- responsible for initiation of neurodegenera-
teolytic cleavage sites, requiring perhaps a tion, prevention of formation of AP fibrils, or
cholesterol-rich environment such as a lipid their disassembly, should have a therapeutic
raft domain (84, 169). Fluorescence micros- effect. If the SPs are just the deposits of assem-
copy studies of PC-12 and SH-SY5Y cell lines bled AP and cellular debris, taking them apart
exposed to exogenous Ap revealed an inverse might do the opposite (173). The division of
correlation between membrane cholesterol
compounds affecting the AP fibril formation
level and AP cell surface binding and subse-
into inhibitors of AP aggregation (self-assem-
quent cell death (170). At present the debate
bly) and accelerators of AP disaggregation is
continues (171a,b) while the clinical trials of
spurious, but practiced by many.
statins for AD indication proceed (172). New,
safer, and significantly superior statins, which
reduce total cholesterol, low density lipopro- 4.4 Inhibitors of AD Aggregation
tein cholesterol, and apolipoprotein B, and in- To inhibit AP aggregation into amyloid is to
crease high density lipoprotein cholesterol prevent misfolding of soluble AP. Once the
more effectively than the presently available misfolding took place the aggregation is only a
five, might be in the NDA pipeline (http:// matter of time and far more difficult to control
www.ndapipe1ine.com). Their effectiveness to (174). So far only nicotine (30, Fig. 13.9) and
reduce the incidence of AD may be hoped to melatonin (31)appear to be affecting the con-
similarly improve. formation of soluble Ap (175-177). Antioxi-
dants, such as antituberculosis antibiotic ri-
4.3 Compounds Affecting Fibril Formation
fampicin (32) or a-tocopherol, were reported
At physiological pH Ap carries seven positive to inhibit aggregation and neurotoxicity of Ap
and seven negative charges in addition to two in vitro (178-180). A monoamine oxidase in-
hydrophobic domains (123). That kind of mol- hibitor, selegiline (33, Fig. 13.9), may also be
4 Prevalent Hypothesis: Amyloid Cascadecum-Cholesterol Et Inflammation
CH3
NH
0
considered to belong to that group. The role of basement membrane, were reported to inhibit
ApoE as antioxidant and fibril formation in- AP fibril formation by inducing a random coil
hibitor is still under investigation. It appears structure (183, 184). Like ApoA-I, laminin
that it has a biphasic effect that inhibits fibril binds AD with high nanomolar affinity.
formation at lower concentrations, while ac- The review by Findeis (185) and the work
celerating at higher micromolar AP concentra- of Bohrmann et al. (117) start a very diversi-
tions (126, 181). A constituent of high density fied list of compounds affecting aggregation of
lipoprotein complexes, apolipoprotein A-I AP in vitro at micromolar concentrations.
(ApoA-I),has a higher affinity to ApoE than to Given the high affinity of Ap for itself it was
AD, which it binds in a saturable, specific, and used as a lead to a number of D-aa analogs,
reversible manner with K, of 6 nM (182). providing stable compounds such as (34) and
Laminin and entactin, the components of (35) (186).
Alzheimer's Disease: Search for Therapeutics
4.5 Accelerators of AP Fibril Disaggregation levels were constant in all brain areas and
were not decreased with AD. That observation
Once an AP fibril is formed and becomes a
led to the conclusion that the lower levels of -
component of SP, it may be impossible to dis-
NEP mRNA observed in hippocampus and the
aggregate it and to return it to its original ran-
temporal gyrus were not related to neuronal
dom coil conformation. Although the process
loss but were preordained. Both areas are the
of assembly of AP fibril appears distinct from
high SP areas of AD brain. In the NEP gene-"
the process of disassembly, the same or similar
disrupted mice, the degradation of exogenous
groups of compounds may affect them both.
and endogenous AP was affected in a gene
The simplest of disaggregation agents 2,4-di-
dose-dependent manner (115). The highest -
nitrophenol and 3-nitrophenol were shown to
levels of AB in NEP-deficient mice were in
cause, at micromolar concentrations, com-
hippocampus and cortex and the lowest in cer-
plete disaggregation and to block neurotoxic-
ebellum, reflecting the AD pattern. Immuno-
ity of AP to rat hippocampal neurons in cul-
histochemical localization of NEP in postmor-
ture (187). In vivo, coinjection of these
tem human brain tissues indicated the same
nitrophenols with AP into rat hippocampus re-
pattern (193). The hippocampus and the asso-
duced (86 +- 17%) the volume of amyloid de-
ciation cortices, the sites most affected in AD,
posits. According to the authors, the hydro-
showed the lowest NEP immunoreactivity
phobic interaction of CTF nonpolar aa
compared to that of the primary somatosen-
sequences responsible for fibril formation is
disrupted by the hydrophobicity of nitrophe- sory and visual cortices. The question remains
whether the observed downregulation of NEP
noles.
activity reflects aging or is particular to AD.
The N-methylated congeners of hydropho-
The upregulation of NEP activity in the af-
bic core domain Ap16-22 (36, Fig. 13.9) were
fected areas of Ap deposition without affecting
reported to inhibit fibrillogenesis and also dis-
the NEP involved in the metabolism of numer-
assemble preformed fibrils (188).
ous regulatory peptides of the nervous, cardio-
vascular, inflammatory, or immune systems
4.6 AP Catabolism and Removal
presents another question. Another metalo-
One assumes that even a slight shift in the protease, endothelinanverting enzyme (ECE-I),
anabolic-catabolic steady state of AP produc- was identified with similar properties of de-
tion and removal, persisting over a prolonged grading Ap (194).
period of time, will eventually produce a Learning and memory deficits developed
pathological change. The hypothesis that the with aging in AP overproducing SAMPB mice
differential vulnerability of distinct parts of have been reversed with intracerebroventric-
brain to Ap deposition and neuronal destruc- ular (i.c.v.) or even i.v. injections of AP anti-
tion is based on altered metabolism of Ap was sense phosphorothiolate oligonucleotide (195).
behind the extensive search for the specific en-
4.7 Modulators of Aj3 Neurotoxicity
dopeptidase catabolizing Ap. Howell et al.
(189)reported extensive catabolism of AP by a APs, soluble as either random coil or a-helix,
neutral endopeptidase that leaves the APP in- do not show any neurotoxicity (196, 197),
tact. A thermolysin-like zinc metalloendopep- which appears to be related to the degree of
tidase, neprilysin (NEP) (1901, was deter- their aggregation (198). The inhibition of var-
mined to be involved in Ap degradation by the ious cholinergic neurotransmitter functions
use of multiple radiolabeled synthetic AP42 by AD takes place at the very low concentra-
injected into rat hippocampus (191). Catabo- tions below the neurotoxic treshold. The
lism of endogenous AP42 was arrested by in- fibrils of AP are toxic to cultured neuronal
fusion of NEP inhibitor and led to pathological cells and lead to their death, probably by ob-
deposition. NEP mRNA levels were measured served rapid production of hydrogen peroxide
in the brain and peripheral organs of AD and and lipid peroxidation (199). Oxidative stress
control cases (192). Simultaneously measured appears to be a proximal event in the AD (200)
were levels of the mRNA for the neuronal and the oxidative damage appears to be the
marker microtubule-associated protein 2. Its greatest in the beginning of the disease and
5 Attenuation of Cholinergic Transmission
decreases with the disease progression and favor of p-secretase in AD. Whether the in-
formation of SP and NFT (201). crease in production of AP40 and/or AP42 is
The recognition sequence of AP leading to achieved at the expense of the a-secretase pro-
aggregation consists of residues 16 to 20, that cess is also unknown. In addition, the process-.
is, KLVFF (135). The residue itself had no ef- ing of APP in human neurons appears to differ
fect on aggregation kinetics and only some ef- considerably from rodent CNS primary neu-
fect on Ap neurotoxicity. Coupling of the rec- ron cultures and continuously dividing celf
ognition sequence with a disrupting lysine types (34). Different sites of a-, P-, and y-secre-
hexamer (KLVFF-KKKKKK)dramatically ac- tase activity require codistribution of APP,
celerated AP aggregation, thus generating in- with respective secretases to affect the relative-
soluble AP precipitate, and significantly im- utilization (209). The removal of APP from
compartments with p-secretase activity to com-
proved protection against AP neurotoxicity
partments with a-secretase activity should in-
(135, 136). The inference from these findings
crease the release of sAPPa and, it is hoped,
is that the insoluble AP aggregate is not the decrease the release of APs (210-212). Phor-
molecule to be blamed for toxicity but, rather, bol esters acting by activation of protein ki-
a soluble intermediate oligomer generated in nase C (PKC) enhance the release of sAPPa
the process (136). from cortical synaptosomes (213, 214). The
The observation that the N-terminal part observation that muscarinic agents may act in
of AP is not associated with the fibril forma- a similar manner (215) provides for collateral
tion and remains outside of the fibril core (202, efficacy of acetylcholine esterase inhibitors. A
203) led to generation of specific monoclonal phosphatidylinositol 3-kinase inhibitor wort-
antibodies (mAbs) against that part of Ap mannin decreased release of both Ap and
(204-206). Used on PC12 cells the mAbs were sAPPa in N2a neuroblastoma cells expressing
shown to disaggregate Ap fibrils, maintain AP either wild-type APP or the Swedish FAD mu-
solubility, and prevent neurotoxic effects tant variant (209). Testosterone was reported
(207). to increase the secretion of sAPPa without af-
fecting the total amount of cellular APP (216).
4.8 APP Gene-Knockdown Agents
5 ATTENUATION OF CHOLlNERGlC
Gene knockdown, unlike gene knockout, per- TRANSMISSION
mits fine-tuning of gene expression. So far
only site-directed antisense oligonucleotides, A failing cholinergic transmission may be at-
directed at the AP region of APP, were tried in tenuated either by (1)prevention of ACh de-
a strain of mice spontaneously overexpressing struction, (2)supplementation of diminishing
APP (208). Administered by i.c.v. injection,
amounts of ACh released with exogenous, or
with or without antibody, the antisense phos-
(3) blocking of feedback mechanism regulat-
phorothiolated oligonucleotides directed at ing the release or synthesis of ACh.
the midregion of Ap reversed deficits in learn-
ing and memory. Given alone, these oligonu- 5.1 Acetylcholine Esterase Inhibitors
cleotides reduced APP levels by 43-68% in the
arnygdala, septum, and hippocampus. It has Acetylcholine esterase inhibitors are the first
been further proved that the phosphorothio- and so far the only group of drugs approved for
lated oligonucleotides can cross the blood- AD indication. With the advancing destruc-
brain barrier, and can be given in effective tion of the cholinergic network, the overall
doses by i.v. route (195). quantity of ACh released decreases below the
level necessary for transmission. Inhibition of
acetylcholine esterase (AChE) may prolong
4.9 aSecretase Shift
the life of Ach in the synapse, modulating the
The a-secretase-processing route is dominant strength and the duration of the signal. The
under normal conditions and it is unknown U.S. FDA has so far approved four AChE in-
whether and how that process is impaired in hibitors. The first on the market, tacrine hy-
Alzheimer's Disease: Search for Therapeutics
-
COOH
HBr
-
HOOC
(39) Rivastigmine tartrate (exelon)(2000) (40) Galantamine hydrobromide (reminyl)(2001)
drochloride (Cognex, 37, Fig. 13.10), was ap- ceptors, if any are left, thus reducing the re-
proved in 1993. It did inhibit the AChE and lease of endogenous ACh. Because of their
butylcholine esterase but the hepatotoxicity nonselective site of action their use eventually
limited its use. Donepezil hydrochloride ( h i - leads to M3 AChR stimulation and untoward
cept, 38) approved in 1997, has a longer half- gastrointestinal effects (nausea and vomit-
life time that permits once-a-day dosing and ing). They do provide some improvement
lower hepatoxicity. Rivastigmine tartrate (Ex- when prescribed to patients with early to mild
elon, 39), approved in 2000, has a similar pro- AD. Their clinical benefits in advanced AD re-
file. Galantamine hydrobromide (Reminyl, main to be proved and seem doubtful. Thus,
40), initially extracted from daffodils, was ap- given the substantial investment in taking
proved in 2001. All four AChE inhibitors have these drugs to the market, the extended pre-
shown in well-controlled trials a statistically and postapproval search for collateral efficacy
significant improvement of at least three of these drugs, such as in affecting APP pro-
points on the Alzheimer's Disease Assessment cessing or allosteric sensitization of nicotinic
Scale, measuring independently the cognitive receptors, seems justified (218-221).
(ADAS-Cog) and noncognitive function. The
ADAS-Cog part of the scale scores between 0
5.2 Muscarinic Acetylcholine Receptor
and 70 points, with zero meaning the patient
Agonists and Antagonists
made no errors at all and 70 meaning the pa-
tient is profoundly demented. The normal in- An MI-selective agonist would be preferred
dividuals will usually make some errors, scor- for two reasons: (1)to activate M1 receptors in
ing between 5 and 10. One can therefore the affected areas and (2)to activate a-secre-
conclude that the relief provided by this group tase, shifting the processing of APP from pro-
of drugs is modest. Thus, the results obtained duction of AP, as M1 muscarinic agonists are
by the approved AChE inhibitors are compa- reported to do (222, 223). The problem with
rable to those produced by the porcine brain- the muscarinic agents is their selectivity. A
derived hydrolysate, marketed outside the muscarinic agonist could be selective to a
United States as Cerebrolysin (217). point in a functional assay and completely
The future of therapy with these drugs is in nonselective in a binding study. Every known
question. Although they prevent ACh from muscarinic agonist tried in preclinical studies
rapid hydrolysis, eventually the saved ACh ac- possessed an agonist, partial agonist, and an-
tivates the presynaptic autoinhibitory M2 re- tagonist portfolio that was impossible to un-
5 Attenuation of Cholinergic Transmission
tangle and was thus dose limiting. Many of properties, provided that the compound could
them, such as sabcomeline (SB-202026, 41, be made CNS selective. Otherwise, the M2
Fig 13.111, milameline (RU35926, 42), xa- AChR in the cardiovascular system would be
nomeline (LY246708, 43), or talsaclidine affected. A number of compounds claim to pos-
(WAL 2014,44), have been tried in the clinic sess that combination. For example, L-687,306
and failed. The reasons for failure were insuf- (45) and L-689,660 (46) were reported to be
ficient efficacy (lack of statistically significant M1 agonists and M2 antagonists in functional
improvement of cognitive functions) and side studies (225) and nonselective in binding stud-
effects. Even the most selective M1 agonist ies (221).
talsaclidine was withdrawn from clinical stud- The antagonists SCH72788 (47) and
ies, thus placing the muscarinic approach to SCH57790 (48) were reported to have greater
AD in doubt (224). affmity to M2 versus M1 AchR, and improved
Even more desired would be a combination performance in rodent models of cognition
of an M1-selective agonist with M2 antagonist (226,227).
Alzheimer's Disease: Search for Therapeutics
duced by Ach and nicotinic agonists, and also potentiation in hippocampus in vivo (250).
decrease the receptor desensitization (230). The mechanism of action of far more potent
LY392098 and LY404187 was suggested to be
different from that of Ampakines, in that the
6 MODULATION OF AMPA RESPONSES
magnitude of the enhancement is time depen-
dent, increasing with continued agonist expo-
Episodic, short-term memory is the first to go
sure (246).In either case, despite the frequent
with the progression of AD and other neuro-
forward-looking statements, compounds like
degenerative diseases (240). Compounds such
the AChE inhibitors are nothing more than
as aniracetam (53,Fig. 13.131, piracetam (54)
palliatives free of disease-modifying proper-
or S18986-1 (55) (2411, CX516 (56) (242),
ties.
CX509 (57) (2431, or CX614 (58) (2441, and
LY392098 (59) (245) or LY404187 (60) (246)
positively modulate a-amino-3-hydroxy-5- 7 ESTROGENS AND ANDROGENS
methyl-4-isoxazole propionic acid (AMPA) re-
ceptors. In addition S18986-1 enhances The belief that estrogen, and especially the
AMPA-mediated release of noradrenaline postmenopausal estrogen replacement ther-
(247). The term nootropic agents is often ap- apy (ERT), may reduce the risk of AD is very
plied to these compounds and one group was strong. The idea was advanced by many short-
even registered as Ampakines (241, 248). term, open-label studies but rejected by the
Compounds such as aniracetam, piracetam, or well-controlled, long-term studies (251-254).
S18986-1 positively modulate the AMPA re- The discrepancy between the in vitro promises
ceptors by suppressing the desensitization and the in vivo hopes is glaring (255,256). In
process (249). The Ampakines also facilitate cultured neurons 17P-estradiolreduced secre-
the induction of NMDA-dependent, long-term tion of AP40142 (257)and protected them from
Alzheimer's Disease: Search for Therapeutics
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CHAPTER FOURTEEN
Cognition Enhancers
CMK N. EID, J R . .
YONG-JIN
WU
Division of Central Nervous System Chemistry
Bristol-Myers Squibb Pharmaceutical Research Institute
Wallingford, Connecticut
GENE G. KINNEY
Department of Neuroscience
Merck Research Laboratories
West Point, Pennsylvania
Contents
1 Introduction, 780
1.1 Dementia, 780
2 Assessing Cognition, 780
2.1 Animal Models of Cognition, 780
2.2 Measurements of Human Cognition, 781
3 Cognition-Enhancing Therapies, 781
3.1 Therapeutic Approaches, 781
3.2 Clinically Approved Agents, 781
3.2.1 Acetylcholinesterase Inhibitors (AChEI),
781
3.3 Exploratory Approaches, 781
4 Ion Channel Modulation of Neurotransmission,
782
4.1 GABAA/BzReceptor Complex, 782
4.1.1 Physiology and Pharmacology of the
GABAA/BzReceptor Complex, 782
4.1.2 Pharmacophore Model for the GABAJ
Bz Receptor Complex, 784
4.1.3 Structure-Activity Relationships with
GABAA/BzRInverse Agonists, 786
4.1.3.1 P-Carbolines and
Pyridodiindoles, 786
4.1.3.2 Imidazobenzodiazepines, 790
4.1.3.3 Pyrazoloquinolinones, 790
4.1.3.4 Miscellaneous Chemotypes, 792
4.1.4 Cognition-Enhancement Experiments,
794
4.1.5 Future Direction, 796
4.2 Nicotinic Acetylcholine Receptor, 797
Burger's Medicinal Chemistry and Drug Discovery 4.2.1 Physiology and Pharmacology of the
Sixth Edition, Volume 6: Nervous System Agents nACh Receptor, 797
Edited by Donald J. Abraham 4.2.2 Structure-Activity Relationships for
ISBN 0-471-27401-1 0 2003 John Wiley & Sons,Inc. nAChR Agonists, 798
779
Cognition Enhancers
2.2 Measurements of Human Cognition these neurotransmitter levels have been the
focus of intense research.
Animal studies are used routinely for predict-
ing the efficacy of cognitive-enhancing phar-
3.2 Clinically Approved Agents
maceutics in humans, but this has its limita-
tions. For example, every marketed agent for Palliative treatment of Alzheimer's disease,
the treatment of dementia has been shown to the most common cause of dementia, has b e 9
enhance cognition in some animal model, but the primary focus of research in cognitive en-
the converse is not always true. Many agents hancement. However, despite these efforts, ef-
that produce positive effects in animal models fective pharmacological interventions remain
fail in clinical trials (22, 23). Increasing the elusive. The most fruitful pharmacological
predictiveness of preclinical behavioral tests strategy pursued in AD research to date has
would greatly improve the development of ef- focused on the relief of cognitive and memory
fective treatments for dementia. Presently, deficits that are attributed to cholinergic dys-
there is no single behavioral test or animal function.
model that increases the likelihood of identi-
fying an effective cognitive-enhancing com- 3.2.1 Acetylcholinesterase Inhibitors (AChEI).
pound in humans, although strategies have There is substantial rationale for exploring
been proposed for achieving this goal (24). cholinomimetic therapies for the treatment of
A fundamental problem with attempting to the symptoms of dementia (3238). Central
quantify cognitive function is that cognition is cholinergic depletion is a hallmark of AD and
multifaceted. This is especially evident in the experimentally induced cholinergic dysfunc-
preclinical setting, where the individual ele- tion produces cognitive deficits both preclini-
ments of attention, learning, and memory can- cally and clinically (39). Acetylcholinesterase
not be adequately measured in a single behav- (AChE)inhibitors suppress the normal break-
ioral test. However, more extensive tests have down of acetylcholine from the synaptic cleft,
become somewhat standardized to measure thereby increasing the overall level of ACh
cognition in humans. The Mini-Mental State available to the relevant postsynaptic recep-
Examination [MMSE (2511 and the cognitive tors. As such, AChE inhibitors represent a
subscale of the Alzheimer's Disease Assess- valid approach in the development of cogni-
ment Scale [ADAS-Cog (26, 2711 are used in tive-enhancing compounds. Indeed, acetylcho-
virtually all clinical evaluations of cognitive linesterase inhibitors, such as tacrine (I),
enhancers. Use of these protocols allows rea- (Cognex; Warner-Lambert Co.), donepezil(2),
sonable comparisons to be made between (Aricept, E2020; Eisai Co. Ltd.), rivastigmine
drugs. (31, (Exelon, Novartis), and galantamine (4),
(Reminyl; Janssen) are the only U.S. FDA-ap-
proved drugs currently marketed in the
3 COGNITION-ENHANCING THERAPIES United States for the symptomatic treatment
of AD (Fig. 14.1). It should be noted, however,
3.1 Therapeutic Approaches that tacrine is no longer widely used because
of the recent advent of safer AChE inhibitors
Alzheimer's disease is the best known age-re-
(e.g., Aricept). The evidence to date indicates
lated disorder for which the primary symptom
that these agents provide only short-term re-
is dementia. Advances have been made in un-
lief, in part by slowing the progression of the
derstanding the etiology of AD, but much re-
disease (40- 43).
mains to be discovered. Clearly, there are doc-
umented neuropathological changes in the
3.3 Exploratory Approaches
brain. For example, there is a broad spectrum
of neurotransmitter dysfunction, where ace- Many other pharmacological approaches are
tylcholine (ACh),serotonin [5-hydroxytrypta- currently being examined to reduce the cogni-
mine (5-HT)], norepinephrine, dopamine tive impairments seen in AD or in other de-
(DA), and glutamate levels are reduced (28- mentia. These approaches include agents that
31). Understandably, therapies that affect act at glutamate receptors [e.g., AMPAkines
Cognition Enhancers
0
I .
(1)tacrine (2) donepezil
(44-46), NMDA receptor modulators (47-50)], agonists; (3) serotonin subtype 3 receptor (5-
monoarnine oxidase B (MA0 B) inhibitors HT,R) antagonists; and (4) potassium M-
(51-55), antioxidants (52, 56-59), nootropics channel inhibitors.
(41, 60), lipid-lowering agents (e.g., statins)
(611, insulin (62), anti-inflammatory agents 4.1 CABAA/Bz Receptor Complex
(63-671, and estrogen supplementation (63,
65,66,68,69). Because several other reviews
are available that compare these therapeutic 4.1.1 Physiology and Pharmacology of the
approaches (70-77), the remainder of the CABAA/Bz Receptor Complex. The neuro-
present chapter focuses on ion channel mod- transmitter y-aminobutyric acid (GABA) and
ulation approaches to cognition enhance- its associated receptors constitute the major
ment. inhibitory pathway in the brain (81). Attenu-
ation of postsynaptic excitatory responses is
achieved when GABA interacts with its recep-
4 ION CHANNEL MODULATION OF tors to stimulate ion conductances that lead to
NEUROTRANSMISSION localized membrane hyperpolarization. In
particular, the conductance of C1- ions is con-
In the remainder of this chapter, we briefly trolled through the ligand-gated GABA, and
review the status of efforts that have in com- GABA, (82-84) receptor subtypes, whereas
mon the potential to enhance the activity of conductance of Ca2+ and K+ ions occurs
multiple neurotransmitter systems through through the GABA, receptor subtype (85).
the modulation of gated ion channels (also see The majority of GABA's inhibitory effect in
reviews in Refs. 10, 78-80). Specifically, we the CNS is mediated by the GAB& receptor
discuss recent advances in the areas of: (1) subtype (86).
y-aminobutyric acid subtype Ahenzodiaz- GABA, receptors belong to the superfam-
epine receptor (GABAA/BzR)inverse agonists; ily of ligand-gated ion channels that include
(2) nicotinic acetylcholine receptor (nAChR) serotonin subtype 3 (5-HT,) receptor, nico-
4 Ion Channel Modulation of Neurotransmission
tinic acetylcholinergic (nACh) receptors, and The GABA, ion channel can be modulated
strychnine-sensitive glycine receptors (81, through ligand interaction with several recep-
87). GABA, receptors are believed to be het- tors (see review in Ref. 108). The GABAA/Bz -
ero-oligomers assembled from eight protein receptor complex has traditionally been differ- -
subunits, drawn from several classes (a1-,, entiated into two general subtypes, based on
Y I - ~8,
, E , T,9, and pl-,) (88-92). The their affinities for the prototypical 1,4-benzo-
subunits of the GABAA/BzRare classified ac- diazepines (Bz) flunitrazepam (23) and diaze- '
cording to the degree of sequence homology. pam (22) (109). Diazepam (22) has high affin-
Each subunit class is defined to exhibit amino ity for the GABAA/Bz receptors composed of
acid homologies of about 60 to 80%, whereas the a,-, and a, subunits and are collectively -
the amino acid homology between the various termed the "diazepam-sensitive" (or "DS")
subunit classes is approximately 30-40% (93, binding sites. The DS binding sites are further
94). categorized as being either Type I or Type I1
Various combinations of these subunits re- BzRs. The Type I receptor has pharmaco-
sult in different GABA, channel isoforms that logical properties similar to those of the
display differential pharmacology, CNS distri- alP,y, isoform, whereas the Type I1 BzR is
bution, and developmental pattern (95). The associated with the a,,,,,&y, subunit-con-
number of different functional isoforms in taining GABA, isoforms. Diazepam (22) does
vivo is unknown, but estimated to be between not have high affinity for the a, and P, sub-
10 (87) and 150 (96, 97). The genetic loss or unit-containing GABAA/BzRisoforms, collec-
mutation of a GABAA channel subunit can tively termed "diazepam-insensitive" (or
have profound neurological consequences. For "DI") sites (110, 111).
example, it has recently been shown that dis- The interaction between the subunits and
rupting the mouse gabrb3 gene, responsible the Bz receptor ligand provides for a wide
for coding the P, subunit, produces EEG ab- range of allosteric regulation of chloride ion
normalities, seizures, poor motor skills, and flux within the associated ion channel (112,
cognitive deficit (98). This research suggests 113). Agonists (e.g., prototypic l,&benzodiaz-
that disruption of the analogous human epines), potentiate GABA-induced C1- flux
GABRB3 gene may contribute to the pathol- and are typically characterized by sedative,
ogy of Angelman syndrome (99), a severe neu- anxiolytic, and anticonvulsant effects (114).
rological disorder characterized by cognitive Partial agonists, antagonists, and inverse ago-
deficits caused by deletionslmutations of ma- nists are also well known (115, 116).
ternal chromosome 15qll-q13 (100, 101). Inverse agonists of the GABA, receptor re-
Positron emission tomography (PET) studies tard GABA-induced C1- flux, thereby indi-
with the high affinity BzR ligand [llC]flu- rectly potentiating the propagation of excit-
mazenil on three Angelman syndrome pa- atory signals. Typically, this type of modulation
tients with maternal deletion of 15qll-q13 produces anxiogenic, somnolytic, and procon-
found a decreased number of GABAA/Bz re- vulsant features (117-121). It deserves men-
ceptors in the frontal, parietal, hippocampal, tion that the anxiogenic pharmacological
and cerebellar regions, which could partially property attributed to inverse agonists has re-
underlie the cognitive deficits of this disorder cently been hypothesized to be a downstream
(102). result of hyperattentional impairments stem-
The GABA, ion channel is formed by a pen- ming from the behavioral assays that involve
tameric assembly of hetero-oligomeric sub- fear- or anxiety-related stimuli (122). Never-
units, with each subunit having four trans- theless, GABAA/Bzreceptor inverse agonists,
membrane-spanning domains (93, 103). The because of their unique ability to reduce the
recombinant GABA, BzR complex assembled inhibitory effects of GABAergic neurotrans-
from two a,, one p,, and two y, subunits most mission, continue to be investigated as thera-
closely resembles the biochemical, electro- pies for several disorders, including dementia
physiological, and pharmacological profile of (123-125) and alcohol addiction (126-129).
native GABAA/Bzreceptors of the mammalian One of the important features of AD'S neu-
brain (104-107). ropathology is the degeneration of cholinergic
Cognition Enhancers
cells within the nucleus basalis of Meynert subunit found amino acid Ile-215 strongly as-
(NBM) with associated loss of afferents to the sociated with the high affinity binding of RY80
neocortex and amygdala (130, 131). The im- (29), an imidazobenzodiazepine selective for
portance of these cholinergic neurons for the GABA, receptors containing the a, sub-
learning and memory in experimental animals unit (155). In another study using chimeric
has been well established (132-139). Because al/a5subunits and site-directed mutagenesis
NMB cholinergic neuronal activity is influ- of the a, subunit, residues a511e215 arid
enced by local GABAergic interneurons (140, a,Thr208 were identified with the high affin-
141), numerous experiments have been un- ity binding of L-655,708 (31),another a, sub-
dertaken to investigate BzR inverse agonist unit-selective ligand (156). -
Within the y, subunit, site-directed mu-
effects on cortical ACh release and perfor-
tagenesis has revealed two residues, Phe77
mance in cognitive tasks. It has been postu-
and Metl30, to be necessary for high affinity
lated that this form of intervention would en- binding of BzR ligands (157, 158). Additional
hance synaptic transmission while preserving investigations using yJa, chimeric subunits
informational fidelity (123). identified two domains of the y, subunit, Lys-
411Try-82 and Arg-1141Asp-161, which to-
4.1.2 PharmacophoreModel for the GABAJ gether are necessary for high affinity binding
Bz Receptor Complex. The Bz receptor, some- (159). Mutagenesis within the y, Lys-411
times referred to as the w binding site, is lo- Trp-82 chimera further identified Met-57,
cated at the interface between the a and y Tyr-58, and Ala-79 as important for this bind-
subunits (95, 121, 142-144). The identifica- ing (160).
tion of conserved residues necessary for drug- Many laboratories have postulated phar-
receptor interaction has been difficult, but macophore models to account for the relation-
progress has been made using site-directed ship between BzR ligand interaction and in-
mutagenesis, photoaffinity labeling, and chi- trinsic activity to modulate the GABAA
meric subunits, as discussed below. receptor function [see Codding (161, 162);
Most notable among the a, subunit resi- Cook (121, 163-172); Crippen (173, 174);
dues to be identified from mutation analyses Filizola (175); Frier (176); Gardner (177);Gilli
as being intricately involved with the and Borea (178); Loew (179, 180); and Wer-
GABAA/Bzreceptor is histidine 101 (rat num- muth (181)l. Among these was a model for un-
bering) or 102 (humadbovine numbering) derstanding binding affinity and pharmaco-
(145,146). Photoaffinity labeling of the bovine logical action of BzR p-carboline ligands,
aHisl02 occurred preferentially with the ago- developed using three-dimensional quantita-
nist [3H]flunitrazepam rather than with the tive structure-activity relationship compara-
inverse agonist [3H]Ro 15-4513, where the tive molecular field analysis (3D QSAR
photoincorporation is associated with an a, CoMFA) (163) and refined employing a proce-
receptor fragment between residues 104 and dure termed GOLPE (generating optimal lin-
the carboxyl terminus (147, 148). This sup- ear PLS estimates) ab initio calculations
ports the view that these structurally distinct (166). This model was later incorporated into
BzR ligands interacted with different amino a unified pharmacophore/receptor model that
acid residues in the binding domains proposed only one binding cleft with multiple
(147-149). It was further postulated from sites for interaction by agonist, antagonist,
these studies that a,H102 contributes to the and inverse agonists (167, 182).
L, lipophilic region, where the pendant phenyl The unified pharmacophore/receptor model
group of diazepam (22) and other 5-phenyl proposed by Cook and coworkers (167, 182)
benzodiazepines are believed to occupy (149). employed CoMFA of the structural parame-
Other amino acids within the a, subunit ters of 136 different BzR ligands, selected
associated with the Bz binding site, affecting from 10 structurally diverse classes of com-
binding affinities by more than 10-fold, are pounds. The model described the BzR site as
Tyr-159, Gly-200, Thr-206, and Try-209 (145, consisting of several key recognition elements,
150-154). Site-directed mutagenesis of the a, including two hydrogen bond donor sites
4 Ion Channel Modulation of Neurotransmission
termed H, and H,; a hydrogen bond acceptor the exact location and function of the amino
site termed 4;four lipophilic regions termed acids in question" (90).
L,, L,, L,, and L,,; and three regions of nega- The unified pharmacophorelreceptor model
tive steric repulsion described as S,, S,, and S, (167, 182, 183) is the most advanced to date
(Fig. 14.2). Another common pharmacophorel and will be used in the following discussion for
receptor model for the a,-, a,-, a,-, a,-, and comparing GABAA/Bz receptor affinities and
a,-containing GABA, isoforms using 19 non- published in uivo pharmacological profiles be-
selective BzR ligands was recently published, tween closely related classes of ligands. De-
which identified similar receptor/ligand inter- tailed data for GABAA/BzR ligand affinity at
actions (175). the various GABA, isoforms have only re-
The Cook group further refined their uni- cently been available, and remain largely in-
fied pharmacophore/receptor model to provide complete for all the many BzR ligands re-
additional insight into the different binding ported to date. Far fewer data have appeared
domains of the GABAA/BzRsubtypes alp3y,, in the literature for a GABAA/BzRligand's abil-
a5P3y2,and a,P,y,. This analysis was based ity to modulate individual GABA, isoforms.
on the affinities of 151 BzR ligands from nine It is difficult to predict the in vivo pharma-
different structural families at five distinct cology of a GABAAiBz receptor ligand based
(a,-3,5,,P3y2) recombinant GABAAIBzR sub- solely on its in vitro binding affinity and mod-
types (183). ulating effect for several different GABA, iso-
Cook and coworkers undertook the difficult forms (114). For example, the in uiuo inverse
task of correlating the data between the amino agonist DMCM (16) is an in uitro negative
acids believed to be involved in ligand binding modulator at the a,-,P,y, and a5@,y2 iso-
and the specific region of their unified phar- forms and a positive modulator at the a5Plyl
macophore1receptor model (90). Some of the isoform (184-187) (Table 14.1). Also, the clas-
amino acid residues likely associated with BzR sic in uivo antagonist flumazenil (Ro 15-1788,
ligand binding are indicated in the unified (26) is an in vitro weak positive modulator at
pharmacophore1receptor model presented in the alP3y2 and a,P,y, isoforms, a positive
Fig. 14.2. There are several key protein-ligand modulator at the a,P, y, and a&, y, isoforms,
interactions that deserve mention, but in the a weak negative modulator at the a5P,y, iso-
author's words, it is clear that "much work form, and a positive modulator at the a,P,y,
remains at the molecular level to determine isoform (187, 188).
786 Cognition Enhancers
Table 14.1 Reported BzR Ligand Modulation of GABA-Induced GABA, Isoform Function
Modulation of GABA-Induced Function
Positive (+); Negative (-1; or Not Determined (ND)
- - - - - -
close structural analogs, the dihydropyridodi- of the pyridodiindole PRI (19) is similarly de-
indoles, at the GABAA/Bzreceptor are shown creased when its indole N(7)-hydrogenatom is
in Table 14.2. Where available, the affinities replaced with a methyl group (IC,, = 4-1163
for specific GABAABz receptor isoforms are nM) (121).
listed. Additional insight into the BzR pharma-
Most P-carbolines have good affinity for re- cophore was gained through the study of a se-
ceptor isoforms containing the a,, a,, as, and ries of rigid p-carboline analogs, the dihydroy
a, subunits. Some selectivity is generally ob- pyridodiindoles (121,203). These BzR ligands
served for the a,-containing isoform and there helped to define the active conformation and-
is usually little affinity for the isoform having binding alignment of the parent P-carboline
an a, subunit. The exceptions for this are
series. The unsubstituted pyridodiindole PRI
BCCT (8)and BCCE (15), which exhibit mod-
(191, an inverse agonist, was believed to bind
erate affinity for the a, subunit (183).
into the BzR active site in much the same way
As shown on the left in Fig. 14.3, p-carbo-
as BCCE (15) (Fig. 14.3) with the centroid of
lines with agonist activity, such as 6-PBC (5),
were proposed to bind into the BzR so as to the E-ring occupying lipophilic region L, and
orient the centroid of their A-ring into the L, its N5 nitrogen lone pair of electrons interact-
region, whereas the pyridyl N(2) nitrogen and ing with H, (167, 191).
the carbonyl oxygen at C3 formed a three-cen- Substitutions at the 1-, 2-, or 4-positions
tered hydrogen bond with hydrogen bond do- were accompanied by diminished affinity, re-
nor site H, (167). Ligands with a C4 alkoxy sulting from the steric constraints of the bind-
moiety, such as 6-PBC (7), are able to form an ing domain (165). Substitution at the 3-posi-
additional hydrogen bond with donor site H, tion helped to define the boundary of the
and can fully occupy the L, region, believed repulsive region S,. Several &position analogs
essential for potent agonist activity (191,201). [e.g., PRI2Me (21), PRI2MeO (201, and
Inverse agonist of the P-carboline series PRI2Cl ( l l ) ] had very good BzR affinities,
also tend to have a vertical alignment when with pharmacological profiles of the &methyl
modeled into the BzR. However, it was hy- and 3-methoxy adducts maintained as inverse
pothesized that for inverse agonist activity in agonist, but the 3-chloro adduct shifting to an
vivo that these ligands bind into the BzR dif- antagonist (167).
ferently, as shown with BCCE (15) on the High affinity binding P-carbolines require
right side of Fig. 14.3, orienting the centroid of moieties at the 3-position that are capable of
their A-ring into the LDi binding pocket. To accepting hydrogen bonds from the receptor's
accomplish this, hydrogen bonds must form hydrogen bond donors H, and H,. Several
between the indole N(9)-H and the A, hy- p-carbolines with esters, amides, and alkoxy
drogen bond acceptor site, and between the groups at the &position have been developed
pyridyl N(2) nitrogen and the hydrogen bond with excellent BzR affinities (Table 14.2). The
donor site H, (163). Additional hydrogen shape and lipophilicity of the 3-position sub-
bonding to H, was thought to be derived from stituent has a direct effect on GABAAisoform
the ligand's carbonyl oxygen. The ligand's C3 selectivity. For example, the t-butyl ester
side chain was also proposed to interact with BCCT (8)is very selective (20-fold) for the a,-
the hydrogen bond donor site H,. containing GABAA isoform (Ki = 0.72 nM),
Both the p-carboline and the structurally thought to be derived from the y-branching of
related pyridodiiodole BzR ligands require the the ester (197), but still maintains some affin-
indole nitrogen of the B-ring to remain unsub- ity for the a,-containing GABA, isoform (Ki=
stituted for interaction with the receptor's hy- 111 nM). The 3-ethoxy analog 3-EBC (lo),de-
drogen bond acceptor site A, (121). For exam- veloped as a long-lived, water-soluble replace-
ple, replacement of the indole N(9)-H on ment for the metabolically labile 3-position es-
P-CCM (14) with a methyl group is accompa- ters (121), also binds tightly with the a,-
nied by a decrease in binding affinity [IC,, = containing GABAA isoform (Ki = 6.43 nM),
5.0 nM for P-CCM (14) and IC,, > 50,000 for but has little affinity for the a,-containing
9-methyl-P-CCM] (202). The binding affinity GABA, isoform (Xi = 826 nM) (191).
Table 14.2 Binding At3hit.y and In Vivo Pharmacological Profile of Several GABAA/BzR p-Carboline and Pyridodiindole Ligands
1
A
(19) Rz = H; PRI
(21) Rz = CH3; PR12CH3
(20) Rz = 0CH3; PR12CH30
(11) Rz = C1; PRI2C1
P-Carboline Pyridodiindole
Ligand Ki ( n W a
R3 R4 R, R6 R7 a1 a2 a3 a5 % Ref?
Agonist
Abercanil(5) C0,i-Pr H OBn H 12.4
ZK 93423 (6) C0,Et H OBn H 4.10
Partial agonist
6-PBC (7) C0,Et H OPr H 0.49
Antagonist
BCCT (8) C0,t-BU H
BCCP (9) C0,n-Pr H
3-PBC (10) OPr H
PRI2Cl(11)
Antagonist/weak
inverse agonist
ZK 93426 (12) COzEt Oi-Pr IC,, = 0.4 nM
BC6OBz (13) C0,Et OBn 169 284 271
Inverse agonist
p-CCM (14) C0,Me H H H 2.4 7.4 72 44
BCCE (15) C0,Et H H H 1.20 4.9 5.7 26.8
DMCM (16) COzMe H OMe OMe 5.70 8.3 4.0 1.04
3-EBC (17) OEt H H H 6.43 25.1 28.2 826.0
FG 7142 (18) CONHMe H H H IC,, = 444 nM
PRI (19) 1.1 1.2 1.1 40.3
PRI2MeO (20) 3.4 11.7 11.0 225
PRI2Me (21) IC,,-.= 10 nM
"Specific amnity data provided when available for a,,a,, a,, a,, and a, subunits coexpressed with Pay, in human cell lines.
bRef. 1: (183);Ref. 2: (199);Ref. 3: (163);Ref. 4: (187); Ref. 5: (200); and Ref. 6: reported as having an "inactive" profile in (169).
4 ion Channel Modulation of Neurotransmission
Figure 14.3. Binding conformation of partial agonist 6-PBC (71, left, and inverse agonist BCCE
(15),right, in the GABAA/BzRpharmacophore/receptor model. (Modified from Ref. 167.)
The pharmacological profile of the p-carbo- inverse agonist profile (169). When inverse ag-
lines also varied with structural changes in the onist P-CCM (14) was modified with methoxy
3-position group. Ligands with smaller 3-posi- groups at the 6- and 7-positions and an ethyl
tion groups, such as P-CCM (14), BCCE (15), group at position 4, the ligand DMCM (16)was
and 3-EBC (17),were more selective for the produced, which maintained binding affinity
a,-containing GABA, isoform and maintained and an inverse agonist profile (163). This ob-
inverse agonist profiles. The N-methyl carbox- servation supported the hypothesis that the
amide analog FG 7142 (18)is also an inverse L,, binding region of the a,-containing
agonist, but with diminished BzR affinity GABA, isoform was larger in size than the
(195, 200). The n-propyl ester BCCP (9) and same region of the other isoforms (191).
the t-butyl ester BCCT (8) provided ligands Addition of a methoxyrnethyl group at the
with overall antagonist profiles (121, 204, 4-position of BC6OBz (13)gave ZK-93423 (61,
205). BCCT (8) was later found to be one of the with greatly improved affinity for all GABA,
most selective ligands for the a,-containing isoforms (201). ZK-93423 (61, with a full ago-
GABA, isoform (197,201). In contrast to 3-EBC nist profile, was believed to orient differently
(171,the 3-n-propoxy-P-carbolineanalog, 3-PBC in the BzR in such a way as to permit a hydro-
(10) was made as a set of compounds to fur- gen bond between the methoxymethyl group's
ther investigate the strict steric requirements oxygen atom and the hydrogen bond donor
of the lipophilic binding region L, (121, 163). site H,. This new orientation would permit
The affinity for this ligand was diminished by the benzyloxy moiety to fully occupy region L,
more than an order of magnitude and found to of the receptor, believed necessary for the ag-
have an antagonist profile (166). onist pharmacology (Fig. 14.3). The ligand
Substitution of the 6-position of inverse ag- 6-PBC (7) was made with the smaller propoxy
onist BCCE (15)with a benzyloxy group gave group at the 6-position to test this hypothesis,
BC6OBz (13). This adduct had an equivalent and resulted in a ligand with increased selec-
afiinity for only the a,-containing GABA, iso- tivity for the a, isoform and a partial agonist
form (K, = 7.2 nM) (183) and a diminished pharmacological profile (170, 171).
Cognition Enhancers
4.1.3.2 Imidazobenzodiazepines. Imida- was well conserved (218). The aliphatic ring of
zobenzodiazepines have been a long-studied the 4,5-pyrroloimidazobenzodiazepine,as in
series of GABAA/BzRligands (167, 189, 206- L-655,708 (311, is believed to play an impor- +
213) that bind with good affinity to the a,, a,, tant role in maintaining an active anti confor- .
a3,a,, and a, subunits containing GABA, iso- mation of the &position ester (183). Of partic-
forms, and often with excellent selectivity at ular interest from this series is the inverse
the a,-containingreceptors (189) (Table14.3). agonist L-655,708 (31), a ligand that has at'
The imidazobenzodiazepine Ro 15-4513 least 50-fold selectivity for the a,-containing
(27) is a partial inverse agonist with about 10- isoform and has been investigated preclini-
fold selectivity toward the a,-containing re- cally by Merck for its cognition-enhancing
ceptor (189). In contrast, substituting a fluo- properties (186). Additionally, L-655,708 (31)
rine atom in the &position on Ro 15-4513 (27) has been radiolabeled with tritium and used as
generates the antagonist flumazenil [Ro 15- a research tool for identification of a,-contain-
1788 (26)], and selectivity is lost. This obser- ing GABA, receptors (214).
vation led researchers to hypothesize that the 4.1.3.3 Pyrazoloquinolinones. The pyrazo-
lipophilic region L, may be smaller in the loquinolinone series of BzR ligands have be-
a,P,y,-containing receptor than the related come generally known as the CGS series be-
a,P,y,-containing receptor, and that selectiv- cause of their discovery in the laboratories of
ity might be achieved with the correctly sized Ciba-Geigy in the early 1980s (220). Like the
8-position substituent (215). Consequently, p-carbolines, this series has been well ex-
several a, subsite-selective imidazobenzodiaz- plored and ligands with a continuum of intrin-
epines were synthesized [e.g., RY24 (30) and sic activities have been discovered (166, 209,
RY8O (29)l that supported this hypothesis 220-223) (Table 14.4).
(215). The pyrazoloquinolinones have unique
Molecular modeling with RY24 (30) and pseudoplanar topographies and high receptor
RY80 (29) in the unified pharmacophorelre- affinities. These properties helped to system-
ceptor model suggested that these ligands atically probe the structure-space of the BzR
bind in an orientation that directs their 8-po- and greatly aided the development of a unified
sition substituent into the L, region, giving pharrnacophore/receptor model (167,183). As
them selectivity toward the a,-containing re- illustrated in Fig. 14.5 with agonist CGS-9896
ceptor (Fig. 14.4) (189, 215). Both RY24 (30) (33),the lone pair of electrons of N1 form a
and RY80 (29) were assayed i n vivo and found hydrogen bond with donor site Hz and the lone
to have an inverse agonist profiles (215). pair of electrons of the C3 carbonyl oxygen
Based on the above findings, an investiga- align to form a hydrogen bond with donor site
tion was launched into the modification of the H,. An additional hydrogen bond is believed to
classic BzR agonist diazepam (22) that inves- form between the proton on the N5 nitrogen
tigated A-ring substituents directed toward and the acceptor site A,. The centroid of the
the L, region (216,217). This led to the deriv- D-ring was believed to occupy the lipophilic
ative QH-11-66 (24), the first benzodiazepine region L,.
selective for the a,-containing receptor. In In the course of investigating substitution
contrast to RY24 (30)and RY80 (29),this BzR patterns of the pyrazoloquinolinones, it was
ligand was reported to have an agonist phar- observed that substituents at both the 6- and
macological profile. 7-positions generally had a negative effect on
Further insight into the topography of the receptor affinity, presumably because of steric
Bz receptor came from a series of chiral, interactions in the binding domain of region
framework-constrained 4,5-substituted pyr- L,,. However, substituents only at the 7- or
roloimidazobenzodiazepines and azetidinyl- 8-position were better tolerated and helped to
imidazobenzodiazepines (218, 219). Only the map out the steric constraints of region L,,
(S) enantiomers of these series of ligands (224,226).
bound to the BzR subtypes with high affinity, The pendant D-ring is the most amenable
suggesting that the conformational topogra- toward manipulation in regard to affinity and
phy at the five recombinant receptor subtypes i n uivo pharmacology (167). In contrast to the
Table 14.3 Binding Affinity and in Vivo Pharmacological Profile of Several GABAA/BzRBenzodiazepine
and Imidazobenzodiazepine Ligands
Ligand Ki bM)"
R R, R, R3 R$R, ff1 ff3 ff5
Agonist
Diazepam (22) 14 20 15 11 >3000
Flunitrazepam (23) 2.2 2.5 4.5 2.1 >2000
QH-11-66 (24) 76.3 42.1 47.4 6.8 >3000
Partial agonist
Bretazenil(25) Cod-Bu -CH2CH,CH2- H(Sj HlBr 0.35 0.64 0.2 0.5 12.7
Antagonist
Flumazenil (26) C0,Et Me H H F/H 0.8 0.9 1.1 0.6 148
(Ro 15-1788)
Partial inverse agonist
Ro 15-4513 (27) C0,Et Me H H N& 3.3 2.6 2.5 0.3 3.8
Sarmazenil(28) C02Et Me H H HICl (DS)Ki= 0.2 nM;(DI) Ki= 20 c 2.4 nM
(Ro 15-3505)
Inverse agonist
RY80 (29) C0,Et Me H H CXHIH 28.4 21.4 25.8 0.49 28.8
RY24 (30) C0,t-Bu Me H H (=CH/H 26.9 26.3 18.7 0.40 5.1
L-655,708 (31) C0,Et -CH,CH,CH,- H(S) OMe/H 48.5 27.4 24.5 0.45 83.2
(MSD; FG8094)
"Specific affinity ~rovidedwhen available for a,, a,, a,, a,, and a, subunits coexpressed with P,y, in human cell lines,
bRef.1: (183); Ref. 2: (210);and Ref. 3: (214).
Cognition Enhancers
Table 14.4 Binding Affinity and in Vivo Pharmacological Profile of Several GABAA/BzR
Pyrazoloquinolinone Ligands
Pyrazoloquinolinone
Agonist
AF'Q3'C1(32) H/CW H H H IC,, = 3.9 nM
Partial agonist
CGS-9896 (33) W/C1 H H H IC,, = 0.6 nM. 5
CGS-9895 (34) H/H/OMe H H H 0.32 1.1 0.28 0.96 ND 7
Antagonist
APQ3'0Me (37) H/OMe/H H H H IC,, = 0.5 nM
APQ2'C1(38) Cl/H/H H H H IC,, = 70 nM
Inverse agonist
I
"Specific affinity data provided for a,, az,a,, a,, and a, subunits coexpressed with P,y2 in human cell lines.
bRef. 1: (224);Ref. 2: (221);Ref. 3: (225);Ref. 4: (223);Ref. 5: (220);Ref. 6: (222);and Ref. 7: (187).
Cognition Enhancers
(48) Imidazoquinoxaline
piperazine urea
Agonist
Zolpidem (45) 26.7 156 383 >10,000 >10,000 1
Zopiclone (46) 28 64 29 46 ND 4
Inverse agonist
L-792782 (47) 1.4 2.7 1.4 0.8 ND 2
Urea (48) Ki = 6.67 nM 3
SX-3933 (49) Not reported 5
"Specific affinity data provided when available for a,, nz, a,, a,, and a6 subunits coexpressed with P3y2in human cell lines.
bRef.1: (191); Ref. 2: (186);Ref. 3: (230); Ref. 4:(187); and Ref. 5: (229).
anxiety and the study was terminated (119). to partially antagonize scopolamine's effects
Nevertheless, FG 7142 remains a valuable on memory and attention (241). NO further
preclinical research tool. development is expected.
In a single clinical study, ZK 93426 (12) /midazobenzodiazempines Ro 15-3505 (28);
was evaluated for its effect on scopolamine- Sarmazenil; Roche Holding AG), RY24 (30), and
induced cognitive impairments and was found L-655708 (31); FG8094; Merck); and the Triazo-
Cognition Enhancers
lopyridazine L-792782 (47; Merck). Ro 15-3505 strated positive results in animal models of
(28) demonstrated convincing efficacy in pre- cognition (249, 250). S-8510 was shown to
clinical measures of arousal and cognitive be- ameliorate the memory impairments of sco- -
havior such as T-maze and delayed match-to- polamine-treated or basal forebrain lesioned
position assays (242-245). However, this rats in a water maze and mice in passive-
compound also demonstrated anxiogenic ac- avoidance behavioral paradigms (251, 252).
r
tivity that precluded its clinical development EEG measurements confirmed the activating
for cognitive impairment (246, 247). effects of S-8510 on rat brain function (253)
An entry into the area of a,-selective li- and to enhance LTP in vitro (254). It was also
gands is represented by the imidazobenzodi- disclosed that S-8510 did not induce anxiety or
azepine RY24 (30). RY24 has been reported to convulsions, even at 10-100 times the thera-
show cognitive-enhancing effects after direct peutically effective dose (255). An additional,
hippocampal injections (Helmstetter et al., interesting feature of this compound is that it
unpublished, as discussed in Ref. 183). How- also displays antidepressant properties in
ever, at similar concentrations this compound mice (254). This finding is made particularly
also produced an increase in freezing behavior interesting, in that there remains a high de-
indicative of anxiogenic activity. Note that no gree of comorbidity between depression and
proconvulsant effects were seen up to the dementia in geriatric populations (see, e.g.,
highest concentration tested (10 pg/pL). In- Ref. 256). 5-8510 has been reported to be in
terestingly, the procognitive and anxiogenic phase 2 clinical trials in Japan as a potential
effects were seen only at lower concentrations treatment for senile dementia (257) and, ac-
and were absent at higher concentrations. cording to a recent press release (PR News-
This combined with the fact that no procogni- wire, July 25,2001) has recently been included
tive effects were noted after peripheral admin- in a joint development venture between
istration indicates that, at higher concentra- Shionogi & Co. Ltd. and GlaxoSmithKline.
tions, this compound may possess agonist The pyrazoloquinoline CGS-8216 (39) was
activity at additional subtypes in multiple reported to be an inverse agonist (258, 259)
brain regions that may have led to its anxio- and to have cognition-enhancing properties
genic profile. for mice in a T-maze behavioral paradigm
Merck has recently disclosed a series of BzR (260).
inverse agonists with greater selectivity for
GABA, receptors containing the a, subunit 4.1.5 Future Direction. The only nonago-
(248). A lead compound, L-655708 (31), dem- nist GABAABzreceptor ligand in clinical use
onstrated good brain-penetrating properties is the antagonist flumazenil (26, Ro 15-1788,
and receptor occupancy (156). However, there Hoffman-LaRoche). Flumazenil has been clin-
is a paucity of data with regard to its activity in ically described as an agent with few intrinsic
behavioral assays of cognitive activity. Subse- properties and is used to reverse the effects of
quent chemistry efforts led to the 3-phenyl- benzodiazepines in conscious sedation, gen-
triazolopyridazine, L-792782 (47), which pro- eral anesthesia, and the management of sus-
duced a marked improvement in a water-maze pected benzodiazepine overdose. Reports from
match-to-position test (186). In this test, rats new clinical studies, however, show that
were placed in a standard water-maze appara- flumazenil (26) does possess intrinsic activity
tus with a hidden platform. After animals had and has significant negative effects on cogni-
"found" the platform on a first trial, a second tion, cardiovascular physiology, and mood
trial followed at either a 0- or 4-h intertrial (261).
interval. L-792782 markedly decreased the Continued research into the identification
time to find the platform on the second trial of subtype-selective GABAA/Bz receptor li-
regardless of intertrial interval. gands can be expected to provide agents with
lrnidazoquinoline S-8510 (44) and Pyrazo- more specific physiological activity. This may
quinolinones CGS-82 7 6 (39). The fused imid- result in new treatments for a variety of disor-
azopyridine S-8510 (44) is a GABAA/Bzrecep- ders, including cognitive deficits, with de-
tor weak inverse agonist that has demon- creased potential for adverse side effects.
4 Ion Channel Modulation of Neurotransmission
4.2 Nicotinic Acetylcholine Receptor have shown that nAChR agonists can act pre-
synaptically to facilitate neurotransmitter re-
4.2.1 Physiology and Pharmacology of the lease. For example, nAChR agonists applied to
nACh Receptor. Acetylcholine (ACh) controls cells expressing either the a,& nAChRs [nic- -
neurotransmission through interaction with otine, carbachollatropine, anatoxin-a, and epi-
both nicotinic and muscarinic types of recep- batidine in mouse thalamus slices (277)l or a,,
tors. The nicotinic acetylcholine receptors nAChRs [nicotine in cultured rat hippocampal
(nAChRs) are ligand-gated ion channels neurons (278, 279)], in the absence of high
(LGIC) that regulate the flux of ions (Na+,Kt, external Ca2+,evoked the release of GABA, .
Ca2+)through the neuronal membrane (262- ACh, and glutamate.
264), whereas the muscarinic receptors are The neurotransmitter release mediated by
members of the G-protein-coupled receptor the a, nAChR subtype was shown to be pre-
superfamily (GPCR). nAChRs are found on dominantly the result of Ca2+ influx through
skeletal muscle at the neuromuscular junc- the activated a, nAChR channel (278-281).
tion, in autonomic ganglia of the peripheral Potentiation of neurotransmission mediated
nervous system, on sensory nerves and some through the a4P2 nAChR subtype was more
peripheral nerve terminals, and at numerous complicated, in that it was facilitated through
sites in the spinal cord and brain. The nAChRs an intracellular increase in Ca2+as a result of
are further subdivided into neuronal nAChRs Ca2+ influx from either the a,& nAChR or
and the nicotinic receptors of the neuromus- from voltage-dependent Ca2+ channels acti-
cular "iunction. The neuronal nAChRs in- vated by a,& nAChR-elicited depolarization
volved in CNS neurotransmission are distinct (277).
from those of the skeletal muscle (265) and The binding affinity to the a,& site is fre-
sympathetic ganglia (266). All nAChRs are quently evaluated in homogenized rodent
thought to be homo- or hetero-oligomeric as- brain tissue using radioligands such as
semblies of five subunits, drawn from several [3H]cytisine, [3Hlnicotine, [3Hlacetylcholine,
classes (267,268). In mammalian or avian spe- [3~methyl~bamylcholine (MCC), and [3Hlepi-
cies, muscle nAChRs consist of a,, P,, y, and 6 batidine. The regulation and functional activ-
subunits, whereas neuronal receptors are ities have been investigated through several in
composed of a, to a, and p, to p, subunits vitro expression systems. Transient expres-
(262, 269-271). In the rodent CNS, the pre- sion of chick, rat, and human a4P, nAChRs in
dominant nAChR suptypes are a,& and the Xenopus oocytes, and stable expression sys-
homoligomer a, (272), and these subtypes tems using chick a,& in a mouse cell line
have been implicated in the functions of learn- (MI0 cells) and human a4P2 in a human cell
ing and memory (273). Numerous nAChRs line (K177 cells) have been used. Studies on
with distinct biophysical and pharmacological endogenous a,& nAChRs have been carried
properties can be generated by the expression out in rodent thalamic tissues, and the re-
of a single a-type subunit (homomeric) or by sponses in these systems have been measured,
coexpression of a- and P-subunits (hetero- either as flux of radioactive ions (e.g., 86Rh+or
meric). The composition and distribution of ',Nat) or as calcium influx detected with cal-
discrete subtypes of nAChRs in the brain still cium-sensitive dyes (273).
remain largely unknown, although it has been Early drug discovery efforts in the nAChR
established that certain subunits will prefer- area were achieved largely through observa-
entially combine to form functional channels tion of the behavioral effect of @)-(-)-nicotine
(263,269, 274). [hereafter designated simply as nicotine (50)
Considerable attention has been given to in Fig. 14.61 and its resulting behavioral ef-
two nAChR subtypes: the heteromer contain- fects in humans. Acute treatment with nico-
ing a4P2 subunits and the a, subunit ho- tine improves cognitive performance in ro-
momer. Both of these receptors have been dents (282, 283) and primates (284) for a
shown to participate in fast excitatory trans- variety of behavioral paradigms. Nicotine is
mission by way of postsynaptic mechanisms also known to generate anxiolytic effects in
(275, 276). In addition, recent experiments humans (285). Recent clinical studies have
Cognition Enhancers
Compound
significantly reduce binding affinity (60 ver- stimulates the release of ACh, DA, NA, and
sus FUR-2403). Third, substitution on the ole- 5-HT as effectively as nicotine in rat neocortex
finic amine side chain also reduces binding af- (317).
finity. Comparison of the binding affinities of RJR-2403 has been described as equal to or
RJR-2403 with (66) and (67) (Fig. 14.7) re- better than nicotine as a cognitive enhancer in
veals a 3 and 254 times reduction in binding behavioral assays (274). This compound ame-
affinity when a methyl and n-pentyl groups liorated scopolamine-induced impairments in
are introduced, respectively, on the carbon ad- passive avoidance as well as working and ref-
jacent to the aliphatic nitrogen. Fourth, a erence memory impairments in a radial arm
chain length of four carbon atoms between maze task caused by NBM lesions in rats
heterocycles and side-chain nitrogen is opti- (318). Later studies using the radial maze
mal for high affinity binding (58versus 61 and showed that the positive cognitive effects of
62). Fifth, the trans double-bond geometry FUR-2403 were long lasting (360 min postoral
provides the best binding activity. Binding af- administration) (319). RJR-2403 also showed
finity follows the trend trans olefin (RJR- significant antinociceptive effects in mice and
2403) > alkyne (64) > cis olefin (63) > satu- rats in a variety of pain models (320). Prelim-
rated analog (65). Finally, the nature of the inary pharmacokinetics studies showed that
heteroaryl moiety is critical to the binding ac- RJR-2403 is metabolically unstable and the
tivity. In general, the binding affinity is in the major metabolic pathway in rats is the oxida-
order of pyridinyl (RJR-2403) > pyrimidinyl tion of the carbon a to the basic nitrogen (321).
(68) > quinolinyl (69) > isoquinolinyl (70) Positive results were reported for clinical tri-
(Fig. 14.7). als of RJR-2403. However, further clinical de-
4.2.2.1.2.1RJR-2403 (57; Metanicotine). velopment was suspended, presumably be-
RJR-2403 (57) was selected for further devel- cause of issues surrounding patent protection.
opment because of its high binding affinity for 4.2.2.1.3 lsoxazole and lsothiazole Deriva-
the a,& nAChR in rat cortex [Ki = 26 nM tives. Bioisosteric replacement of the pyridine
(314)l and equivalent efficacy to that of ACh ring in nicotine generated a series of novel
for human a,& nAChRs expressed inxenopus isoxazole compounds that are selective and po-
oocytes, with fourfold greater potency (EC,,: tent neuronal nAChR agonists, as exemplified
FUR-2403 = 16 2 4.6 a; ACh = 57 -t 13 p M ) by ABT-418 (71) (Table 14.8) (299). Among
(315, 316). The compound was about 9 times the variety of substituents examined at C3 of
less potent and slightly less efficacious than the isoxazole, methyl turns out to be optimal,
nicotine in stimulating DA release from rat even though other substituents, such as C,-C,
striatum (314). However, in vivo microdialysis linear alkyl, CF,, Br, and benzyl (not phenyl),
experiments have demonstrated that RJR2403 still provide potent analogs. The 3-des-methyl
I Figure 14.14. Example of pharmacophoric element selection for molecular modeling of nicotine.
Pharmacophoric elements A and B are nitrogens and element C is on the nAChR receptor with
which element B would optimally connect. Carbon, green; nitrogen, light blue; hydrogen attached
to the basic nitrogen, white; putative nAChR binding site, dark blue.
4 Ion Channel Modulation of Neurotransmission
analog of ABT-418, (75), was shown to possess The binding activity of the reverse isox-
50 times lower binding affinity than that of azole (76) is significantly reduced relative to
ABT-418 for the [3H]cytisine binding site that of ABT-418 (Fig. 14.8). Replacement of
(294). Substitution of the pyrrolidine moiety oxygen in ABT-418 with sulfur generates
in ABT-418 generally reduces activity (323). isothiazole (79) with reduced binding affinity
The enantiomer of ABT-418, (72), has 13 (Table 14.9). It is interesting to note that the
times less binding affinity than that of ABT- N-unsubstituted isothiazole (77) is sixfold
418, compared with the 14-fold difference in more active than the isoxazole counterpart
binding affinity for the corresponding nicotine (73), but still 12 times less active than ABT-
analogs. ABT-418 has approximately 80-fold 418, in terms of binding activity, and also less
more potent binding affinity than that of the efficacious with respect to stimulation of dopa-
desmethyl analog (731, whereas nicotine has a mine release from striatal synaptosomes.
binding affinity 15-fold greater than that of 4.2.2.1.3.1 ABT-418 (71). From a series of
the desmethyl analog. Also of note is that the isoxazole analogs, ABT-418 (711,was selected
opposite trend is observed with the two (R)- for further evaluation (324-326). It is a full
enantiomeric isoxazole counterparts. The (R)- agonist at the brain a&, nAChR, with re-
NH-isoxazole (74) has a binding affinity sev- duced affinity for ganglionic-like nAChRs
enfold greater than that of the N-methyl (327, 328). In vitro, ABT-418 stimulated the
analog (721, but binds with an affinity 2 times
less than that of ABT-418. Isoxazole (74) also Table 14.9 Binding Data of Isothiazoles
demonstrated comparable efficacy to that of
ABT-418 and nicotine in stimulation of DA
release from striatal synaptosomes.
I
R1
Compound n Stereochemistry R1 RZ K, (nM)"
release of [3H]ACh from rat hippocampal syn- using transdermal administration of ABT-418
aptosomes (EC,, = 2.6 p M ; nicotine EC,, = 1 have been reported to be in progress (78).
piV0 (328) and [3Hldopaminefrom rat striatal 4.2.2.I .4 Pyridyl Ether Derivatives. A series
slices (EC,, = 380 nM;nicotine EC,, = 40 of 3-pyridyloxymethyl heterocyclic ether com-
nM) (327). Like nicotine, ABT-418 has poor pounds have been identified with subnanomo-
oral bioavailability in dogs and monkeys lar affinity for brain nAChRs (336) (Table
(<5%) and moderate oral bioavailability in 14.10). Of particular note are A-85380 (81)and
rats (27%) (329). ABT-418 was shown to be A-84543 (87).A-85380 exhibits high binding af-
effective in animal behavioral models de- finity (Ki = 52 pM), for a4P2nAChRs, compa-
signed to access cognition enhancement (re- rable to that of epibatidine, the most potent
viewed in Ref. (328)).Positive effects were ob- nAChR ligand reported to date. This com-
served for both mice and rats in passive pound stimulated ion flux at the human a,P2
avoidance and Morris water-maze tasks. Stud- nAChR subtypes at 163% relative to nicotine.
ies with nonhuman primates have demon- A-85380 (81)is also a full nicotine agonist
strated positive effects on cognition using the at recombinant human a, nAChRs and yet
delayed matching-to-sample task (330) and a possesses potent activity at ganglionic-like
task designed to measure distractibility (331). nAChRs, as evidenced by 113% of the nicotine
An initial clinical trial of ABT-418 in AD pa- response in an assay examining cation flux
tients yielded positive results in both selective through IMR-32 cells. The IMR-32 assay
reminding tasks (332) and nonverbal learning serves as a model for activity at human periph-
tasks (333).A controlled clinical trial also sug- eral ganglionic receptors, which are believed
gested that ABT-418 may be potentially useful to partially mediate undesired cardiovascular
in the treatment of attention deficit hyperac- and gastrointestinal effects of nicotine. Not
tivity disorder (ADHD) (334). Phase I1clinical surprisingly, a radioiodinatedanalog of A-85380
trials with oral ABT-418 were reported to has demonstrated utility as a research tool for
have had positive effects in enhancing acute in vivo studies of central nAChRs (337339).
attention in AD patients, but the compound's A-84543 (€471,another full agonist at hu-
development was terminated for undisclosed man (Ki= 150 pM), has been shown to
reasons (335). More recently, clinical studies have 32-fold selectivity to stimulate ion flux at
4 Ion Channel Modulation of Neurotransmission
human a,P, nAChRs compared to human than the unsubstituted analog (97) (Fig. 14.9).
ganglionic nAChRs, and less efficacy (77% of Both (97) and (98) are less efficacious than
nicotine response) at human sympathetic gan- A-84543 (<40% of nicotine response) at human
glionic nAChRs (340).Various substituents at recombinant a,& and ganglionic-type receptom.
the C5 position of the pyridyl moiety of SAR studies on the heteroaryl moiety in
A-84543 have been evaluated. According to A-84543 (87) revealed that the pyridyl nitro-
the binding data, large substituents are well gen in the 3-position is important, and that
tolerated at the C5 position. However, func- additional nitrogens in the ring are generally
tional activity can change from agonist to an- detrimental to activity. It is interesting to note
tagonist by means of varying the substituents that the 3-methyl-5-isoxazole moiety, which
on the pyridine ring (341). served as a bioisostere for pyridine in ABT-
It should be noted that the N-methylation 418, is a poor substitute for pyridine in
for the pyridyl ether series has a different im- A-84543 (see 99, Fig. 14.9). With respect to the
pact from that in the nicotine series (Table N-methyl analogs, the pyrrolidinyl analog (87)
14.10). For example, the N-H pyrrolidine ana- is threefold more active than azetidine (83)
log (85) possesses comparable affinity to that and 480-fold more active than piperidine (93).
of the corresponding N-methyl analog A-84543 Extensive SAR studies were conducted
(87). In the case of corresponding azetidines, with the aim of reducing propensity for pe-
the unsubstituted analog A-85380 is ninefold ripheral ganglionic-like nAChRs, and it was
more potent than the N-methyl counterpart discovered that the 2-methyl substitution of
(83).With regard to stereochemistry, the N- pyridine moiety results in the reduced activity
methyl compounds with ( S )configuration ex- at both ganglionic-like and human a4P2recep-
hibit more potent binding affinity than that of tors. For example, both (85) and (88) pos-
their (R)counterparts, whereas the unsubsti- sessed efficacy comparable to that of nicotine
tuted N-H enantiomers have similar affinity in the IMR-32 assay, whereas their respective
regardless of the stereochemistry. Structure- 2-methyl analogs, (89) and (921, generated
activity studies on the methyleneoxy moiety in only 8% and 11% response, respectively, com-
A-84543 revealed that replacement of the ox- pared with that of nicotine. The 2-methyl pyri-
ygen atom with 4- (94), 4 H 2 - (95), and dine analogs also have 100 to 300 times lower
-CH,O- (96) (Fig. 14.9) reduces affinity at the affhity than those lacking the 2-methyl sub-
rat a,& nAChR by about 2000-, 150-, and 120- stituent for native a,& nAChRs and much re-
fold, respectively. Replacement of the pyridine duced efficacy at human recombinant a4P2re-
moiety in A-84543 (87) with a phenyl ring (97) ceptors. The pattern of binding affinities
decreases binding affinity by 280 times, but regarding stereochemistry and N-methylation
the corresponding m-fluorophenyl derivative parallels that observed for the corresponding
(98, Ki = 5 n M ) is about eightfold more potent 2-desmethyl series.
804 Cognition Enhancers
"Rat brai~~/[~H]cytisine.
bMinimumeffective dose (MED) in mouse hot-plate assay, i.p. (294, 342).
Of potential relevance to the SAR of these that a different nAChR subtype is responsible
compounds for cognition enhancement is their for the analgesic activity, although other fac-
SAR in antinociceptive assays. In that regard, tors such as species differences and pharmaco-
it is important to note that A-85380 (81) (Ta- kinetic properties cannot be ruled out.
ble 14.10), which possess affinity for a,& re- With respect to the activation of cation flux
ceptors comparable to that of (?)-epibatidine, in IMR-32 cells, compounds (100) and ABT-
failed to demonstrate a significant antinoci- 594 were shown to be more potent and (in the
ceptive effect in the mouse hot-plate model at case of ABT-594) more efficacious than the
doses up to 6.2 pmol/kg (Table l4.ll), whereas corresponding deschloro analog. The 6-bromo
epibatidine shows a robust activity at 0.1 analog (102) maintains both binding activity
pmolkg. Compound (82), an enantiomer of and analgesic activity, whereas the 5-chloro
A-85380, also did not show a significant anal- analog (103) failed to demonstrate analgesic
gesic activity at doses up to 6.2 pmol/kg, de- activity despite a binding affinity similar to
spite its high affinity for central a4P2nAChRs that of compound (102). The N-methyl analog
(Table 14.11) (342). In contrast, the 6-chloro of ABT-594, (104), dramatically reduces both
analogs (100) and ABT-594 (101) not only binding and analgesic activity.
maintained high affinity in a L3H1cytisine dis- The SAR studies on both the azacycle ring
placement assay but also demonstrated anal- size and azetidine N-methylation have been
gesic effects at 0.62 pmol/kg comparable to carried out (Fig. 14.11) (342). Pyrrolidine an-
that of epibatidine. Like epibatidine, both en- alogs (105) and (106) failed to demonstrate
antiomers have similar analgesic activity. As significant activity in the hot-plate assay, even
shown in Table 14.11, the binding affinity to at doses 10-fold higher than that at which
the i3H1cystine site (i.e., the putative a,& (100) and ABT-594 (101) showed robust ef-
nAChR) does not necessarily correlate well fects. The in vitro tests showed only a modest
with analgesic activity. For example, both decrease in potency with the increased ring
ABT-418 and ABT-594 are a,& agonists, but size. N-Methylation of ABT-594 gave (104),
profound analgesic-like effects were not ob- with substantially reduced the [3H]cytisine
served for ABT-418, whereas ABT-594 dem- binding affinity and ganglionic-like activity,
onstrated significant analgesic activity (343). and loss of analgesic activity. Also of note is
The difference in activity between these two that one or two methyl substituents at the
agonists may reflect interactions with differ- 3-position of the azetidine ring in ABT-594
ent nAChR states, but this has yet to be (107 and 108) (Fig. 14.10) as well as one-car-
verified (344,345). It has also been postulated bon homologation of the linkage (109) signifi-
4 Ion Channel Modulation of Neurotransmission
(107) R' = Me, R2= H ; 4:7.6 nM a; hot plate MED: >62 mrnol/kgb
Figure 14.10. "Binding data: rat braid
(108) R' = R2 = Me; 4:37 nMa; hot plate MED: >62 rnrnol/kgb
[3H]cytisine; bminimum effective dose
(109) 4:11 nMa; hot plate MED: >62 rnmol/kgb (MED) in mouse hot-plate assay (346).
cantly reduces binding affinity and analgesic (101)because the latter shows less cardiovas-
activity in the mouse hot-plate assay. It will be cular toxicity in dogs (294).The separation be-
of interest to determine whether a similar tween antinociceptive doses and lethal doses
SAR will be found when examining the poten- in mice is fivefold greater than that for epiba-
tial efficacy of these compounds in behavioral tidine (351). However, a recent study on the
models of cognitive function. analgesic and toxic effects of ABT-594, nico-
4.2.2.1.4.1 ABT-089 (89). ABT-089 (89) tine, and epibatidine indicates that the acute
was identified as the backup compound to safety profile of ABT-594 is not significantly
ABT-418. The agonist ABT-089 is nearly as improved over that of other nicotinic analge-
potent as nicotine for stimulating ACh release sics (351). ABT-594 has the affinity for a,&
from rat hippocampal tissue. However, ABT- neuronal nAChRs comparable to that of (?)-
089 was 25% less potent and only 70% as effi- epibatidine, but its affinity for neuromuscular
cacious as nicotine in stimulating dopamine nAChRs is 4000 times less than that of (?)-
release from rat striatal tissue (314). ABT-089 epibatidine. Moreover, this compound is about
has also been characterized as a weak agonist 30 times less potent and less efficacious at gan-
at the human a, nAChR subtype (1.5% re- glionic-like nAChRs in IMR-32 cells. The high
sponse at 1 a) and to inhibit the response to selectivity for neuronal a&, nAChRs is re-
a)
ACh (IC,, = 48 (347).In vivo studies with sponsible for an improved therapeutic index
ABT-089 demonstrated cognition enhance- over that of (2)-epibatidine. ABT-594 is also
ment in a variety of rat and monkey behav- less potent than epibatidine in assays of acute
ioral models (348, 349) and has recently been and persistent pain and in the rotarod assay.
shown to reduce distractibility in adult mon- However, it displays a clear separation be-
keys (331). As previously described, ABT-089 tween its motor and analgesic effects (352).
is a relatively poor activator of ganglionic ABT-594 shows good oral bioavailability
nAChRs, suggesting a reduced propensity for across species (30-SO%), with oral half-lives
negative side effects at high doses (350). ABT- ranging from 1.4 to 4.2 h. ABT-594 rapidly
089 showed good oral bioavailability (30-70% enters the brain, exhibiting a brain-to-plasma
across rat, dog, and monkey), superior to both ratio of about 2 within 1.5 h (294,342). Recent
ABT-418 and nicotine (294). It is noteworthy studies on ABT-594 showed an increase in
that the secondary amine functionality serves FGF-2 expression in various rat brain regions,
to enhance the oral bioavailability compared suggesting a therapeutic significance in neu-
to the corresponding N-methyl analog (91) rodegenerative disorders (353),but no preclin-
(62% versus 6%in dog), presumably enhanc- ical data for cognitive enhancement have been
ing its stability to first-pass metabolism (336). reported. ABT-594 is currently in phase 111
ABT-089 is currently in phase I clinical trials trials as an antinociceptive agent (292, 314,
as a potential treatment for Alzheimer's dis- 354).
ease, schizophrenia, and attention deficit hy- 4.2.2.1.5 Phenylthioether Derivatives
peractivity disorder. 4.2.2.1.5.1 SIB-1553A (111). Based on the
4.2.2.1.4.2 ABT-594 (101). A-98593 (100) shift of nAChR subtype selectivity from SIB-
was originally selected for further develop- 1508Y (51)to ligand (56) (Table 14.61, Merck
ment and was later replaced with ABT-594 (then SIBIA Neuroscience) scientists designed
Cognition Enhancers
glia (266) mediate many of the undesired func- with (?)-epibatidine (365). The deschloro de-
tional effects of (2)-epibatidine. Nevertheless, rivative of epibatidine (123) is comparable to
the antioceptive properties of epibatidine have epibatidine in terms of binding affinity for
led to a search for novel agents with enhanced a,P, nAChRs and functional activity in mod-
selectivity for the a,& receptor subtype over els of ganglionic (PC12) and muscle (TE671)
that of other nicotinic receptor subtypes, and nAChR function, but its analgesic potency is
these efforts culminated in the discovery of significantly reduced in the formalin test but
ABT-594 as a novel antinociceptive agent comparable to epibatidine in the tail-flick
(294). It remains to be determined whether model (366, 367). N-Ethylation (126) reduces
these compounds will display efficacy as cogni- the binding activity by 486 times (368). A
tive-enhancing agents. number of substituents at the 2'-position have
N-Methylation of natural epibatidine (112) been evaluated. In general, electron-with-
and its enantiomer (113)reduced affinities for drawing groups and small alkyl groups (115,
rat a4P2nAChRs by 6 and 2 times, respec- 116, 117, 118) maintain activity, whereas
tively. With respect to functional assays, the electron-donating groups (OH, NMe,) (119,
impact of N-methylation on the activities of 121) reduce activity with the exception of NH,
the two enantiomers is relatively small but dif- (120). The 6'-chloro analog (127) is 55 times
ferential, and the N-methyl products (124 and less active in terms of binding affinity (367).
125) showed modest enantioselectivities (358) The racemic 8-azabicyclo[3.2.lloctane ho-
I (Table 14.12). N-Methyl (?I-epibatidine (114) moepibatidine derivatives were found to have
demonstrated the analgesic activity in the analgesic activity in the hot-plate model (369,
I mouse tail-flick model similar to that observed 370) (Fig. 14.12). Compound (128) was shown
:
Cognition Enhancers
Figure 14.12.
to have the same activity as that of epibatidine a4p2and a,/3,6y(~)was relatively unchanged.
at fourfold higher dosage. The N-methyl ana- The in vivo studies of these pyridazine analogs
log (129) possesses activity comparable to that have not yet been reported (373). Recently,
of epibatidine, but the N-isopropyl derivative identified epibatidine analogs include (138)
(130) is 15 times less potent. The tropinone [Ki = 0.03 nM (rat brai~d[~HIcytisine) (37411
analog (131)was shown to be inactive even at and (139) [Ki = 0.26 nM (rat brair~/[~HIepiba-
high doses. The synthesis of the bis-homoepi- tidine) (37511 (Fig. 14.12).
batidine derivative (132) was reported, al- 4.2.2.3 Anabaseine Derivatives. The natu-
though its biological activity has not yet been ral product anabaseine (140) (Fig. 14.13) was
disclosed (371). isolated from the marine worm Hoplonem-
Replacement of the chloropyridyl ring of etines hydrobiologia (376). In vitro analysis
(?I-epibatidine with methylisoxazole pro- found anabaseine to have approximately 20
vided (?)-epiboxidine (133) as a potent times lower affinity than that of nicotine for
nAChR agonist (372) (Fig. 14.12). This analog rat brain nAChRs, with a fivefold selectivity
is 10 times less active than natural epibatidine for the a,& subtype over the a, subtype (377,
and about 17-fold more potent than ABT-418 378). In Xenopus oocytes, however, ana-
in inhibiting [3H]nicotine binding to a4& baseine had a twofold higher intrinsic potency
nAChRs in rat cerebral cortical membranes. than that of nicotine at the expressed a,
Although (?)-epiboxidine exhibits potent ac- nAChR subtype.
tivity at ganglionic-type nAChRs in PC12 DMAB-anabaseine and GTS-21 (141 and
cells, it is less toxic than epibatidine in mice. 142, respectively) are derivatives of ana-
Bioisosteric replacement of the chloropyr- baseine (1401, obtained through condensation
idinyl moiety in epibatidine with pyridazine with the appropriate benzaldehyde (379,380)
generated (136) (Fig. 14.12). Both (136) and (Fig. 14.13). DMAB-anabaseine was more po-
its N-methyl derivative (137) maintained tent than nicotine, but had similar efficacy, at
much of the potency of natural epibatidine, the a, nAChR subtype. Studies in rat brain
but the selectivity between a,& and a3P4sub- indicate GTS-21 binds 20-fold more potently
types was enhanced by eight- and 25-fold, re- than nicotine to the a,& nAChR subtype (Ki
spectively. However, the selectivity between = 19 ? 4 nM)and 6 times less potently than
4 Ion Channel Modulation of Neurotransmission
drogen bond may form (399). This element can A-85380 based nAChR ligands possess st;able
be exemplified by the pyridine ring centroid or conformers featuring similar spatial arrmnge-
the carbonyl carbon. ment of the Sheridan pharmacophoric eleme!nts.
The Sheridan model suggested the optimal The most recent model developed by -
distances between the three elements: A-B Olesen et al. suggests the essential groups are
(internitrogen distance), 4.7 0.3 A; A-C, 4.0 as follows: (1)site point a, correspondin)g to
2 0.3 A; B-C, 1.2 A. The Sheridan models also the protonated nitrogen atom; (2)site poinlt b,
suggested the Beers-Reich distance of 5.9 A. corresponding to the electron negative a.tom
The discovery of epibatidine as an exception- capable of forming a hydrogen bond; (3) site
ally high affinity ligand for nAChRs led to the point c, which is the center of a heteroiaro-
proposal that the optimal internitrogen dis- matic ring or a C----0 bond (Fig. 14.15). 'The
tance for high affinity binding may be close to site points a and b are placed 2.9 A from the
5.5 A as the lowest energy conformer of epiba- corresponding atoms in the direction of the
tidine had an internitrogen distance of 5.51 A lone pairs. Optimal pharmacophoric parame-
(400).A-85380, which possesses affinity simi- ters were estimated as follows: a-b, 7.3-8.( 9 A;
lar to that of epibatidine, showed an interni- a-c, 6.5-7.4 A; Aabc, 30.4-35.8". This th:ree-
trogen distance of 6.1 A in the minimum en- element model was able to explain 65%of the
ergy conformer (340). However, based on the variation seen in the p(IC,,) values (295:). It
argument that the receptor-bound conforma- should be emphasized that it is oversimplified
tion of ligands may not be the same as the to assume that the binding affinity of nAC:hR
lowest energy conformations either in a vac-
uum or in solution, Koren et al. conducted
computational studies on higher than mini-
mum energy conformers of compounds such a-b: 7.3-8.0 A
as epibatidine and A-85380. These studies j b a-C:6.5-7.4 A
show that the internitrogen distances for Aabc: 30.4-35.t
these stable conformers with relative energies
not exceeding 0.42 kcdmol are about 4.4 A,
which is close to the proposed ranges in the
Sheridan model (401).Therefore, it can be con- Figure 14.15. Olesen nicotinic pharmacopl.lore
cluded that epibatidine and the high affinity model. (Modified from Ref. 295.)
4 Ion Channel Modulation of Neurotransmission
Table 14.13 Shared Pharmacological Properties Between 5-HT, and nACh Receptors
nACh Receptors
5-HT3
Receptors Agonist Antagonist
Agonist N-Methylbufotenine Iodide (432,433) Ethanol (440), Halothane (434) and
(at ganglionic nAChR) Isoflurane (434,435) (at the a,
.T
nAChR)
Serotonin (5-HT) (443,444)
ceptors have been observed to mediate inhibi- was found to coassemble with the 5-HT,, sub-
tion of ACh release in human cerebral cortex unit inxenopus oocytes to form a Ca2+perme-
synaptosomes (4231, in rat cortical synaptosomes able channel (430). However, analysis of na-
(424,4251, and in the rat frontal cortex (418). tive 5-HT, receptors purified from porcine
The in vitro affinity of compounds for central cerebral cortex failed to reveal the presence of
5-HT, receptor sites is often determined by ra- nACh receptor subunits (a, to a, or P2) (431).
dioligand binding assay, or more specifically, by Nevertheless, the prevalence and regional dis-
the ability to displace radioligands from 5-HT, tribution of this subunit coassembly remain to
sites of rat entorhind cortex. Several radioli- be determined.
gands have been used, including [3HlLY278584 The structural similarity between 5-HT,
(426), [,HIGR 65630, and [,HIBRL 43694 (427). and nACh receptors provides for an interest-
The in vitro antagonistic activity at the 5-HT, ing but complex pharmacological overlap (Ta-
receptors is carried out on the rat isolated vagus ble 14.13). An overlap of agonist activity is
nerve (RVN) or guinea pig isolated ileum (GpI). known with N-methyl bufotenine iodide [the
5-HT, receptor agonists cause a rapid depolar- quaternary salt of serotonin; 5-HT,, Ki = 75
ization of the vagus nerve. Antagonists cause nM (432, 43311. General anesthetics such as
parallel, rightward displacements of the agonist isoflurane increase the apparent agonist affin-
concentration response curve. The functional ity for both 5-HT, and nACh receptors, but
activity is expressed by the pA, value, which is also cause channel blockade of the nACh re-
the negative logarithm of the molar concentra- ceptor (434, 435). Mutual receptor antago-
tion of an antagonist that necessitates the dou- nism has been observed with chlorpromazine
bling of the agonist dose to counteract the effect and quaternary linocaine derivative QX222
of that antagonist and restore the original re- (436), tropisetron (437,438), and also with d-
sponse (428, 429). The most frequently used tubocurarine (439).Ethanol has been found to
method for assessing the in vivo activity of potentiate agonist activity at the 5-HT, recep-
5-HT, receptor is accomplished through moni- tor (440) while inhibiting the nACh receptor
toring a transient, dose-dependent reflexive fall (441). The nACh receptor agonists ACh, (S)-
in heart rate and blood pressure (von Bezold- and (R)-nicotine, epibatidine, 1,l-dimethyl-4-
Jarisch reflex) evoked by 5-HT, or a 5-HT, re- phenylpiperazine (DMPP), and GTS-21 (142
ceptor agonist, in urethane-anesthestized rats. in Fig. 14.13) have been shown to function as
This effect can be blocked by prior administra- 5-HT, antagonists (442). 5-Hydroxytrypt-
tion of a 5-HT3 antagonist such as ondansetron. amine (5-HT), the endogenous agonist of the
Recently, the nicotinic a, receptor subunit 5-HT, receptor, is an antagonist of nACh re-
4 Ion Channel Modulation of Neurotransmission
ceptors (443,444). The 5-HT, antagonist qui- rat vagus nerve (PA, = 6.5) through selective
pazine and tropisetron have been reported to screening. The N-imidazolyl (147) was found
be an agonist of the a, nACh receptor subtype to be at least 10-fold more potent (PA, = 7.61) -
(445).It seems likely that further examples of than the parent dimethylamino derivative .
pharmacological overlap will continue to be (146) and became a template for further elab-
uncovered. oration. The 1-methylindole analog (148),
Currently, 5-HT, antagonists have found which was marginally more potent than (147)"
their greatest therapeutic value in the treat- in vitro, demonstrated oral activity in the BJ
ment of cancer chemotherapy-induced emesis test in the rat (ED,, = 0.11 mg/kg). However,
(406). Release of 5-HT from the enterochro- it potentiated the pentobarbitone sleeping -
mafin cells in the gastrointestinal track often time in the mouse, presumably because of the
results from cancer chemotherapy with cyto- imidazole moiety, which can bind to and in-
toxic agents, such as cisplatin (446, 447). hibit the hepatic cytochrome P450 oxidase
Blockade of 5-HT, receptors in the CNS or on system. This side effect was overcome by the
peripheral vagal afferent fibers prevents the introduction of a methyl group at the 2-posi-
initiation of the vomit reflex. tion of the imidazole moiety. The resulting
There is a considerable literature indicat- 2-methyl derivative (149) maintained the in
ing that 5-HT, agonists impair, whereas vitro activity (PA, 7.61), but its oral activity
5-HT, antagonists facilitate, learning and (ED,, = 2.5 mgkg in the BJ test in the rat)
memory (448-451). The mechanisms by was substantially reduced, presumably attrib-
which 5-HT, antagonists achieve their posi- utable to first-pass metabolism. Incorporating
tive effects on cognition are not clear. It would the side chain into the tetrahydrocarbazolone
be reasonable to hypothesize that this is at- system generated the conformationally re-
tributable to positive modulation of acetylcho- strained compound, ondansetron (144, GR
line release in the neocortex (but not hip- 65630) (PA, = 8.6), which was more potent
pocampus), where such agents would be than the acyclic ketone (148) on rat vagus
expected to have no effect or inhibit ACh re- nerve and had much improved oral activity in
lease at behaviorally effective doses (418). An- the rat (ED,, = 7 g/kg, p.0.). Like its acyclic
other alternative is that 5-HT, antagonists counterpart (148), it had no effect on the cy-
achieve their effects on cognition indirectly, tochrome P450 oxidase system. Ondansetron
through inhibition of GABAergic interneu- displayed more than 1000-fold selectivity for
rons that regulate the release of many neuro- the 5-HT, receptor over any other receptors
transmitters, including glutamate (451-453). examined, including 5-HT2- and 5-HT,-like
However, as mentioned earlier, there is grow- receptors. Ondansetron is a racemic com-
ing evidence that multiple 5-HT, receptor pound and both of its enantiomers have simi-
subtypes exist that may have distinct distribu- lar in vitro activity. The quaternary derivative
tion or functional properties, which may ac- (145) maintained activity, suggesting that the
count for their effects. Several 5-HT, receptor imidazole moiety is protonated in the binding
antagonists are described that have been re- interaction with the receptor.
ported to improve cognition in various behav- Considerable efforts have gone into the
ioral models. SAR studies on tetrahydrocarbazolones. First,
substituting the indole nucleus at C6 (e.g.,
4.3.2 Structure-Activity Relationships for 151,152), a potential site of metabolism, gen-
5HT,r Antagonists erally reduces potency, with the exception of
4.3.2.1 lmidazolyl lndolyl Derivatives. The the 6-F derivative (150). Second, substitution
SAR studies of imidazolyl tetrahydrocarbazo- at the 9-position is well tolerated (e.g., 153,
lones, exemplified by ondansetron (144) as 154), but some long-chain lipophilic substitu-
5-HT, antagonists, were presented in a review ents reduce activity (e.g., 155). Third, the
by Oxford et al. (428) (Fig. 14.16). The discov- carbonyl function in this series is critical to
ery of ondansetron began with the indolylpro- potent 5-HT, antagonist activity. The corre-
panone (146), which was identified as a weak sponding alcohols (156, 157) and the tetrahy-
antagonist of 5-HT,-induced depolarization of drocarbozole (158) are less potent than the
Cognition Enhancers
I
Me
(144) ondansetron
pA2 8.6 K, 1.6
I
R
(150) R = F, pA2 8.6 (153) R = Et, pA2 8.7
(151) R = Br, pA2 7.5 (154) R = CH2Ph, pA28.2
(152) R = OMe, pA2 6.1 (155) R = (CH&Ph, pA26.8
Me
(156) X: B0H;pA2 6.9
(157) X: a OH; pA2 7.1
(158) X: H; pA2 6.4
tetrahydrocarbazolone (144) by about 30 and tron as potent and selective 5-HT, antagonists
100 times, respectively. Finally, the indole nu- (Table 14.14) (429). Similar to ondansetron,
cleus is superior to the benzofuran (159) and the l,7-annelated indoles show little stereose-
benzothiophen (160) moieties. lectivity. The (-)-isomers are only slightly
Researchers from Solvay reported a series more potent than the (+)-isomers. In this se-
of l,7-annelated indole derivatives of ondanse- ries the five- and six-membered ring analogs,
4 Ion Channel Modulation of Neurotransmission
Compound X KT
(+)-I61
(+)-I62
(+)-I62
(- )-I62 (cilansetron)
(+)-I63
(+)-I63
(-1-163
(?)-I64
(?)-I65 (168) X = CH pA2 10.2
(?)-I66 (169) X = N, Ki0.16 n M (alosetron)
"Rat brain corte~/[~H]GR-65630
(Ki for (2)-ondanse-
tron: 1.6 nM) (429).
Figure 14.17.
ated the scopolamine-induced changes. In an- gation task (477). However, no improvement
other pilot study from the same investigators, in spatial workinghhort-term memory was ob-
elderly humans demonstrated no cognitive en- served in untreated adult rats (478).
hancement from ondansetron alone, nor had Itasetron (171; DAU 6215; Boehringer In-
cognitive deficits induced by scopolamine gelheim Corp.) is a selective and high &nity
treatment attenuated (469). A recent study antagonist of the 5-HT, receptor (Ki = 3.75
conducted in 189 patients with dementia of nM) currently in clinical trials for the tr&t-
the Alzheimer's type, ondansetron (10 or 50 ment of emesis (452). In scopolamine-im-
pg bid for 24 weeks) failed to show any cogni- paired rats, acute administration of itasetron
tive improvement over placebo (470). Ondan- attenuates memory deficits in the Morris wa-
setron is currently not approved by the FDA ter maze and the passive-avoidance task (438,
for use in dementia, but is marketed as an 479, 480). Positive effects for aged rats were
antiemetic. also observed in an avoidance task (479,481)
4.3.2.1.2 Alosetron (169; GR-68755). Alo- and in the Morris water maze (479,482).
setron (169 in Fig. 14.17) is apotent (Ki = 0.16 Tropisetron [ICS-205,930, Novoban, (172)
nM) and selective 5-HT, antagonist (471),and in Fig. 14.18; Novartis] is a potent 5-HT, re-
- duration of action and a wider
it has a longer ceptor antagonist (Ki = 0.38-3.1 nM) (483-
effective dose range than that of ondansetron 485) that has recently been found to possess a,
(472). Studies in the marmoset have shown nAChR subtype antagonist activity (437) (see
alosetron to improve cognitive performance in Table 14.13) and a , nAChR partial agonist ac-
an object discrimination reversal task (10 ng/ tivity (445). In rat behavioral studies, tropise-
kg, s.c. bid) (473) and to attenuate scopol- tron attenuated scopolamine-induced mem-
amine-induced impairment in the acquisition ory deficits in the passive-avoidance test (438),
of an object discrimination task (1mg/kg, i.p.1 and spatial navigation deficits in the Morris
(474). In healthy humans, alosetron (10 and water-maze task (463), and improved the re-
250 pg) showed significant improvement in re- tention of a conditioned response in rats with
ducing spatial and verbal memory deficits in- p-chloroamphetamine-induced deficits (486).
duced by scopolamine (475). In another clini- Tropisetron is currently marketed for the
cal study, alosetron (20 mg orally) enhanced treatment of nausea and vomiting associated
accuracy on a computerized attention task but with chemotherapy treatments (485).
failed to reverse d-amphetamine-induced def- GYKI-46903 (173; Egis Gyogyszergyar) is a
icits in healthy male subjects (476). potent 5-HT, antagonist (Ki = 20 ? 2 nM)
Alosetron was approved for U.S. use in Feb- reported to be under development as a poten-
ruary 2000 for the treatment of irritable bowel tial treatment for dementia (487,488).In rats,
syndrome (IBS)in women whose predominant GYKI-46903 was shown to attenuate scopol-
bowel symptom is diarrhea, although the amine-induced memory impairments in the
-product was withdrawn in November 2001 be- stepdown passive-avoidance task and the
cause of serious side effects, particularly isch- eight-arm radial maze (489).
emic colitis. Earlier studies in men for anxiety Mirisetron (174; SEC-579; WAY-100579;
disorder and schizophrenia, and Phase I1stud- American Home Products Corp.) is a 5-HT,
ies for nonulcer dyspepsia, have also been dis- antagonist (IC,, = 1.2 nM) (490) that im-
continued. proved spatial learning of lesioned rats in the
4.3.2.2 Other Chemotypes. Several other water maze (466) and enhanced acquisition of
5-HT, antagonists have demonstrated cogni- a visual object discrimination for aged mon-
tion-enhancing effects in animal models. How- keys (467). This compound had reached phase
ever, data from human studies have yet to be I Clinical trials for use as a potential treat-
reported. (R)-Zacopride (170 in Fig. 14.18; ment for anxiety, but no further development
Synthelabo) is undergoing clinical develop- has been reported.
ment as a -potential treatment for cancer
chemotherapy-induced emesis. In atropine- 4.3.3 Future Direction. 5-HT, receptor an-
treated rats, (R)-zacopridesignificantly atten- tagonists were initially hailed as potential
uated memory impairments in a spatial navi- treatments for anxiety, Alzheimer's disease,
4 Ion Channel Modulation of Neurotransmission 817
Figure 14.18.
schizophrenia, pain, and drug dependency. AP) and 3,4-diaminopyridine (3,4-DAP), were
Despite some early encouraging results, no initially evaluated for cognition enhancement
5-HT, antagonists have been approved for the in AD, but gave poor results (493). New com-
treatment of cognitive dysfunction. It would pounds that are selective inhibitors of the K t
be important to know whether 5-HT, antago- M-current, or the channels that underlie this
nists produced any significant improvement current, appear to offer better promise as cog-
over placebo in the clinical trials. At present, nitive enhancers.
their therapeutic application for cognition en- The M-current [I,(,,] is an outward K t
hancement remains unknown. conductance through M-type voltage-gated
Kt channels. This current, which is induced
4.4 Potassium M-Channel
by membrane depolarization and is relatively
slowly activating and deactivating, modulates
4.4.1 Physiology and Pharmacology. Potas-
the subthreshold electrical excitability of neu-
sium (Kt ) channels serve a modulatory func-
tion in excitable and nonexcitable cells by reg- rons in response to synaptic inputs (494). M-
ulating K t conductance (491,492). Within the current is so named because it was inhibited
CNS, many typesof K+ channels are known by activation of muscarinic acetylcholine re-
with differential patterns of distribution. ceptors (495). Inhibition of M-current results
Opening of neuronal Kt channels generally in enhancement of the release of excitatory
acts to dampen neuronal excitability by pre- or neurotransmitters and their postsynaptic ef-
postsynaptic mechanisms. Nonspecific Kt fects (496,497). This net amplification of sig-
channel blockers, such as Caminopyridine (4- nals that originate through normal synaptic
Cognition Enhancers
activitv
" makes the M-channel an attractive vous system. If true, agents that selectively
target for the development of cognitive en- inhibit M-current by directly interacting with
hancers (123,498). the M-channel may enhance cognition with-.
The molecular identity underlying the M- out the adverse side-effect profile typically
current had been established as the coassem- observed with direct muscarinic agonists or
bly of channel subunits (499, 500), KCNQ2 acetylcholinesterase inhibitors. Several candi-
(501) and KCNQ3 (502), and it now appears date M-channel modulators are discussed be?
that KCNQ5 may play an additional role in the low.
generation of an M-type current (503). Muta-
tions in the genes encoding either of these 4.4.2 Structure-Activity Relationships for
channel subunits can result in inactive chan- M-Channel Blockers
nels, causing a form of idiopathic generalized Linopirdine (DuP 996; 175), XE997 (180),
epilepsy (500-502, 504). and DMP 543 (181). The M-current blocker
Several endogenous molecules are known linopirdine (3,3-bis(4-pyridinylmethy1)-1-phe-
to inhibit the M-current, including acetylcho- nylindolin-2-one; DuP 996; 175) is one of a
line, uridine and adenosine triphosphate series of cognition-enhancing agents devel-
(505), bradykinin (506-5091, angiotensin I1 oped by DuPont-Merck (527, 528) that has
(510),endothelin 1 (5111, substance P, and lu- been the focus of intense investigation (Fig.
teinizing hormone-releasing hormone (512). 14.19) (reviewed in Refs. 496, 529). In slices
On the other hand, M-current is increased (po- from rat brain cerebral cortex, hippocampus,
tentiated) by somatostatin and P-adrenore- and caudate nucleus, linopirdine was found to
ceptors through a CAMP-dependent process increase ACh (EC,, value of 4.5 and other
(513). Recently, the novel anticonvulsant reti- neurotransmitters in response to a depolariz-
gabine has been shown to activate human ing stimulus, but had no effect on basal release
KCNQ2 and KCNQ213 channels expressed in (530,5311,suggesting use dependency. In vivo
CHO cells, perhaps contributing to the phar- microdialysis studies have shown an increase
macology of this compound (514). in the release of ACh from rat hippocampus at
Of the five known classes of muscarinic a dose of 10 mg/kg, S.C. (532).
ACh receptors, M-current is preferentially in- Mechanistic studies have indicated that li-
hibited by m l or m3 subtypes (515, 516). Ex- nopirdine (175) inhibits M-current (IKcM,) by
periments with mice lacking the m l receptor channel blockade from the extracellular side
exhibit a loss of muscarinic regulation of M- (529,533,534). Interestingly, linopirdine's ef-
current and are resistant to pilocarpine-in- fect on neurotransmitter release was demon-
duced seizures (517). Recently, specific anti- strated to be brain region specific, indicative of
sense plasmids have been used to strongly a complex in vivo mode of action (535). The
suggest the G-protein GCY,as the primary mechanism underlying the effects of linopir-
transducer of muscarinic inhibition of IK(M, dine on neurotransmitter release, in particu-
(518). lar the potentiation of ACh release from basal
M1 mAChR agonists (that likely inhibit M- forebrain cholinergic neurons, is not known,
current. in addition to their other actions) but could be explained by an action on presyn-
have received considerable attention as possi- aptic KCNQ2 and/or KCNQ3 channels (536).
ble cognitive enhancers (519-521). Although Several substitutions were made for li-
many m l agonists have produced positive re- nopirdine's phenylindolinone "core" to iden-
sults in animal models (522-5251, most of tify more active analogs (530). This was done
these compounds generate unacceptable side in relation to three regioisomer variations of
effects in humans (e.g., 526). It is possible that the pyridinylmethyl "pendent" moiety, while
the cognition-enhancing properties of m l ago- attempting to maintain asymmetry in the con-
nists (and, perhaps, cholinesterase inhibitors) structs. Two compounds worth noting are
are the consequence of a downstream M-cur- xanthene (176) and acenapthenone (177) de-
rent inhibition, whereas the side effects result rivatives that were found to be approximately
primarily from muscarinic receptor mediated equipotent with linopirdine for their ability to
over stimulation of the parasympathetic ner- enhance depolarization-induced ACh release
4 Ion Channel Modulation of Neurotransrnission
(175)
0
linopirdine
from rat brain hippocampal slices. Further core structure of linopirdine was substituted
SAR studies focused on substitution of only with an anthrone moiety to provide XE991
one of the pair of pyridinylmethyl groups (180),a more lipophilic analog. XE991 showed
while maintaining the phenylindolinone core approximately 10-fold greater potency than
(537). Of the racemic compounds generated, that of linopirdine in inhibiting M-current in
the (?)-ethylbutpate (178) and (2)-valeroni- cultured superior cervical ganglion neurons
trile (179) derivatives deserve mention. Each and in KCNQ2/3 channels expressed in Xeno-
analog replaced one of the two pendant groups pus oocytes (297). Further structural modifi-
with an isostere capable of acting as a hydro- cation was done to reduce in vivo N-oxidation
gen-bond acceptor and was found to be as good by replacing the pendant groups with 2-fluoro-
as, or superior to, linopirdine as enhancers of 4-pyridinylmethyl groups, generating DMP
ACh release. Furthermore, when the racemic 543 (181). Both compounds were superior to
ethylbutyrate derivative (178) was resolved, linopirdine for enhancement of ACh release
the biological activity of the (+)-isomer was from rat brain slices (EC,,: linopirdine = 4.2
found, surprisingly, to be only slightly higher f l ;XE991 = 490 nM, DMP 543 = 700 nM)
than that of the racemic mixture. The (-1- and had greater in vivo potency and duration
isomer was inactive and reported not to be an of action (538). An improved synthesis of
antagonist to ACh release. DMP-543 was later reported (539).
Further research to develop a second-gen-
eration clinical candidate with improved phar- 4.4.3 Cognition-Enhancement Experiments.
macokinetic and pharmacodynamic proper- Linopirdine (175) has been assayed in a wide
ties has focused on core and pendant group range of animal models for cognitive enhance-
modifications of linopirdine (175) (530). The ment. In mice, linopirdine enhanced perfor-
Cognition Enhancers
mance in an active avoidance task (540) and cellular Ca2+ dynamics. As such, this class of
improved working memory deficits caused by compounds may normalize the normally
ethylcholine aziridium (AF64A)in the T-maze pathological levels of Ca2+ seen after cerebral -
(541). In rats with and without hypoxia-in- insult, which may otherwise lead to cell death .
duced memory deficits, linopirdine improved and cognitive dysfunction. One compound
performance in the passive-avoidance task, from this class, MEM 1003, is currently in late
suggesting augmentation of task acquisition preclinical trials and may enter European
and consolidation of information skills (540, clinical trials in 2002 (SCRIP, September 20,
542). Linopirdine was also reported to im- 2001).
prove the spatial learning of septal lesioned
rats in the Morris water maze (543), but has
produced conflicting results for untreated rats 5 CONCLUSION
in similar tasks (543-545). In contrast, li-
nopirdine did not improve performance in As the world's population ages, effective treat-
matching-to-sample paradigms using pigeons ment for the cognitive impairments of demen-
or squirrel monkeys (546). Initial clinical tri- tia becomes an increasingly important goal.
als conducted with linopirdine in AD patients The limited success of currently marketed ace-
have been reported to show trends toward cog- tylcholinesterase inhibitors may be partially
nitive improvement (78). However, in phase attributed to factors such as potency, selectiv-
I11 clinical trials linopirdine failed to show ity, or brain penetration. However, it is also
consistent efficacy (547) and continued devel- likely that the potential of these drugs is lim-
opment was terminated (548). ited because of their focused targeting of only
Phase I clinical studies with DMP 543 one of the several neurotransmitter systems
(181),up to a single dose of 55 mg, demon- known to be disturbed in dementing illnesses.
strated that it was well tolerated and had an An additional potential limitation of these me-
improved mean elimination half-life of 30-57 dicaments is that they do not necessarily in-
h (linopirdine was 0.4-3.2 h) (549). To date, crease the fidelity of the signaling event, but
preclinical studies describing cognitive en- instead simply increase the overall back-
hancement with these compounds have not ground level of central ACh. This approach
been published. The development of DMP 543, remains viable only until a critical number of
after reaching phase I1 clinical trials as a po- ACh producing neurons degenerates, at which
tential therapy for the treatment of Alzhei- time a rapid decrease in central ACh can be
mer's disease, has now been discontinued (Du- expected.
Pont Pharm, 2000 Annual Report). The purpose of the present review has been
to consider alternative, ion channel-based ap-
4.4.4 Future Direction. Potassium chan- proaches to the development of cognitive en-
nels, especially the KCNQ family, are widely hancers. Existing preclinical data indicate
involved with the physiology and pathophysi- that these approaches are at least as effective
ology of the human body. Research into their as cholinesterase inhibitors in enhancing
biology will continue to blossom as more cognition. The agents described herein all fa-
cloned KCNQ subunits are available, along cilitate synaptic transmission in several neu-
with selective agents to modify their action. rotransmitter systems, primarily through en-
An additional area of potentially fruitful re- hancement of release. Thus, they work as
search is in the area of central Ca2+ channel positive modulators of normal brain circuit
modulation. Nimodipine, a centrally acting functioning, rather than affecting neurotrans-
Ca2+ channel antagonist marketed in the mitter levels without regard for temporal pa-
United States by Bayer AG for the treatment rameters that are likely critical to meaningful
of subarachnoid hemorrhage, is known to pro- signaling. Another potential advantage of the
tect against the cognitive impairment that mechanisms discussed above is that they
normally follows cerebral insult (550). This ef- present a multifaceted secondary pharmaco-
fect is thought to be attributed to the ability of therapy that would seem to be more appropri-
nimodipine to normalize the function of intra- ate for treating the complex neuropathology of
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CHAPTER FIFTEEN
Weight Disorders -
PHILIP A. CARPINO
JOHN R. HADCOCK
Pfizer Global Research & Development-Groton Labs
Department of Cardiovascular and Metabolic Diseases
Groton, Connecticut
Contents
1 Introduction, 838
1.1Eating and Body Weight Disorders:
Definitions, Causes, Comorbidities, 839
1.1.1Obesity, 839
1.1.2 WastingICachexia Secondary to Other
Diseases, 840
1.1.3 Eating Disorders, 840
1.2 Guidelines for Treatment of Eating
and Body Weight Disorders, 842
1.2.1 Obesity, 842
1.2.2 Eating Disorders~WaatingDisorders,
842
2 Clinical Applications, 843
2.1 Overview: Current Drugs, 843
2.1.1 Drugs to Treat Obesity, 843
2.1.1.1 Fat-Absorption Inhibitors, 843
2.1.1.2 Appetite Suppressants, 843
2.1.1.3 Lipolytic and Thermogenic
Agents, 857
2.1.2 Drugs to Treat Eating Disorders, 857
2.1.3 Drugs to Treat Wasting Diseases, 858
2.2 Adverse Effects and Precautions, 858
3 Drug Metabolism, 860
3.1 Absorption/Metabolism of Antiobesity Drugs,
860
3.1.1 Orlistat, 860
3.1.2 Sibutramine, 860
3.1.3 Phentermine, 861
3.1.4 Benzphetamine, 861
3.1.5 Phendimetrazine, 863
3.1.6 Diethylpropion, 863
3.1.7 Mazindol, 866
Burger's Medicinal Chemistry and Drug Discovery 3.2 Metabolism of Appetite-Stimulant Drugs,
Sixth Edition, Volume 6: Nervous System Agents 866
Edited by Donald J. Abraham 4 Physiology and Pharmacology, 867
ISBN 0-471-27401-1 O 2003 John Wiley & Sons, Inc. 4.1 Body Weight, 867
837
Drugs to Treat Eating and Body Weight Disorders
bulimia nervosa, and cachexia, although there The incidence of obesity has dramatically
is still a great medical need for new agents increased over the past decade, reaching epi-
with improved properties. demic proportions in industrialized countries
This chapter describes (1) the physiology throughout the world (2). In the United
and pharmacology of the most common forms States, the disease varies significantly by age,
of eating and body weight disorders; (2) clini- race, and gender and is more prevalent in cer-
cal guidelines for treatment; (3)current drugs tain socioeconomic groups that are predomi-
and their mechanisms of action, adverse effect nantly poor, less educated, and less affluent
profiles, structure-activity relationships, and (3). The proportion of obese American men
metabolic degradation pathways; and (4) new and women has increased by approximately
agents in development.
50% over the past decade, from 12% of the
1.1 Eating and Body Weight Disorders:
population in 1991 to 17.9% in 1998 (3). A ma-
Definitions, Causes, Comorbidities jority of adult Americans and a significant
number of children are now considered either
1.I .I Obesity. Obesity is a condition of ab- overweight or obese (4). Interestingly, more
normal body weight resulting from an accu- people in the world are classified as over-
mulation of extra adipose tissue, generally in weight and obese rather than as malnour-
response to a state of positive energy balance ished, according to the World Health Or-
that occurs when energy intake exceeds en- ganization.
ergy expenditure. Obesity is characterized by Excess adiposity and above-average body
an elevated body mass index (BMI), a com- weight, two distinguishing hallmarks of obe-
monly used descriptor of body composition sity, are associated with numerous health
that is defined as the weight of an individual in risks, resulting in the designation of obesity as
kilograms divided by the square of the height a serious disease (5, 6). According to recent
in meters, or kg/m2 (1).Three discrete classes epidemiological data, obesity increases sus-
of obesity have been established based on ceptibility to a wide range of both cardiovas-
BMI, depending on the risks of associated co- cular and metabolic diseases (7, 8), including
morbidities (see Table 15.1). A preobese state hypertension (91, non-insulin-dependent dia-
is called overweight, with above-average risks betes mellitus or NIDDM (10, ll),and hyper-
of comorbidities. Morphological features of lipidemia (121, and is an independent risk fac-
obesity are usually distinct and often gender tor for certain cancers, including breast,
specific, although there is considerable indi- colorectal, and endometrial cancers (13, 14).
vidual variability. Adiposity in men forms High BMIs also cause debilitating musculo-
mostly in the abdomen, resulting in a unique skeletal (15) and respiratoryhleep disorders
"apple" shape; in women, excess fatty tissue (16) that result in diminished quality of life
generally accumulates in the upper body (17). Because of these comorbidities, the risk
and/or in the hips and the buttocks, giving a of mortality for individuals with BMIs greater
unique "pear" shape. than 30 kg/m2 is nearly twice that for individ-
840 Drugs to Treat Eating and Body Weight Disorders
Metabolic/Endocrine
Cardiovascular Diseases Diseases Joint Diseases CNS
Congestive heart failure Diabetes Osteoarthritis Depression
Hypertension Insulin resistance Rheumatoid arthritis Pain
Deep vein thrombosis Glucocorticoid imbalance Bone fractures T
Coronary artery disease Gall bladder disease Lower back pain
Stroke Hypercholesterolemia Carpal tunnel syndrome
Hypertriglyceridemia
Gout
Pancreatitis
Liver disease
Cancers Respiratory diseases Immune impairments Other
Breast cancer Asthma Impaired wound healing Infertility
Colorectal cancer Chronic obstructive Impaired immune response Incontinence
pulmonary disease (COPD)
Renal cell cancer Chronic bronchitis Renal failure Renal failure
Endometrial cancer Sleep apnea
uals with normal BMIs (18). Obesity is now 1 .1 .2 Wasting/Cachexia Secondary to Other
the second leading cause of preventable death Diseases. Wasting diseases such as cachexia
in the United States after smoking, with ap- are body weight disorders that are character-
proximately 300,000 deaths a year directly at- ized by excessive loss of lean body mass, espe-
tributable to the disease or its comorbidities cially muscle tissue, resulting in profound
(19). A comprehensive listing of the comor- weight loss. The decrease in body weight that
bidities associated with obesity is provided in occurs with these diseases fails to stimulate an
Table 15.2. adaptive response that would normally en-
The causes of obesity are complex, but a hance appetite. Profound loss of lean body
combination of behavioral, genetic, hormonal, mass ultimately leads to organ failure and
economic, and environmental factors all play death. Wasting diseases can be caused by a
critical roles in the origin and progression of variety of factors such as change in taste per-
the disease. Expression changes in genes and ception, decreased food intake, increased sati-
single-nucleotide polymorphisms can predis- ety, or malnutrition, and often occur second-
pose individuals to become overweight or arily in diseases such as AIDS and cancer or
obese. However, lifestyle choices (i.e., low with certain injuries such as severe burns.
physical activity), food preferences (i.e., high Some eating disorders such as anorexia ner-
fat diets, energy-dense foods), and certain be- vosa and several forms of cancer (i.e., lung
haviors (i.e., overeating) hasten the actual cancer) tend to cause severe wasting, which
pathogenesis, generally when caloric intake requires separate therapeutic management
exceeds energy output. The body has devel- apart from the primary disease.
oped numerous methods for managing physi-
ological states of negative energy balance and 1.1 .3 Eating Disorders. Eating disorders
enhancing survival, but few mechanisms for such as anorexia nervosa, binge eating disor-
coping with sustained states of positive energy der, bulimia nervosa, and night-eating syn-
balance and preserving long-term health. It drome are primarily behavioral diseases that
should be noted that only a small percentage are associated with irregular eating patterns
of individuals develop obesity as a result of and/or weight control. The two major eating
specific genetic defects in essential metabolic disorders, anorexia nervosa and bulimia ner-
processes (20). vosa, are often caused by unrealistic percep-
1 Introduction 841
Table 15.3 Symptoms Used to Define Anorexia Nervosa and Bulimia Nervosa as Determined
by the American Psychiatric Associationa
-- -
Anorexia nervosa
1. Refusal to maintain weight greater than the lowest weight considered normal for age and height.
2. Intense fear of gaining weight or becoming fat, even though underweight.
3. Distorted body image in women.
4. Three consecutive missed menstrual periods without pregnancy. T
Bulimia nervosa
1. Recurrent episodes of binge eating (minimum average of two binge-eating episodes a week for at least
3 months). -
2. A feeling of lack of control over eating during the binges.
3. Regular use of self-induced vomiting, laxatives, diuretics, or vigorous exercise to prevent weight gain.
4. Persistent preoccupation with body shape and weight.
"Ref. 5.
tions of ideal body shape and body weight. An- caused by excessive fears of body weight or fat
orexia nervosa results from extreme food gain, leading to dieting and self-imposed star-
restriction (starvation) and less frequently vation. AN is difficult to diagnose in clinical
from other behaviors such as extensive exer- practice because of the extreme measures pa-
cise, and is characterized by a body weight tients undertake to hide the disease. Loss of
that is less than 85% of expected relative body weight, especially body fat, leads to
weight (21). Binge eating disorder consists of amenorrhea, which results in decreased estro-
episodic compulsive eating behavior that re- gen production and diminished bone mineral
sults in body weight gain (22). Bulimia ner- density. Several other side effects of AN in-
vosa is a binge eating disorder that is accom- clude hypotension, hypothermia, pancreatitis,
panied by inappropriate compensatory actions and metabolic abnormalities associated with
such as vomiting, laxative use, or less fre- protein and electrolyte insufficiencies. The
quently, excessive exercise (21). Night-eating death rate from AN is 12 times higher than
syndrome occurs with many sleeping disor- that for other age-matched, young women in
ders (apnea) and results in excessive caloric the population. Leading causes of mortality
intake during nighttime periods of wakeful- include ventricular tachyarrhythmias that re-
ness (23). Anorexia nervosa, bulimia nervosa, sult from electrolyte imbalance (hypokalemia)
and binge eating disorder occur frequently or protein malnutrition (cachexia).
with other diseases of the CNS such as depres- Bulimia nervosa (BN) is an eating disorder
sion, anxiety, and various personality disor- that is becoming increasingly prevalent in
ders and are listed as psychiatric/behavioral Western societies, often occurring in approxi-
disorders in the Diagnostic and Statistical mately 1 3 %of women over the course of their
Manual of Mental Diseases, 4th edition (24). lifetime. Unlike anorexia nervosa, BN is not
Several reviews describe these illnesses in associated with an abnormal state of body
great detail (21,25). weight, in that most women with the disease
Anorexia nervosa (AN) is a chronic disease are within 15% of their ideal body weight.
that is associated with many life-threatening Common symptoms of BN are listed in Table
comorbidities (26). This disorder, which af- 15.3. Risk factors for BN include a previous
fects predominantly young women, often experience with anorexia nervosa, low self-es-
starts in middle adolescence, with a mean on- teem, and certain personality traits. In the
set of action of 17 years of age, and is likely to earliest stages of the disease, patients begin to
occur in approximately 0.5% of women over diet, but the diet-induced hunger cannot be
the course of their lifetime. The clinical crite- controlled. This failure then leads to binge
ria used to define AN are listed in Table 15.3. eating behavior, followed by purging actions
Body weight is controlled by restriction of food (vomiting, laxative, or diuretic use) or non-
intake, although extensive exercise is some- purging behavior (excessive exercising, fast-
times used as a substitute. The disease is often ing) to remove excess calories. As the disease
Drugs to Treat Eating and Body Weight Disorders
progresses, binge eating and the compensa- treatment program. The successful mainte-
tory behavior(s) become mechanisms for cop- nance of lower body weight generally requires
ing with and seeking temporary relief of co- rigid adherence to strict behavioral modifica-
morbidities such as depression or anxiety (26). tions (31).
A typical binge eating episode consists mostly Several drugs for weight loss and weight
of high carbohydrate meals. The average du- maintenance are currently available. These
ration of a meal can be greater than 1 h, with drugs are recommended for patients wit?^
more than 4000 kcal ingested in a single meal. BMIs greater than 30 kg/m2 without addi-
In extreme instances, the amount of food in- tional risk factors or with BMIs greater than
gested at a single setting can be more than 27 kg/m2 with two or more risk factors (29):
20,000 kcal and can last for hours (27). The Obesity is now considered a chronic disease
physical complications of bulimia nervosa that must be managed accordingly.
tend to be limited to hypokalemia, metabolic
alkalosis, arrhythmias, and gastric and esoph- 1.2.2 Eating DisordersNVasting Disorders.
ageal rupture and rarely result in death (25). Few clinical guidelines have been established
Binge eating disorder is the only major eat- for the treatment of eating and wasting disor-
ing disorder that is found appreciably in both ders. Disease-associated cachexia is usually
sexes (35%men; 65% women) (28).A majority managed with nutritional therapy and phar-
of binge eaters are overweight and experience macotherapy involving either anabolic agents
one or more symptoms of classical depression. or appetite stimulants. Eating disorders are
Binge eating behavior occurs alone and ap- treated with a combination of nutritional
pears to be uncontrollable, although it may be therapy, pharmacotherapy, and/or cognitive
soon followed by feelings of embarrassment or behavioral therapy (CBT). For anorexia ner-
guilt. vosa, oral or p a r e n t e d nutritional therapy is
first used to restore a healthy normal weight.
1.2 Guidelines for Treatment of Eating This is followed by behavioral therapy to re-
and Body Weight Disorders move unhealthy preoccupations with weight
loss and to change perceptions about body fat
1.2.1 Obesity. An expert panel of physi- and appearance. Severe cases of anorexia, es-
cians has published several recommendations pecially after onset of cachexia, are best man-
for the treatment of obesity (29, 30). The aged within an in-patient hospital setting.
guidelines call for a 5-10% decrease in body Pharrnacotherapy is generally used to ease
weight by first decreasing total caloric intake fear of food intake, enhance nutritional ab-
and increasing physical activity. This overall sorption, and protect against comorbidities,
decrease in body weight, although small in although it is not very successful in treating
magnitude, is normally adequate to restore the disease itself. For bulimia nervosa, the ini-
"metabolic fitness" by decreasing blood pres- tial goal of treatment is to remove the under-
sure, blood glucose, and serum triglyceridel lying cause of any overeating behavior such as
cholesterol levels. It is recognized that exer- diet-induced hunger or depression and elimi-
cise and diet often prove difficult to implement nate the psychological need for and depen-
on a routine basis and often prove ineffective dency on compensatory behaviors. Pharmaco-
over the long term in causing weight loss, pri- therapy has proven remarkably effective in
marily because weight loss by diet alone re- the treatment of this disease. The manage-
sults in compensatory increases in energy ef- ment of binge eating disorder requires behav-
ficiency and decreases in basal metabolic rate. ioral therapy to restore restraint in eating be-
In such cases where lifestyle changes fail to havior ("self-denial")and pharmacotherapy to
produce the desired reductions in body weight, address the underlying psychiatric compo-
antiobesity pharmacotherapy is subsequently nents of the diseases (32). Because a majority
recommended. Surgery is an option for pa- of patients with binge eating disorder are also
tients with clinically severe obesity (i.e., BMIs overweight, lifestyle changes are necessary to
> 40 kg/m2 or BMIs > 35 kg/m2 with coexist- stimulate weight loss. For all eating disorders,
ing conditions) who do not respond to any prevention of relapse is still a major challenge.
2 Clinical Applications 843
The role of drug therapy in the treatment of weight loss therapy, including nonsystemic fat
eating disorders has been reviewed elsewhere absorption inhibitors, appetite suppressants, .
(21, 33). and thermogenidlipolytic agents (see Table
15.4). These drugs can either decrease energy -
2 CLINICAL APPLICATIONS expenditure or enhance fat metabolism. They
generally decrease body weight by producing,
2.1 Overview: Current Drugs
negative states of energy balance. Fat absorp-
A limited number of safe and efficacious drugs tion inhibitors and lipolytic agents act primar-
are available for the pharmacological treat- ily at molecular targets in the periphery, -
ment of eating and body weight disorders. whereas appetite suppressants generally me-
Compounds (1-9) are used with varying fre- diate their effects in the central nervous sys-
quencies as antiobesity drugs, generally as tem (CNS).Most centrally acting appetite sup-
part of a comprehensive treatment program pressants have low molecular weights and are
involving lifestyle and behavioral changes (see highly lipophilic, properties that promote
Table 15.4). These drugs generally do not transcellular diffusion across the blood-brain
modulate the pathophysiology of obesity be- barrier into the brain.
cause cessation of therapy usually results in Several recent reviews describe in greater
the return of any lost weight. Several antiobe- detail the different pharmacological treat-
sity agents have been removed from the mar- ments for obesity and the clinical efficacies of
ket or are now no longer prescribed as weight both old and new agents (34-36). Worldwide
loss drugs, primarily because of safety con- and U.S. sales of selected drugs used for the
cerns. These include thyroid hormone (10) treatment of eating and body weight disorders
and appetite suppressants such as dexamphet- are presented in Table 15.8 (37).
m i n e (ll),aminorex (12),fenfluramine (131, 2.1.1.1 Fat-Absorption Inhibitors. Orlistat
dexfenfluramine (14), and phenylpropanol- (1, Ro 18-0647, tetrahydrolipstatin) is a non-
m i n e (15) (see Table 15.5). systemic fat absorption inhibitor approved for
Few drugs are available to specifically treat chronic use as a weight loss treatment. It pre-
eating disorders, in part because these dis- vents the breakdown of triglycerides in the
eases are so complex, with features of eating, gastrointestinal tract and blocks the intestinal
psychiatric, and body weight disorders. In gen- absorption of fatty acids (38). It acts as an ir-
eral, antidepressant medications such as (16- reversible inhibitor of gastric and intestinal
24) are used to treat the underlying psychiat- lipases by reacting with specific serine resi-
ric symptoms associated with bulimia nervosa, dues found in the catalytic sites (39). Orlistat
binge eating disorder, and, less frequently, an- is modestly effective at reducing both body
orexia nervosa, and to prevent relapse of many weight and decreasing frequency of comor-
of these disorders (Table 15.6). For anorexia bidities such as diabetes, hypertension, and
nervosa, a variety of different drugs are used hyperlipidemia. In one Pyear clinical trial,
to overcome the side effects generally associ- obese patients with BMIs greater than 30
ated with malnutrition and the fears associ- kg/m2 lost 5-10% of their body weight in their
ated with food intake and body weight gain first year on orlistat therapy and showed sig-
(see 25-29, Table 15.6). nificantly less weight regain in their second
Several drugs (30-33) have recently been year of the study (40).
approved as appetite stimulants or anabolic1 2.1.1.2 Appetite Suppressants. Appetite sup-
growth-promoting agents in wasting diseases, pressants induce satiety mechanisms and, less
generally as short-term treatments until a frequently, increase energy expenditure by in-
normal body weight is achieved (see Table teracting with sympathetic pathways in the
15.7). These agents are often prescribed in CNS. These drugs decrease food intake by ac-
conjunction with other drugs used to treat the tivating doparninergic, sympathomimetic (ad-
primary illness such as cancer or AIDS. renergic), and/or serotonergic systems in the
brain. Modulations of these pathways occur
2.1.1 Drugs to Treat Obesity. Three classes through direct and indirect mechanisms. Indi-
I
E
of antiobesity drugs are currently used in rect-acting drugs increase concentrations of
Table 16.4 (Continued)
Trade DEA Dose
Generic Name Structure Name(s) Originator(s) Class Class (rnglday)
Bontril; Amarin Appetite 111 70-210
Plegine Pharmaceuticals; suppressant
Wyeth
Thermogenicl No label 75
lipolytic
agent
Table 15.5 Miscellaneous Antiobesity Drugs
Generic Name Structure Class Status
Thyroid hormone (10) I Thermogenic agent Rarely prescribed as weight loss
I agent
" because of
hyperthyroidism
Trade Dose
a
Generic Name Structure Name Originator Indication Class (mdday)
-
p H 3
H
0,
CH3
HO
(27)
Equilin (-30%):
0)
tn
W
"SSRI, selective serotonin reuptake inhibitor; TCA, tricyclic amine antidepressant; MAOI, monoamine oxidase inhibitor.
*Withsymptoms of depression.
"Off-label.
dSecond-linetherapy.
2 Clinical Applications 855
Table 15.8 Worldwide and U.S.Sales in Year 2000 of Selected Drugs used for the Treatment
of Eating and Body Weight Disordersa
Worldwide Sales U.S. Sales
Indication ($, millions) ($, millions) -
Sibutramine Obesity (OBI
Phentermine OB
Phendimetrazine OB
Orlistat OB
Mazindol OB
Ephedrine OB
Diethylpropion OB
Oxandrolone Cachexia (C)
Megestrolb C
"Adapted from IMS Health Global Services, 2001.
bMost of the sales for Megestrol were for other indications.
dopamine (DA, 341, norepinephrine (NE, 351, petite. Sympathomimetic drugs have been his-
and serotonin [5-hydroxytryptamine (5-HT), torically classified as either direct- or indirect-
361 at postsynaptic neuronal receptors by (1) acting agents.
The majority of appetite-suppressant drugs
are congeners of 2-phenethylamine (P-phen-
ethylamine, 37), a compound with close struc-
(37) 2-Phenylethylamine, R = H
(38) Amphetamine, R = CH3
long-term use in weight loss therapy (41). In that reduces the central excitatory activity as-
clinical trials, sibutramine has been shown to sociated with the Bphenethylamine class of
reduce total daily energy intake, decrease fat appetite suppressants, without affecting ano-7
intake, and lower feelings of hunger (42). It rectic activity. Diethylpropion is recom-.
also has been reported to increase energy ex- mended for obese patients with elevated blood
penditure, although the data are somewhat pressure or cardiovascular diseases. These
conflicting (43). Sibutramine has been found drugs all decrease appetite by increasing N E ~
to prevent body weight regain after weight release from nerve terminals.
loss (44). The long-term effectiveness of si- Mazindol (6) is the only non-2-phenethyl-
butramine in reducing obesity-related comor- m i n e indirect-acting syrnpathomimetic drug
bidities has yet to be conclusively established, approved as a short-term appetite suppres-
but sibutramine has been shown to decrease sant. The drug was the principal anorectic
cholesterol and triglyceride levels after 2 years agent used in many countries outside the
of therapy (44). An extensive review of the hu- United States until the introduction of Orli-
man clinical trials with sibutramine was re- stat and Sibutramine. Mazindol is a weak in-
cently published (45). hibitor of NE reuptake and a potent inhibitor
Phentermine (3),a close-in analog of am- of both 5-HT and DA reuptake, although other
phetamine with an extra methyl group adja- activities may be partially responsible for the
cent to the primary amino group, is approved anorectic effect. This drug is postulated to
for short-term weight-loss therapy, generally bind at the "cocaine site" on the DA trans-
lasting up to 3 months. Phentermine stimu- porter, yet it exhibits few of the undesirable
lates synaptic NE release, without affecting pharmacological activities associated with co-
dopamine release or reuptake. Phentermine caine administration (46). Mazindol exists in
was often combined off-label with either fen- two tautomeric forms, with the open-ring ben-
fluramine (13)or dexfenfluramine (14), stim- zophenone tautomer (39) preferred under
ulators of 5-HT release and inhibitors of 5-HT acidic conditions and the cyclic form preferred
reuptake, to give extremely effective weight- under neutral conditions.
loss combinations called "fenlphen" or "d-fen/
phen." These combinations, which never re-
ceived U.S. Food and Drug Administration
(FDA) approval, exhibited greater efficacy
than any of the agents alone, probably because
they targeted two important pathways in-
volved in the regulation of food intake. Both
"fenlphen" and "d-fen-phen" were associated
with cardiac side effects (valvulopathy), par-
tially attributed to the fenfluramine or dexfen-
fluramine part of the combination. Fenflura-
mine and dexfenfluramine have been removed
from the market, but phentermine is still used
as monotherapy for weight loss, although gen-
erally as an adjunct to diet and exercise. The
anorectic effects of phentermine generally de-
crease with time.
Benzphetamine (6), diethylpropion (4), The biological activities of the different ap-
and phendimetrazine (7) are amphetamine- petite suppressants are summarized in Table
like drugs that have been available for several 15.9. Because these drugs can increase synap-
decades, but that are now considered second- tic levels of multiple neurotransmitters, they
tier agents for weight loss. These drugs pro- often exhibit complex polypharmacology by
duce modest reductions in body weight and interacting with entire families of biogenic
are indicated for short-term use only. Benz- m i n e receptors that often have multiple func-
phetamine contains a bulky N-benzyl group tions.
2 Clinical Applications 857
2.1.1.3 Lipolytic and Thermogenic Agents. 2.1.2 Drugs to Treat Eating Disorders. Clas-
Caffeine (8),a methylxanthine derivative oc- sical antidepressants drugs, such as selective
curring naturally in coffee and tea, is a central 5-HT reuptake inhibitors, tricyclic mines,
stimulant occasionally prescribed for short- and monoamine oxidase inhibitors, are used to
term weight reduction. Caffeine has been treat selected eating disorders, manage the
shown to increase energy expenditure and underlying psychiatric symptoms associated
stimulate lipolysis. The lipolytic activity of caf- with most atypical feeding behaviors (depres-
feine is mediated through antagonism of aden- sion, anxiety), and to prevent relapse of all
osine receptors (mainly A,-R) in adipose tis- eating diseases after treatment (see Table
sue. Some of the pharmacological activities of 15.6). These agents are generally used in con-
caffeine may also be attributed to inhibition of junction with cognitive behavioral therapy
phosphodiesterases, which increase intracel- and nutritional counseling. Selective seroto-
lular concentrations of a cell-signaling mole- nin reuptake inhibitors (SSRIs) prevent re-
cule called cyclic adenosine 3',5'-monophos- uptake of synaptic 5-HT by transporters lo-
phate (CAMP). Caffeine is often combined cated on neuronal membranes. The SSRI
with D-(-)-ephedrine (91, a naturally occur- fluoxetine (16) is approved for the treatment
ring direct-acting sympathomimetic agent of bulimia nervosa (50) and is used off-label in
found in the plant Ephedra equisedina, which the treatment of binge eating disorders (51).
is available as an over-the-counter drug. Other marketed SSRIs such as sertraline (171,
Ephredrine is a weak p,-adrenergic receptor paroxetine (181,and fluvoxamine (19) are con-
agonist that increases intracellular CAMP lev- sidered therapeutically equivalent to fluox-
els. The caffeinelephredine combination has etine and sometimes used interchangeably
been shown to significantly increase energy with fluoxetine to treat bulimia nervosa, with
expenditure (thermogenesis) and fat metabo- the choice depending on the adverse effect pro-
lism in both obese women and adolescents file of the particular SSRI. Sertraline has been
(47-49). This combination is more efficacious shown to decrease frequency of binge eating
than either agent alone, possibly because it disorder behavior (52); fluvoxamine has been
has synergistic effects on the stimulation of found to prevent relapse of bulimia nervosa
CAMP. after psychotherapy (53). SSRIs are generally
Drugs to Treat Eating and Body Weight Disorders
not effective in treating anorexia nervosa, pri- crease risk of fractures, and prevent adult
marily because malnutrition and low body onset of osteoporosis, a potential concern for
weight cause drastic reductions in synaptic young, anorectic women with prolonged epi-
levels of 5-HT (21,541. SSRIs are efficacious in sodes of amenorrhea and decreased endoge-
preventing relapse of anorexia nervosa after nous estrogen production.
weight regain and normalization of 5-HT lev- The pharmacological mechanisms of the
els (55). prototypical drugs used to treat eating disoT-
Tricyclic m i n e antidepressants (TCAs) ders are summarized in Table 15.9.
are nonselective 5-HT and NE reuptake inhib-
itors. Two TCAs, imipramine (20) and desi- 2.1.3 Drugs to Treat Wasting Diseases.
pramine (211,are used to treat bulimia ner- Several centrally and peripherally acting ap-
vosa, primarily as second-line therapies. petite stimulants and anabolic agents are used
Imipramine, a tertiary amine, is a 5-HT re- in the treatment of wasting diseases (see Ta-
uptake inhibitor with weak NE reuptake ac- ble 15.7). A progesterone derivative, meges-
tivity. Desipramine, a secondary m i n e and trol (301, has been shown to enhance caloric
the des-ethyl analog of imipramine, is a NE intake and weight gain in anorectic and ca-
reuptake inhibitor. Other TCAs used less fre- chectic AIDS patients. The exact mechanism
quently to treat BN include amitriptyline (22) by which this agent stimulates appetite is not
and its N-desmethyl analog nortripyline (23). yet known. An anabolic steroid and testoster-
Monoamine oxidase inhibitors are a class of one analog called oxandrolone (31,a class I11
antidepressant drugs that inactivate enzymes controlled substance) has been found to stim-
responsible for degrading biogenic amines, ulate weight gain, improve physical strength,
thus elevating extracellular or synaptic con- and increase lean body mass in anorectic AIDS
centrations of neurotransmitters such as NE patients (59). Dronabinol [32, A9-tetrahydro-
and 5-HT. Two forms of the enzyme are cannabinol (A9-THC)], a synthetic derivative
known: monoamine oxidase (MAO) A, which of one of the active components in Cannabis
is found within nerve terminals and is highly satiua L (marijuana), stimulates appetite
selective for NE and 5-HT; and monoamine through activation of central cannabinoid re-
oxidase B, which is found extracellularly, pri- ceptors, most likely the cannabinoid receptor
marily in platelets, and is highly specific for 1 (CB,-R) subtype, which is found almost ex-
nonphenolic 2-phenethylarnine derivatives clusively in the brain. Dronabinol has been ap-
-
(56). Phenelzine (24) is a non-selective MA0 proved for use in body wasting disorders in
inhibitor that forms an irreversible adduct AIDS patients. Recombinant human growth
with the enzyme through a phenethyl radical hormone (33, rhGH, somatotropin) is also
intermediate. It is used occasionally to treat used to stimulate protein anabolism and in-
bulimics, generally when all other therapies crease body weight gain (lean body mass), with
fail. one marketed rhGH preparation called seros-
Several drugs are used to restore a normal tim, approved for the treatment of AIDS wast-
body weight in anorectic patients and to pre- ing (60). Somatotropin mediates many of its
vent onset of comorbidities (26, 57). Metoclo- pharmacological effects through a hormone
pramide (251, a prokinetic dopamine receptor synthesized by the liver, insulin-like growth
antagonist and a muscarinic agonist, de- factor 1 (IGF-1).
creases bloating during nutritional therapy by
2.2 Adverse Effects and Precautions
increasing gastric emptying, thereby enhanc-
ing nutrient absorption (33, 58). Anxiolytic Side-effect profiles of many antiobesity drugs
agents such as alprazolam (26) are helpful in have led to high attrition rates, lessening the
easing fear of food intake and body weight overall appeal of pharmacotherapy as a treat-
gain and in reestablishing normal eating pat- ment option. In general, anti-obesity drugs
terns. Estrogen replacement therapy through should have fairly mild and non-life-threaten-
use of conjugated estrogens (estrone, 27; equi- ing side effects, because of the potential off-
lin, 28) or ethinyl estradiol (29) is often pre- label use of such compounds for cosmetic pur-
scribed to increase bone mineral density, de- poses in relatively healthy individuals.
2 Clinical Applications
erally considered one of the safest antiobesity Caffeine causes several centrally mediated
drugs, primarily because it shows low sys- side effects including nervousness, irritability,
temic exposure, which generally minimizes and sleeplessness. Caffeine also acts on kid-
the likelihood of general compound-related or- neys to increase diuresis. Convulsions and in-
gan toxicity. creased heart rate can occur with particularly
The adverse effects of amphetamine and high doses of the drug.
related sympathomimetic appetite suppres- The SSRIs used in the treatment of eating
sants are well documented. All of these agents disorders such as bulimia nervosa are gener-
are classified by the U.S. Drug Enforcement ally well tolerated. In clinical trials with fluox-
Administration (DEA) as controlled sub- etine, patients reported increased incidences
stances (classes 11-IV) according to their po- of dry mouth, anxiety, nervousness, insomnia,
tential for causing addiction (see Table 15.4). and sexual dysfunction. Interestingly, two
Class I1 agents such as amphetamine are commonly reported side effects of fluoxetine
highly abused, with prescription restricted to treatment are anorexia and weight loss (63).
special circumstances; class IV anorectic However, fluoxetine is not approved as a ther-
drugs such as sibutramine, phentermine, di- apy for the treatment of obesity, primarily be-
ethylpropion, and mazindol have minimal cause such SSRI-induced weight loss cannot
abuse potential. be maintained. In one controlled trial with flu-
Sibutramine is contraindicated in obese pa- oxetine, severely obese patients (BMIs > 35.6
tients with preexisting cardiovascular dis- kg/m2) reportedly lost significant body weight
eases because it causes a small but significant during the first 20 weeks of the study, but re-
increase in both systolic blood pressure and gained most of the lost weight in the second 20
supine heart rate (62). Sibutramine elevates weeks, despite continuation of drug treatment
synaptic levels of NE, resulting in activation of (64).
sympathomimetic pathways involved in blood The tricyclic amine antidepressants such
pressure regulation. Sibutramine does not ap- as desipramine and imipramine have been rel-
pear to cause valvular heart disease, unlike egated to second-line therapy, mainly because
other agents that potently stimulate 5-HT re- they cause hypotension, tachycardia, blurred
lease like dexfenfluramine. vision, dry mouth, constipation, fatigue, and
The principal side effects of phentermine sedation. attributed in part to their adrener-
A
this agent, resulting in discontinuation of phenelzine causes numerous side effects re-
therapy. The combination of phentermine sulting from its irreversible inhibition of both
with fenfluramine or dexfenfluramine was as- brain and liver monoamine oxidases and its
sociated with increased incidences of both pri- nonselective inhibition of other enzymes. The
mary pulmonary hypertension (PPH) and car- liver activity of phenelzine prevents degrada-
diac valvulopathy, but it is unlikely that tion of sympathomimetic amines in food prod-
phentermine alone causes these same prob- ucts, leading to elevated levels of potentially
lems. Phentermine, nonetheless, contains a toxic norepinephrine-like pressor substances.
warning label listing PPH and cardiac valve Adverse effects of phenelzine include severe
lesions as possible adverse events. hypertensive crises, nausea, and vomiting.
Drugs to Treat Eating and Body Weight Disorders
These side effects can last for weeks, depend- are also illustrated. The pharmacokinetic
ing on the rate of resynthesis of new enzyme. properties and metabolic degradation path-
Drugs used in the treatment of wasting dis- ways for antidepressant drugs that are used in
eases exhibit several side effects, some of the treatment of eating disorders (i.e., SSRIs,
which are mechanism related. Oxandrolone is TCAs, MAOIs) have been summarized in pre-
an anabolic steroid with potent androgenic ac- vious volumes (67).
tivity that disrupts the hypothalamo-pitu-
3.1 Absorption/Metabolism of Antiobesity
itary-gonadal axis. It can adversely affect
Drugs
growth and sexual development in children
and suppress gonadotrophic function in
3.1.1 Orlistat. Orlistat acts in the GI tract,
adults. It also causes masculinization (i.e.,
where it inhibits pancreatic lipases located in
hoarseness, hair growth) in women; acne and
the lumen. Systemic exposure of the drug is
decreased spermatogenesis in men; and
not required for pharmacological activity.
edema, jaundice, and cholestatic hepatitis in
With negligible oral bioavailability (F < 5%),
both sexes (65). Dronabinol(32) causes a wide
orlistat is primarily excreted unchanged in the
variety of psychotropic effects including dys-
feces. However, several metabolites including
phoria, anxiety, and hallucination that are at-
(40) and (41) have been identified in the
tributed to activation of cannabinoid recep-
plasma of both normal and obese volunteers.
tors within the CNS. This drug can also cause
These compounds are formed by hydrolysis of
tachycardia and orthostatic hypotension (66).
both the N-formyl leucine ester moiety and
Megestrol is associated with respiratory ail-
the lactone ring and do not exhibit any inhib-
ments, elevated liver enzymes, and hypergly-
itory activities toward pancreatic lipases (68)
cemia (66).
(see Fig. 15.1).
methyl compound BTS 54 354 (42) and the In humans, after an oral dose, phentermine is
di-desmethyl analog BTS 54 505 (43; see Fig. converted into the N-hydroxy and N-nitroso
15.2) (70). The R- and the S-enantiomers of derivatives (52) and (53), whereas in rats,
both sibutramine and BTS 54 354 are demeth- phentermine is oxidized to 4-hydroxyphenter-
ylated at different rates, to give a complex mine (54), which is then conjugated and ex-
plasma mixture of both primary and second- creted (73, 74).
ary Bphenethylamine metabolites (44-47).
The half-lives of the R-enantiomers of the two 3.1.4 Benzphetamine. The short-acting ano-
desmethyl and di-desmethyl metabolites (44) rectic benzphetamine is first metabolized in
and (46) are considerably longer than the half- humans to norbenzphetamine (55) through
life of the parent drug. The two enantiomers of N-demethylation and then to 1-(4-hydroxy-
BTS 54 505 (46), (47) are further deactivated pheny1)-2-(N-benzy1amino)propane (56)
through oxidation and conjugation, to give through phenyl ring oxidation (75). Metham-
compounds that are cleared through the phetamine (57) and amphetamine (38) are
kidney.
The pharmacological activities of sibutra-
mine metabolites have been evaluated. The
R-enantiomers of BTS 54 354 and BTS 54
505 (44 and 46) show potent NE, 5-HT, and
DA reuptake inhibitory activities, with IC,,
values less than 13 and 140 nit4 for NE re-
uptake inhibition and 5-HT reuptake inhibi-
(55) Norbenzphetamine
tion, respectively (see Table 15.10) (71). The
S-enantiomer of BTS 54 354 (45) exhibits
weak inhibitory activity for inhibition of up-
take of NE, 5-HT, and DA, whereas the S-
isomer of BTS 54 505 (47) displays good ac-
tivity for inhibition of NE and DA reuptake.
Sibutramine metabolites may be primarily
responsible for the potent anorectic activity
of the parent drug.
"Ref. 71.
also formed, but only as minor metabolites. The secondary amino group in (55) is then
Interestingly, only the D-enantiomers of (57) oxidized further to give the N-hydroxyl deriv-
and (38) are detected in urine (76). In hepatic ative (68),which is converted by a flavoprotein
microsomes obtained from rats pretreated mixed-fundion amine oxidase into the nitrone
with phenobarbital, benzphetamine initially derivative (59). Degradation of the nitrone
undergoes demethylation to give norbenz- metabolite, followed by oxidation yields 2-ni-
phetamine (55), as shown in Fig. 15.5 (77). troso-1-phenylpropane (60).
3 Drug Metabolism
/
- WH
Major
Pathway /
___)
/
0
NH2 NH2
Amphetamine (38) (48) (49)
1 Minor pathway
Conjugates +
NH2
w3
primary metabolites, phenmetrazine (61) and
phendimetrazine N-oxide (621, are formed by
N-dealkylation and N-oxidation and are
cleared through the kidney (79). / I
moderately potent substrate for the NE trans- conjugate called hippuric acid (65) (81). A pro-
porter (IC,, = 99 nM) and an inhibitor of NE posed pathway for the conversion of (64) to
uptake (IC,, = 360 nM), in contrast to dieth- (65) is shown in Fig. 15.6. In one study,
ylpropion, which shows only weak activity in norephedrine analogs (66) and (67) were
these assays (see Table 15.11) (80). Ethylpro- found in only neglible quantities in urine, sug-
pion is further metabolized by P450 enzymes gesting reduction of the keto group is not a
to the di-des-ethyl derivative (64) and benzoic major route of elimination for compound (64)
acid (50),which exists in plasma as a glycine (see Table 15.11) (82).These norephedrine an-
O \ 0
Ethylpropion
Diethylpropion
1
glycine glycine N-acyltransferase
N-
Ethylpropion
(63)
Drugs to Treat Eating and Body Weight Disorders
3.1.7 Mazindol. Mazindol exhibits a rapid 20%), attributed in part to high first-pass
onset of action and a long duration of action in metabolism (86). In humans, dronabinol un-
humans, primarily because of its slow absorp- dergoes allylic oxidation of the terpene ring,
tion and elimination (83, 84). The majority of to give 11-hydroxy-As-tetrahydrocannabinol
drug is excreted unchanged in urine, although (70). This metabolite is oxidized by alcohol
two metabolites, (68) and (69), have been dehydrogenase enzymes in the liver to yield
identified that are formed by oxidation of the ll-nor-A9-tetrahydrocannabinol-9-carbox-
ylic acid (711,which occurs as a conjugate in
plasma (see Fig. 15.7) (87). The C-3 pentyl
chain in (71) undergoes additional oxida-
tion, to give 4'-hydroxy-ll-nor-A9-tetrahy-
drocannabinol-9-carboxylic acid (72), which
is degraded further to the dicarboxylic acid
derivative (73). Several other metabolites
(74, 75) are formed in small amounts
through allylic oxidation of the C8 position
on the dronabinol A ring, followed by oxida-
tive carboxylation of the C-3 side chain (88).
An interesting metabolite of As-THC is
formed in mice through aromatization of the
terpene ring, to give cannabinol (76) (see
Fig. 15.8) (89). The biological activities of
many of these metabolites have not been
fully described in the literature.
Megestrol (30) is metabolized into three
products in humans, as shown in Fig. 15.9,
although the majority of drug is excreted un-
changed. Megestrol undergoes allylic oxida-
tion, hydroxylation adjacent to the carbonyl
group on the A-ring, and conjugation, to give
metabolites (771, (78), and (79) (90).
1.
i
ii 4 Physiology and Pharmacology
(79) (78)
energy homeostasis. Elucidation of the under- the excess calories are stored as adipose tissue,
lying mechanisms involved in obesity and leading to obesity. Several recent reviews de-
related disorders has come primarily from re- scribe the regulation of body weight in exten-
ceptor and hormone knockout mice and trans- sive detail (94-96).
genic animal models (92, 93). Body weight is
controlled through an integrated process that 4.1 .I Neuroendocrine Regulation of Energy
balances energy intake with energy expendi- Homeostasis by the Hypothalamus. The hypo-
ture. This system encompasses many overlap- thalamus, a collection of neuronal bodies lo-
ping interactions between adipose tissue, cated near the thalamus at the base of the
muscle, the adrenal glands, the GI tract, and brain, coordinates signals from the autonomic
the CNS (hypothalamus, nucleus tractus soli- nervous system and regulates many bodily
taris) (see Fig. 15.11). It also includes numer- functions, including metabolism and temper-
ous satiety, anorectic, and orexigenic factors, ature. The role of the hypothalamus in the
several neurotransmitters (5-HT, NE, DA), control of energy balance was discovered
and various circulating nutrients (glucose, lip- through neuronal lesion studies: ablation of
ids) (see Table 15.12). As a central integrator neurons located in the lateral hypothalamic
of these signals, the hypothalamus responds to area (LHA) was shown to cause leanness,
the body's varied energy needs by shifting be- whereas destruction of neurons in the ventro-
tween anabolic and catabolic pathways, that medial nucleus (VMN) was found to result in
is, stimulating food intake and increasing en- obesity (97). Several hypothalamic regions
ergy reserves or inhibiting food intake and de- have now been shown to be involved in the
creasing body weight. The intricate mecha- regulation of energy homeostasis, including
nisms that control these processes can the LHA, VMN, and arcuate nucleus (ARC),
sometimes be disrupted. When energy input located adjacent to the third ventricle. The
exceeds energy output for long periods of time, ARC contains a discrete set of neurons that
870 Drugs to Treat Eating and Body Weight Disorders
Table 15.12 Hormones and Neurotransmitters that Alter Food Intake or Energy Expenditure
Substances that Affect Energy
Anorectic Substances Orexigenic Substances Expenditure
Cocaine- and Agouti gene-related Glucocorticoids
amphetamine-related peptide
transcript product
-?
Cholecystokinin Anandamide Histamine
Corticotropin-releasing y-Butyric acid Leptin
hormone
Enterostatin Galanin a-Melanocytc+stimulating hormone
Gastrin release peptide Ghrelin Neuromedin U
Glucagon-like peptide 1 Growth hormone- Norepinephrine
releasing hormone
Glucagon-like peptide 2 Glucocorticoids Serotonin
Insulin Melanin-concentrating Thyroid hormones
hormone
Leptin Neuropeptide Y
a-Melanocyte stimulating Norepinephrine
hormone
Neuromedin B Opioids
Neuromedin U Orexin
Neurotensin Pancreatic polypeptide
Serotonin
coexpress two orexigenic hormones, called ag- immune, and stress responses. Both the PVN
outi gene-related peptide (AGRP) and neu- and the VMH contain mRNA for a CRH recep-
ropeptide Y (NPY), and another grouping of tor subtype, termed CRH,-R, that is involved
neurons that coexpress anorectic hormones in decreasing food intake and increasing en-
such as a-melanocyte-stimulating hormone ergy expenditure in rats (101). Neurons in the
(a-MSH) and peptidic hormone derived from LHA secrete an orexigenic hormone called
cocaine and amphetamine-related transcript melanin-concentrating hormone (MCH) that
(CART) (see Fig. 15.12). a-MSH is a proopio- acts at specific receptors in the PVN. A newly
melanocortin (P0MC)-derived peptide in the discovered 33 amino acid neuropeptide, called
melanocortin (MC) family. Neurons in the orexin A, is synthesized in the PFA and LHA
ARC project to other sites within the hypo- and may mediate its orexigenic activities
thalamus, including the dorsomedial nuclei through receptor subtypes found in the PVN
(DMN), paraventricular nucleus (PVN), peri- and the VMN. More complete descriptions of
fornical area (PFA), and LHA, and to other the anatomical distribution of hypothalamic
regions of the brain. The hypothalamic path- neurons that secrete neuropeptides and their
ways involved in food intake and energy ho- receptors have been described elsewhere (97,
meostasis have been reviewed (98-100) 100).
Several hypothalamic regions outside the A functional melanocortinergic system in
ARC also synthesize neuropeptides involved the hypothalamus is essential for maintaining
in the regulation of energy homeostasis or normal body weight. This system consists of
contain receptors that respond to ARC hor- two opposing hormones, AGRP and a-MSH,
mones (see Figs. 15.13 and 15.14). The PVN and two MC receptor subtypes, MC,-R and
contains multiple subtypes of receptors for MC4-R (see Fig. 15.12). The neuropeptide
NPY and POMC-derived peptides such as a-MSH, an endogenous agonist for both
a-MSH. Neurons in the PVN synthesize and MC3-R and MC,-R, decreases food intake and
release corticotrophin-releasing hormone increases energy expenditure in rodents.
(CRH), an important central hormone that is AGRP, an inverse agonist of MC3-R and
involved in the regulation of many endocrine, MC4-R, stimulates food intake by antagoniz-
4 Physiology and Pharmacology 871
t t
ing t h e actions o f a-MSH (102). AGRP trans- knockout mice can be reversed by peripheral
genic animals, POMC gene KO, MC3-R KO, administration o f a l o w dose o f a-MSH. T h e
a n d MC4-R KO mice are all obese. Interest- obese phenotypes in MC3-R and MC4-R KO
ingly, t h e obesity observed in t h e POMC mice are caused by distinct mechanisms. T h e
ARC PVN
Key:
-
I POMC Proopioimelanocortin
AGRP - Agouti-related protein
NPY - Neuropeptide Y
\
CART - Cocaine- and amphetamine-related transcript
MC - Melanocortin
MSH - Melanocyte stimulating hormone
OX1 - Orexin 1
MCH - Melanin concentrating hormone
Figure 15.14. LHA neuronal projec- o b --~Leptin receptor
tions.
morbid obesity in MC,-R knockout mice is at- 4.1.2 PeripheralICentral Adiposity Sig-
tributed to hyperphagia, whereas the obesity nals. Several peripheral signals including the
associated with MC3-R knockout mice is at- pancreatic hormone insulin and the adipocyte-
tributed to metabolic deficiencies. MC3-R derived hormone leptin are transported across
knockout mice have decreased lean body mass the blood-brain barrier into the arcuate nu-
in conjunction with increased adipose mass; cleus and play important roles in the regula-
however, they are not hyperphagic (103). tion of energy balance. Leptin provides a crit-
AGRP transgenic mice are similar to MC,-R ical connection between energy reserves
KO mice. (adipose tissue) and the brain (108). Leptin
The functional importance of other hypo- mediates its effects through specific receptors
thalamic neuropeptide systems in the physio- (Ob-R) on ARC neurons (109) that either in-
logical regulation of body weight in humans is hibit synthesis/secretion of orexigenic neuro-
less clear. Many neuropeptides including hormones in the ARC, including NPY and
MCH. NPY. and orexin A stimulate food in- AGRP. or stimulate secretion of anorectic hor-
take upon exogenous administration. MCH mones such as a-MSH, neurotensin, and the
knockout mice are lean and mice overexpress- peptide product from CART (see Figs. 15.12
ing MCH in the hypothalamus are obese (104, and 15.13). It also stimulates MCH and orexin
105). NPY gene knockout mice show normal neurons in the LHA (see Fig. 15.14). Leptin
feeding behavior, whereas mice lacking NPY levels fall during periods of starvation and in-
receptors (NPY,-R, NPY,-R) develop late-on- crease during periods of excess energy, in pro-
set obesity. NPY appears to be critical for portion to adiposity. Any disruption in the
stimulating food intake during periods of en- centrally mediated signaling of either leptin or
ergy deprivation (starvation), but may not be insulin can cause obesity. Leptin-deficient (obl
necessary for normal body weight regulation. ob) mice are hyperphagic, obese, and diabetic
The primary phenotype of orexin gene knock- (108). The discovery of leptin underscored the
out mice is narcolepsy, not changes in body important role that molecular factors play in
weight. However, icv administration of anti- regulation of body weight homeostasis and set
bodies to orexin A or antagonists to the OX,-R off the search for other candidate genes.
decrease food intake (106, 107). Studies with The pancreatic hormone insulin is primar-
selective peptide or nonpeptide pharmacologi- ily involved in the regulation of glucose ho-
cal probes are necessary to fully decipher the meostasis. However, insulin inhibits food in-
complex roles of these neuropeptides and their take when administered directly into the
receptors. brain (110). Furthermore, insulin levels rise
4 Physiology and Pharmacology
with increasing adiposity, contributing to the satiety signals, generally released into sys-
pathophysiology of obesity-related comorbidi- temic circulation after food intake. CCK is also
-
ties. Neuron-specific deletion of either the in- synthesized in the brain where it is colocalized '
sulin receptor or insulin receptor substrate-2 with dopamine in selected neurons. The pe- -
in mice produces an obese phenotype (111, ripheral pharmacological activities of the CCK
112). peptides, which include stimulation of gastric
The recently identified gut/brain hormone emptying and release of digestive enzymes,
ghrelin may be another important integrator are mediated by a subtype of the CCK family of
of peripheral and central signals (113). Ghre- receptors called CCq-R, found mainly in
lin, an endogenous ligand for the growth hor- nerve terminals in the periphery. The anorec-
mone secretagogue receptor type la (GHS- tic effects of CCK fragments require an intact
Rla), is synthesized in the stomach, gut, and vagal nerve that serves as the main conduit
hypothalamus and is regulated by feeding and between the stomach and the brain, primarily
fasting. Ghrelin promotes hyperphagia, in- the NTS. Otsuka Long-Evans Tokushima
creased adiposity, and obesity in rodents Fatty (OLETF) rats lack CCK,-R and are hy-
(114). The orexigenic actions of ghrelin are perphagic and obese (117). Interestingly,
blocked by NPY antagonists and immuno-neu- CCK,-R knockout mice are not obese. A recent
traliiation of AGRP, supporting a role for ghre- review describes the biological activities of
lin in energy balance in rodent models (115). CCK and analogs (118).
Adipose tissue was once considered to be Several other gut peptides such as en-
primarily a storage site of triglycerides that terostatin or gastrin-releasing-peptide (and
could be released as free fatty acids upon sym- other bombesin-like peptides) are also in-
pathetic stimulation. However, it is now rec- volved in initiating satiation responses after
ognized that adipose tissue is an important food intake. The roles of these peptides in reg-
endocrine organ that secretes many hormones ulating food intake have been reviewed else-
and cytokines that are involved in energy ho- where (34).
meostasis, including leptin and other factors
such as angiotensinogen, resistin, adipsin, 4.1.4 Adrenergic and Serotonergic Regula-
ACRP30, tumor necrosis factor a, IL-6, and tion of Food Intake. Both the hypothalamus
plasminogen activator inhibitor 1. The roles of and NTShrain stem are innervated with NE
these adipogenic hormones in the regulation and 5-HT fibers that are involved in the regu-
of energy balance and carbohydrate metabo- lation of food intake. NE stimulates food in-
lism have been reviewed elsewhere (116). take through activation of a,-adrenergic re-
ceptors (a,-AR) in the PVN and decreases
4.1.3 Nucleus Tractus Solitaris (NTS) and feeding through a,-ARs that are also in the
Peripheral Satiety Signals. The nucleus tractis PVN (119). Many sympathomimetic agents
solitaris (nucleus of the solitary tract, NTS) is that increase synaptic NE release actually de-
a collection of neurons found in the region of crease appetite by indirectly stimulating al-
the brain stem (hindbrain) that integrates adrenoceptor pathways. Serotonin acts on
many signals from the GI tract that are re- postsynaptic 5-HT,, and 5-HT,, receptors in
leased into the circulatory system after food the LH, VMH, and NTShrain stem, to de-
intake. The NTS receives signals through va- crease food intake and alter meal size and
gal nerve afferents and then communicates meal number (120, 121). The anorectic effects
with the hypothalamus to modulate the activ- of 5-HT, however, are primarily mediated by
ities of important neuropeptides involved in 5-HT,, receptors located in the brain stem.
stimulating or inhibiting food intake. The For example, fourth ventricle administration
NTS is also activated by the neurotransmit- of small molecule 5-HT,, receptor agonists is
ters NE and 5-HT, which can stimulate satiety sufficient to mediate all the anorectic effects of
mechanisms and increase energy expenditure. such agents (122). Synaptic levels of 5-HT are
Cholecystokinin (CCK) and related peptide controlled by a variety of signals, including
fragments such as CCK-33 and CCK-8 are a such peripheral satiety factors as CCK. Acti-
class of gut peptides that act as short-term vation of hypothalamic 5-HT receptors results
Drugs to Treat Eating and Body Weight Disorders
blocked by administration of centrally active membrane transporters. Such compounds all con-
-
serotonin receptor antagonists (metergoline), tain a 2-phenethylamine moiety, in which a basic
a,-receptor antagonists (prazosin), and &-re- amino group is separated from a lipophilic aryl*
ceptor antagonists (metoprolol) (138). ring by an alk.yl spacer consisting of two carbon
Sibutramine failed to show antidepressant atoms. The unsubstituted derivative,2-phenethyl-
activity in humans, but was found to be a potent amine, shows weak sympathomimeticand serobP
anorectic agent. A structurally distinct com- nergic activity and exhibits no CNS exposure
pound, venlafaxine (811,with similar in vitro because of its rapid metabolism by MAOs. Intro-
duction of one or two -1 substituents at the C1.
position adjacent to the amino group decreases
metabolism by MAOs and enhances CNS expo-
sure (140).Both amphetamine (38)and phenter-
mine (3),which differ from 2-phenethylamine by
the addition of one or two methyl groups, exhibit
good CNS exposure.
The structure/activity relationships of
2-phenethylamines on serotonin uptake in-
hibitory activity have been investigated using
(81) Venlafaxine platelet-rich plasma from human volunteers
(141). The uptake activity of amphetamine is
pharmacology at both the NE and 5-HT re- enhanced by alkyl substituents on the amino
uptake transporters, proved to be an efficacious group and by the incorporation of methyl sub-
antidepressant drug, without displaying sigdi- stituents adjacent to the nitrogen atom (see
cant anorectic activity in humans. Venlafaxine Table 15.13). Tertiary m i n e s such as sibutra-
has been shown to inhibit food intake in rodent mine exhibit weak 5-HT uptake activity. In-
models, but only at very high doses. The reasons troduction of lipophilic electronegative sub-
for the disparities in the pharmacological activ- stituents (i.e., C1, CF,) on the aryl ring results
ities between the two structurally different com- in a significant increase in activity, whereas
pounds are not yet fully known. introduction of polar groups (i.e., OH) leads to
a decrease in activity. The dextrorotatory iso-
mers are more active than the corresponding
6 STRUCTURE-ACTIVITY RELATIONSHIPS levorotatory isomers.
Hansch and Caldwell have analyzed the
Structure-activity relationships (SARs) for quantitative structure/activity relationships
several drugs used in the treatment of body (QSAR) of a series of amphetamine and
weight disorders, including some 2-phenethyl- 2-phenethylamine analogs, to discern the role
m i n e derivatives and dronabinol, have been of steric and hydrophobic aryl substituents on
described in the literature. For other drugs the inhibition of 5-HT uptake (142). From the
such as sibutramine and diethypropion, the biological data of 19 compounds, including
only close-in compounds for which biological those in Table 15.13. and some additional an-
data exist are metabolites of the parent drug alogs, the following equation was derived for
(see Section 3). Analogs of sibutramine are de- inhibition of uptake activity, where C is the
scribed in the patent literature, but no data IC,, concentration, MR, is the molar refrac-
are provided (139). The SARs of antidepres- tivity value of the aryl substituent scaled by
sant medications have been described else- 0.1, and n3 is the hydrophobicity of the meta
where in this series. substituent on the aryl ring:
6.1 SAR of 2-Phenethylamines
Appetite suppressants such as amphetamine, fen-
fluramhe, and sibutramine stimulate 5-HT, NE,
and/or DA release or inhibit their reuptake by
i
876 Drugs to Treat Eating and Body Weight Disorders
(-)-Amphetamine (82)
Phentermine (3)
(2)-4-Chloromethylarnphetamine(85)
Chlorphentermine (86)
An indicator variable I was added to the was low (s = 0.219). The data suggest that
above equation to differentiate substituted lipophilic substituents at the meta position
2-phenethylamine derivatives such as am- on the aryl ring in phenethylamine analogs
phetamine from unsubstituted 2-phenethyl- fit into an important hydrophobic pocket in
amine compounds. The correlation coeffi- the 5-HT transporter and that the introduc-
cient ( r ) for the equation was high (r = tion of such groups should enhance the in-
0.965), whereas s, the standard deviation, hibitory activity.
6 Structure-Activity Relationships
"Ref. 141.
The SAR for norepinephrine uptake inhibi- poor CNS exposure. Substitution of the termi-
tion by amphetamine analogs is similar to that nal amino group with one alkyl group de-
for inhibition of 5-HT reuptake. The protypi- creases NE activity and substitution with two
cal unsubstituted derivative, 2-phenethyl- alkyl groups essentially eliminates activity
amine, is a weak uptake inhibitor in isolated (see Table 15.11). In general, as the size of the
rat heart membranes (ID,, = 1.1 CLM) (143). alkyl group on the basic nitrogen group in-
Introduction of a methyl group at the C1 posi- creases, the indirect syrnpathomimetic activ-
tion adjacent to the amino group results in a ity decreases. Serotonin release, in contrast, is
10-fold increase in potency (i.e., dexamphet- generally enhanced by electron-withdrawing
amine, (a), ID,, = 0.18 p M ) (143). Sibutra- substituents on the phenyl ring (see Table
mine, a tertiary amine, shows moderate NE 15.9). Thus, fenfluramine shows no NE releas-
uptake activity (Ki = 350 nM), but its des- ing activity but is a potent 5-HT releasing
methyl and di-desmethyl metabolites, (R)- agent. Likewise, amphetamine and phenter-
BTS 54 354 (44) and (R)-BTS 54 505 (461, mine do not stimulate 5-HT release but show
exhibit potent activity (Kivalues <20 nM)(see weak to moderate activity for stimulating NE
Table 15.10) (71). release. Sibutramine has little effect on either
The SAR for NE and 5-HT releasing activ- NE or 5-HT release.
ity of amphetamine derivatives is less well de-
6.2 SAR of Dronabinol Analogs
fined (144). NE release is increased by the
presence of hydroxyl groups on the aryl ring The structure-activity relationships of dron-
and decreased by substitution with halogen or abinol (32) on cannabinoid receptor activity
CF, groups. Phenolic and catechol derivatives have been investigated (see Table 15.14). Both
of 2-phenethylamine, however, generally un- the phenolic hydroxyl group and the C3 alkyl
dergo rapid glucuronidation and thus show chain in (32) are critical for binding affinity.
878 Drugs to Treat Eating and Body Weight Disorders
Transposition of the olefin from the A9-posi- not lead to a significant drop in potency. Sev-
tion to the As-position as in compound (91) eral bicyclic compounds such as (94) and (95)
(ARTHC)results in a threefold loss of potency have been prepared that exhibit moderate ac-
(145).Compound (92) with a C3 dimethylhep- tivity.
tyl (DMH) chain is 100-fold more potent than Dronabinol(32)and analog (92) exhibit lit-
the analogous compound with a C3 pentyl tle selectivity for CB,-R vs. CB,-R. These two
chain. The binding affinities of compounds receptors show 44% sequence homology, with
with either endocyclic or exocylic olefin groups modest homology in the transmembrane do-
in the cyclohexane ring of the tricyclic core mains (68%).Replacement of the phenolic hy-
template in the DMH series are similar. Aro- droxyl group in the aromatic ring of (92) by a
matization of the terpene ring in As-THC does phenyl methyl ether moiety as in L-759633
6 Structure-Activity Relationships 879
(96) results in a 10,000-fold decrease in CBl-R regions were found in the CBl-R, where steric,
activity, but only a 40-fold loss of CB2-Raffin- electronic, and lipophilic interactions between
ity, providing CB,-R-preferring compounds ligands and receptor appeared to be critical for
(146). CB2-R-selectivecompounds may show activity. Additional interaction regions were
anti-inflammatory and immunosuppressive identified that conferred selectivity for one of
properties. the receptors. All compounds used in the
Fichera et al. used a series of 20 different study, including the endogenous cannabinoids
cannabinoid compounds from several differ- such as anandamide, were aligned against
ent chemical series to develop 3D-QSAR mod- dronabinol.
els for activities at CB,- and CB,-receptors Several natural and synthetic dronabinol
(147). The QSAR study was performed by use analogs have been clinically evaluated as safer
of GOLPE methodology (148). Seven spatial alternatives to AS-THC, although primarily as
880 Drugs to Treat Eating and Body Weight Disorders
"Ref. 145.
*Ref. 146.
(100) Cannabidiol, R =
A
6.3 Mazindol Analogs
Mazindol is a potent inhibitor of both dopa-
(99) Nabilone, CB1-R Ki = 2.19 nM mine and serotonin uptake that was first de-
CB2-RKi = 1.84 nM scribed in 1975 (149). The compound exists in
two tautomeric forms, although it is believed
that most of the pharmacological activity lies
CB,-R and CB,-R agonist, contains a carbonyl in the closed, tricyclic form where the hy-
group at the C9 position and a C3 dimethyl- droxyl group can act as both a hydrogen-bond
heptyl group. Cannabidiol (loo), a bicyclic de- acceptor and donor. The open form has been
rivative of (32)in which the central pyran ring shown to exhibit much weaker anorectic activ-
has been opened up, is a natural constituent of ity in rat models compared to that of the closed
Cannabis sativa L that exhibits no psycho- form tautomer (150).In one report on the SAR
tropic properties, although it shows neuropro- of Mazindol analogs, replacement of the
tective activities. C4'-C1 substituent on the B-ring with a C4'-F
7 Future Directions 881
4'
IC50, nM
[3H]Dopamine
Compound W X Y Uptake"
(109) Lipstatin, IC50 = 0.14 pg/mL (1) Orlistat, IC50= 0.4 pg/mL
",
; vation that occurs after weight loss.
New weight loss drugs must meet certain
n-C9H19 guidelines established by the FDA before re-
ceiving marketing approval (153). These
(110) Compound, IC50 = 1.7 pg/mL guidelines require that weight-loss agents
Table 15.16 Antiobesity Drugs in Late-Stage Clinical Development
Mechanism of Development
Name Structure Action Stage
Axokine (111) 1-185-ciliary neurotropic factor (CNTF) Activation of STAT Phase I11
signaling
Rimonabant, CB,-R antagonism Phase I11
SR-141716A
(112)
C1
0, ,
S
No
7 Future Directions 883
C1
JJ+ /
N
inhibition obesity
'Z
H
0
BW1555U88 F
I DA/NE reuptake
demonstrate efficacy for at least 1 year, de- given that withdrawal of treatment does not
fined as greater than 5% weight loss vs. pla- result in any rebound weight gain as is ob-
cebo, generally in double-blind clinical trials served with most other weight-loss agents. In
in patients with BMIs greater than 30 kg/m2 a 3-month clinical trial, obese patients who
or BMIs greater than 27 kg/m2 with at least were administered 2 pg kg-' day-' reportedly
one associated comorbidity. The clinical trials lost 10 extra pounds compared to the placebo
should preferably last at least 2 years, al- group (156). This weight loss was equivalent
though the second year need not be blinded. to that observed for sibutramine and orlistat
The guidelines also require that such drugs over the same time period. In earlier trials for
reduce comorbidities, especially in overweight another indication, amyotrophic atherosclero-
patients with BMIs less than 30 kg/m2. Any sis, lean patients reportedly lost 14% of their
drug used for the chronic treatment of a dis-
body weight. However, the dose in this trial
ease must also exhibit an exceptional safety
was much greater than that used in the obe-
profile, with acceptable benefit-to-risk pro-
sity trial (15or 30 pg/kg three times per week
files.
vs. a maximum dose of 2 pg kg-' day-') (157).
7.1 Antiobesity Compounds and New Drugs The precise mechanism of action of CNTF
to Treat Binge Eating in Clinical Development in the promotion of weight loss has not been
defined, but its effects appear to be mediated
7.1.1 Axokine. Axokine (111) is a recombi- by various hypothalamic pathways. The
nant form of ciliary neurotrophic factor weight loss from a low dose of CNTF in ro-
(CNTF), a 200 amino acid member of the cy- dents and humans does not appear to be
tokine family, which also includes proteins caused by cachectic cytokines such as interleu-
such as leptin, gp130, interleukins, prolactin, kin-1 because no muscle wasting was observed
and growth hormone (154). CNTF promotes in the CNTF-treated groups. CNTF may me-
weight loss in several different animal models diate some of its effects by coupling to down-
of obesity including oblob mice, diet-induced stream signaling events in the leptin pathway.
obesity, and fatfa Zucker rats (155). Interest- In a diet-induced model of obesity, CNTF, but
ingly, CNTF appears to change the set point not leptin, was shown to stimulate phosphor-
for body weight in both rodents and humans, ylation of the cytoplasmic transcription factor
Table 15.18 Peripheral Antiobesity Drugs
Mechanism of Development Stage
Name Structure Action (status)
P3-AR agonism Ph I1 (abandoned)
P3-AR agonism
CCK-R
agonism
7 Future Directions
called signal transducer and transcription fac- creases body weight is unknown, although its
tor 3 (STAT-3) in the arcuate nucleus and anticonvulsant properties are attributed in .
thereby activate downstream signaling path- part to increasing gamma-aminobutyric acid
.
ways. In lean control animals, however, both (GABA), a neurotransmitter that inhibits ex-
CNTF and leptin stimulated phosphorylation citation of nerve cells in the brain (163). Topi-
of STAT-3. These data support a hypothesis ramate may alter both energy intake and en- r
that diet-induced obesity causes "leptin resis- ergy expenditure, decrease food intake, and
tance" and that CNTF may overcome the re- increase energy utilization (164).
sistance observed in this model of obesity.
been shown that many obese patients actually 7.2 New Targets
have elevated plasma, but low CSF levels of In the past decade, many new genetic targets
leptin, suggesting defects in leptin transport' involved in regulation of energy homeostasis, '
across the blood-brain barrier, possibly be- especially in rodent models, have been identi-
cause of saturation of the transporter (173). fied. A number of these molecular targets are
considered "druggable" or suitable for phar- ,
7.1.6 p,-Adrenergic Receptor Agonists. P3- macological modulation, including the 5-HT,,-R
Adrenergic receptor agonists (P3-AR) are pe- that mediates the anorectic effects of fenflur-
ripherally acting thermogenic agents that are amine, the NPYl- and NPY,-receptor sub- .
currently undergoing clinical evaluation for types that are involved in the stimulation of
the treatment of obesity and diabetes. P3-AR food intake, the melanocortin-4 receptor sub-
agonists increase thermogenesis by activation type, and the orexin-1 receptor subtype. Stim-
of P3-ARson brown adipocytes, which leads to ulation of NPYl-R, NPY,-R, and OXl-R may
increases in intracellular CAMPlevels, stimu- be useful in the treatment of wasting diseases
lation of uncoupling proteins, and increases in (anorexia nervosa, cachexia), whereas inhibi-
fatty acid acid oxidation and nonshivering tion of these receptors may show utility in the
therrnogenesis. These compounds also show a management of body weight disorders such as
pronounced insulin-sensitizing, antidiabetic obesity. Activation of the melanocortin sys-
effect in diabetic rodent models, an effect that tem, in particular the MC,-R subtype, should
is independent of weight loss. A first-genera- decrease food intake and antagonism of the
system should increase food intake.
tion P3-ARagonist, CL-316243 (117),has been
Selective ligands for 5-HT,,, NPY, and
reported to increase glucose disposal, whole
orexin receptors are currently undergoing
body fat oxidation and plasma free fatty acids
preclinical and clinical evaluation for the
after 4 weeks in lean males (174). CL-316243 treatment of body weight disorders, primarily
also has been found to increase whole body obesity. Ro 60-0175 (120) and ORG-12962
lipolysis in obese subjects after 3 months. No (121) are potent 5-HT,, receptor agonists cur-
increase in thermogenesis, however, was ob-
served in the study. Another P,-AR agonist,
LY-377604 (lls),has been shown to increase
energy expenditure and decrease respiratory
quotient. The decrease in respiratory quotient
indicated a shift from carbohydrate oxidation
to fat oxidation. Several P3-AR agonists also
reported to increase energy expenditure in
monkeys (175).
intraperitoneal administration, but does not activity at several receptors that may influ-
affect food intake in NPY,-R knockout mice ence food intake(179-181).L-152804 inhibits
(178).CGP 71683A(123)and L-152804(124) the orexigenic effects of NPY5-R, preferring
are potent NPY Y5-R antagonists, although peptidic agonists, but not the effects of NPY
CGP 71683A is not particularly selective, with itself. An interesting series of pyrrolo[3,2-
dlpyrimidine derivatives such as compound
(125)have been reported as Y,-R antagonists,
with nanomolar affinity for the receptor(182).
A selective OX,-R antagonist, SB-334867-A
Drugs to Treat Eating and Body Weight Disorders
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Index
P
Terms that begin with numbers Acetaminophen (APAP) with for neurodegenerative condi-
are indexed as if the number were codeine, 335 tions, 675
spelled out; e.g., "3D models" is Acetaminophen (APAP) with phosphorus-derived cholin- -
located as if it were spelled hydrocodone, 335 ergics, 90-93
"ThreeD models." Acetaminophen (APAP) with reversible noncovalent cholin-
oxycodone, 335 ergics related to 1,2,3,4-tet-
A-68930 Acetaminophen (APAP) with
for Parkinson's disease, 727, propoxyphene, 335
728 Acetorphan reversible quaternary ammo-
A-77636 opioid peptide metabolism in- nium cholinergics, 84-86
for Parkinson's disease, 727, hibitor, 442 Acetylcoenzyme A, 40
728 Acetyl-L-carnitine Actiq, 334
A-84543 for Alzheimer's disease, 768 Acyclic analgesics, 334
cognition enhancer, 802-803 Acetylcholine, 110. See also An- structure-activity relationship,
A-85380 ticholinergics; Cholinergics; 385-387
cognition enhancer, 802,804 Muscarinic acetylcholine Adatanserin, 572
A-86929 receptors; Nicotinic acetyl- Adderall, 3,170
for Parkinson's disease, 727, choline receptors Addiction
728 cholinergics based on, 43-49 narcotic analgesics, 336
Abercarnil, 558 and constrictor muscle fibers, Adenosine antagonists
binding affinity with GABA,I 114 for Parkinson's disease, 733
Bz, 788 dissociation constant, 151 Adinazolam, 552
Absorption, distribution, metab- role in nerve transmission, 40 Adipex, 844
olism, and excretion substitution of ester group by Adipex-P, 171
(ADME) other groups, 47-49 Adiphenine
adrenergics and adrenergic- variations of acyl group, anticholinergic, 134
blocking agents, 16-21 45-46 influence of cyclic radicals on
anticonvulsants, 273-291 variations of ethylene bridge, anticholinergic activity, 143
opioid analgesics, 339-341 46-47 local anesthetic activity, 154
sedative-hypnotics, 211-233 variations of quaternary am- relative anticholinergic activ-
ABT-089 monium group, 43-45 ity, 156
cognition enhancer, 805 Acetylcholine-atropine antago- Adipose tissue, 873
ABT-418 nism, 152-153 Adipost, 171
cognition enhancer, 799-800 Acetylcholine receptors, 41-43. ADME studies, See Absorption,
ABT-431 See also Muscarinic acetyl- distribution, metabolism,
for Parkinson's disease, 727, choline receptors; Nicotinic and excretion (ADME)
728 acetylcholine receptors Adrenaline, 2
ABT-594 Acetylcholinesterase, 40 applications, 9-10
I cholinergic, 53 catalytic site, 84-85 general adrenergic agonist, 3
! cognition enhancer, 804-805 Acetylcholinesterase inhibitors, history, 26-27
Abyssinian tea, 168 40 metabolism, 16-17
Acebutolol for Alzheimer's disease, synthesis, 23-25
P-antagonist, 5 761-762, 781 Adrenergic agonists, 2, 3
applications, 16 carbamate-derived cholin- ADME, 16-21
Aceclidine ergics, 88-90 applications of a,-agonists,
cholinergic, 65, 78-79 for cognition enhancement, 12-13
Acetaminophen, 331 781 applications of a,-agonists,
first tier of cancer pain treat- miscellaneous cholinergics, 13-14
ment, 332-333 93-97 applications of P-agonists, 14
895
Index
Antianxiety agents (Continued) stereoisomerism and activity, recent and future develop-
physiology and pharmacology, 147-150 ments, 516-518
537-546 structure-activity relation- side effects, 490-496 -
structure-activity relation- ships, 133-151 structure-activity relation:
ships, 548-578 synthetic, 126-133 ships and metabolism,
Anticholinergics, 110-111 therapeutic uses, 153-154 510-516
anticholinergic activity as side tricyclic benzodiazepines, Antihistamines F
effect of drugs, 115 128-129 ADME, 223
antispasmodic activity assay, Anticholinergic syndrome, currently used drugs, 212-214
116-118 115-116 discovery, 236 -
as antispasmodics, 111 Anticholinersterases,40. See pharmacologic effects, 230
antiulcer activity assay, also Acetylcholinesterase side effects and drug interac-
118-119 inhibitors tions, 211
as antiulcer agents, 111-114 a,-Antichymotrypsin (ACT), 767 Antihypertensives
atropine analogs, 126-128 Anticonvulsant receptor models, adrenergics, 2
benzothiazepines, 129 319-320 Anti-inflammatory agents
biocomparative assay, Anticonvulsants, 264-265 for Alzheimer's disease, 767
116-120 ADME, 273-291 Antimalarial agents
classification based on sub- currently used drugs, 265 for Alzheimer's disease, 767
types of muscarinic recep- future developments, 315320 Anti-obesity drugs, 844-849
tors, 115-116 GABA, receptors, 295-296 absorption and metabolism,
developed for specific uses, GABA,receptors, 296 860-866
154-158 glutamate receptors, 296-297 clinical applications, 843-860
diphenylacetyloxy derivatives, history, 297-304 CNS stimulants for, 173, 193
132-133 ion channels, 292 new drugs in development,
dissociation constants, 150 NMDA receptor antagonists, 881-886
dual cholinergic/anticholin- 694,696-697 new targets for drugs,
ergic compounds, 150-151 with opioid analgesics, 333 886-888
himbacine, 133 physiology and pharmacology, structure-activity relation-
indene derivatives, 131-132 291-297 ships, 875-881
interaction at muscarinic re- recently developed agents, Antioxidants
ceptors, 151-153 311315 for Alzheimer's disease, 674,
kinetic basis for mechanism of side effects, adverse effects,
675, 758,767-768
action, 152 and drug interactions,
Antipsychotic agents, 600-602
miscellaneous activity assays, 265-273
120 animal models of efficacy and
structure-activity relation-
mydriatic and cycloplegic ac- ships, 304-311 side effects, 611-617
tivity assay, 119-120 T-type calcium channels, 297 anticholinergic effects,
as mydriatics and cycloplegics, Antidepressants, 484. See also 608- 609
114-115 Monoamine oxidase inhibi- cardiovascular effects, 609
for Parkinson's disease, tors; Serotonin-selective cognitive impairment, 608
731-733 reuptake inhibitors; Tricy- dermatologic effects, 611
polymethylene tetramines, clic antidepressants discovery, 617-619
131 clinical efficacy, 486-490 drug discovery strategies
in premedication during anes- CNS stimulants in terminal based on dopaminergic ap-
thesia, 115 illness, 173 proaches, 640- 651
quinuclidine-based antago- currently used drugs and clas- drug discovery strategies
nists, 129-131 sification, 484-486 based on non-dopaminergic
receptor-subtype-selective, for eating disorders, 843, approaches, 651-655
128-133 849-851 extrapyramidal symptoms,
relative activities, 156 efficacy relative placebo, 489 600,605-607
side effects, 154 history, 508-510 hematologic effects, 610
sila-difenidols, 132 for Parkinson's disease, 725 hepatic effects, 610-611
solanaceous alkaloids, pharmacokinetics, 496-498 neuroleptic malignant syn-
120-126 physiology and pharmacology, drome, 607-608
specificity of antagonism, 152 498-508 for Parkinson's disease, 725
Index
Epilepsy, 264-265. See also An- Ethynyl estradiol Fight or flight response, 526
ticonvulsants dose for eating disorders, 853 FK 506
new causative agent for, 320 for eating disorders, 858 for Alzheimer's disease, 766 .
porcine embryonic GABAergic 5-Ethynylnicotine, 52 Flesinoxan, 568-569
cell transplants, 320 Etizolam, 554 Flomax, 4
Epimuscarine, 57 Etodroxizine, 212,214 Flumazenil, 563-564
Epinephrine, See Adrenaline Etonitazene cognition enhancer, 796 T
/
tors, neuronal, 42 Obesity, 838, 839-840. See also
Night-eating syndrom, 840 inhibition by depressants, 500 Anti-obesity drugs
Nimetazepam, 207 metabolism, 16-17 CNS stimulants for, 173, 193
physiological significance,
1 Nipecotic acid analog anticon-
21-22
comorbidities associated with,
vulsants 840
ADME, 286-291 role in CNS stimulant action, guidelines for treatment, 842
Niravoline 183-184 Obsessive-compulsive disorder,
analgesic, 389 role in CNS stimulant effects, 527
Nitrazepam, 207 181,183-184 Octamethylpyrophosphoramide
ADME, 282 Norepinephrine-selective re- (OMPA), 91,92
anticonvulsant, 266, 267, 307 uptake inhibitors, 486,494, Ocupress, 5
side effects, 277 496 Ohmfentanyl, 383,384
Nitric oxide synthase, 695 combined with dopamine re- Olanzepine
Nitric oxide synthase inhibitors, uptake inhibitors, 885 dose, 601
695-706 serotonin-selected contrasted, for Parkinson's disease, 725
Nitrites 501 receptor binding and side ef-
antispasmodic, 111 structure-activity relationship, fects, 606
6-Nitrodopamine 510,511-512 Oleamide, 254-255
nitric oxide synthase inhibi- OM991
Norepinephrine transporter,
tors, 705 for Alzheimer's disease, 754
498- 499
6-Nitronorepinephrine OM992
nitric oxide synthase inhibi- inhibition by antidepressants,
for Alzheimer's disease, 754
tors, 705 500 Onchidal
5-Nitrouracil Normison, 207 anticholinesterase, 94
PARP inhibitor, 681 Normodyne, 5 Ondansetron, 573
NMDA receptor antagonists, Nornicotine, 49 cognition enhancer, 813,
689-695 Noroxyphenonium, 123 815-816
analgesics, 449 Norpramin, 485 OPC-4392,650
antidepressants, 516 dose for eating disorders, 850 OPC-14597,650
NMDA receptors Nortriptyline Opioid agonists, 333
and anxiety, 546 affinities for monoamine re- delta-receptor selective,
and schizophrenia, 604 ceptors, 495 395-400
Nocistatin, 357 antidepressant, 485,486,487 kappa-receptor selective,
Noctal, 205 dose for eating disorders, 851 387-395
Noctamid, 207 for eating disorders, 858 opioid receptor affinities, 348
Noctec, 209 inhibition of serotonin, norepi- Opioid analgesics, 331-333
NoDoz, 170 nephrine, and doparnine ADME, 339341
Nonsteroidal anti-inflammatory transporters, 500 central nervous system, 341
drugs pharmacokinetic parameters, currently used drugs, 333,
for Alzheimer's disease, 767 497 334-335
first tier of cancer pain treat- side effects profile, 493 cytochrome P,,, substrates
ment, 332-333 Novaltropine, 117 and inhibitors, 339
Index
Opioid analgesics (Continued) Opioid receptors, 331. See also pediatric indications, 274
endogenous opioid peptides, Delta opioid receptors; side effects, 279
341-354 Kappa opioid receptors; Mu Oxotremorine analog cholin-
future developments, 448-450 opioid receptors ergics, 71-78
history, 358360 characterization, 343-350 Oxotremorine-M
miscellaneous nonpeptide opi- cloning, 341-342,350 cholinergic, 75
ates, 407 computational models, 352 Oxycodone, 332,363 9
radioligand binding assays, dimerization, 354,443-444 dose, 334
344-347 mutagenesis studies, 351-352 OxyContin, 334
recent developments, 443-448 nomenclature, 341-342 Oxymetazoline
receptor types, 341354 nonpeptide affinity labels used a,-agonist, 3
side effects, adverse effects, to study, 400-407
applications, 13
and drug interactions, signal transduction mecha-
333339 structure-activity relationship,
nisms, 342-343
signal transduction mecha- 30-31
splice variants, 352-354
nisms, 342-343 structure and molecular biol- Oxymorphone, 363
structure-activity relation- ogy, 350-354 dose, 334
ships of nonpeptide ligands, subtypes, 331,352-354 0xyph'encyclimine
360-407 Opium, 331,358-359 anticholinergic, 136
in vitro efficacy assays, OptiPranolol, 5 relative anticholinergic activ-
347-349 Orap, 601 ity, 157
in vivo evaluation, 349350 Orexin A, 872 Oxyphenonium
Web resources, 450-452 ORG-12962,572,581,886 anticholinergic, 137
Opioid antagonists, 333 Orlaam, 334 mydriatic and cycloplegic ac-
delta-receptor selective, Orlistat tivity, 153
367-370 absorption and metabolism, relative anticholinergic activ-
kappa-receptor selective, 860 ity, 156
365-367 dose, 844
opioid receptor affinities, 348 structure-activity relation- Pagoclone, 558-559
structure-activity relation- ships of analogs, 880-881 Pain, 331-333. See also Narcotic
ships, 364-370 Orphanin FQ/Nociceptin, 342, analgesics; Opioid analge-
Opioid bowel dysfunction, 337 357,358,444-445 sics
Opioid peptide analogs, 407-409 structure-activity relationship, cholinergics for, 41
from amphibian skin, 446-447 Pamelor
409-410 Web resources dealing with, dose for eating disorders, 851
P-casomorphin and endomor- 451 Pamine, 117
phin analogs, 422-425 Orphenadrine Panadiplon, 564565
deltorphin analogs, 432-437 for Parkinson's disease, 731, Panic disorder, 527
dermorphin analogs, 428-432 732 Papaverine, 361
dynorphin analogs, 418-422 Oxandrin, 854 antispasmodic, 111
enkephalin analogs, 410-418 Oxandrolone Paraoxon, 91,93
other peptides with high affin- dose, 854 Parasyrnpathomimetics, See
ity for opioid receptors, metabolism, 867 Cholinergics
437-440 for wasting, 858,860 Parathion, 91,93
peptidase inhibitors, 440-443 Oxazeparn, 530 Paregoric, 337338
peptide affinity label deriva- dose for anxiety, 529 Parkin
tives, 440 Oxazolam, 551-552 and Parkinson's disease, 716
TIPP and related peptides, Oxazolidinedione anticonvul- Parkinsonian dementia, 780
425-428 sants, 271 Parkinsonism, 712,716
with Tyr-D-aa-Phesequence, ADME, 282-284 in Alzheimer's disease, 745
428-437 Oxcarbazepine and MPTP (designer heroin),
Opioid-receptor-like 1 (ORL1) ADME, 275,286 717-721
receptor, 341-342,358,444 anticonvulsant, 265, 268, 269, Parkinson's disease, 674,
nonpeptide ligands for, 298,306 712-713
447-448 clinical efficacy, 273 cholinergics for, 41
Web resources dealing with, development, 302-303 diagnostic agents for, 734-736
451 dose, 270 etiology, 715-721
Index
Arecoline analogs, 1:693-694 Arrayscan HCS System, 2:626, Artificial organs, 5526
as cholinergics, 6:62-71 2:627 Arylalkylamine calcium channel
Arenaviruses Arrhythmias, See Antiarrhyth- blockers, 3:16-20
antiviral agents, 5:430 mic agents; Cardiac ar- Arylesterase, 2:538
Argatroban, 3:29,3:285 rhythmias Aryloxy acetic acid high ceiling
ADME, 3296 Arsumax, 5:935 diuretics, 3:93-94
clinical trials, 3:293 Artabotrys uncinatus, 1:887 Aryloxypropanolamines, 6:5, t
development of, 3314-315 Artane 6:16
formula weight, mechanism of for Parkinson's disease, 6:731, metabolism, 6:19-21
action, and route of admin- 6:732 structure-activity relationship,
istration, 3286 Arteannuin, 5:961 &30-31
structure, 3:288 Arteether, 1:887,5:964 Arylpropionic acids
structure-based design, 1:442, antimalarial, 5:974-975 inversion in metabolism,
1:444 dosage forms, 5:935 2:460
Arginase inhibitors, 1:736-737 Arteflene Arylsulfatase B, 2:219,2:220
Arginine antimalarial, 5:984-985 N-Arylsulfonamides
chemical modification re- Artelinic acid, 5:964,5:974-975 selective COX-2 inhibitors,
agents, 1:755 Artemether, 1:887-888,5:964 4241-242
Arginine vasopressin, See Vaso- antimalarial, 5:973-974 Arylsulfonamidophenethanol-
pressin dosage forms, 5:935 amine analogs, 1:703
Aricept Artemisia annua (sweet worm- p-Arylthio cinnamide antago-
for Alzheimer's disease, 6:762 wood), 1:886, 1:888 nists, 1:566-567
for cognition enhancement, Artemisinin, 1:849, 1:886-888, Aryplase, 2:219,2:220
6:781 5:960-963 Arzoxifene, 3:668
Arimidex, 3632 dosage forms, 5:935 estrogen receptor affinity,
Aripiprazole, 6:650 first generation, 5:970-976 3:659
Arixtra, 3:286,3:310 mechanism of action, ASA with codeine, a 3 3 5
ARL17477 5966-970 ASCII (American Standard Code
nitric oxide synthase inhibi- structure-activity relationship, for Information Exchange),
tor, a706 5:963-966 1:398
Armed antibodies, 5:83, 5:92-94 9-epi-Artemisinin, 5:965 Ascomycin
Aromasin, 3:632,3:728 Artemotil, 5935 binding to FKBP, 1:552,
Aromatase Arterial thrombosis, 3:290 1:553-554
in androgen metabolism, antithrombotic treatment reg- Ascorbic acid, 4:399. See also
3:692 imen, 3298-299 Vitamin C
in estrogen metabolism, 3642 Arteries, 5:216 Asendin, 6:485
Aromatase inhibitors, 1:717, Arteriography, 4567. See also Asendis, 6:485
1:770,3:604,3:642-643 Radiopaques Asepsis, 5539
C-20 steroids as, 3:726-728 CT application, 4568 Ashimycin A, 2:172
currently marketed, 3632 Artesunate, 5:964,5:972-973 Ashimycin B, 2:172
nonsteroidal, 3:668-669 dosage forms, 5:935 Asimadoline
Aromatic aldehydes Arthrobactin, 3:483,3:486 analgesic, 6:394, a 3 9 5
hemoglobin allosteric effec- synthesis, 3:524 Asinex catalog, 1:385
tors, 3:394-396 Artificial intelligence, 1:398 Asparenomycin A, 5:670,5:671
Aromatic-aromatic interactions, in automated high-throughput in vitro activity, 5:692
1286 screening, 2:65 Aspartate transcarbamoylase
Aromatics Artificial neural networks (ATCase)
analogs based on substitution anti-inflammatory steroids functional domains in,
of aromatic for saturated application, 3:791-792 2:307308,2:310
ring; or the converse, for druglikeness screening, Aspartate transcarbamoylase
1~699-704 1:247-248, 1250 (ATCase)inhibitors,
complexation, 2:670 in molecular modeling, 1:126 1:743-744
growth inhibition by, 1:38 in QSAR, 1:53, 1:62, 1:67 Aspartic acid
molecular comparisons, 1:139 for structural genomics study, chemical modification re-
ArrayExpress, 1:345 1:353 agents, 1:755
Cumulative Index
i
I
role of insulin, 4 2 genetic algorithm study of ac- Cardrase, 3:70
I thyroid hormone effect on, tive site, 1:89 Cardura, 6:4
/ 3:571
Carbo index, l:202
Carbonate
molecular modeling, 1:116
Carbrital, 6:209
Carbromal, 6:208,6:209
Caretakers, 5 2 6
Carfecillin, 2:510, 5:609
Carfentanil, 6:382
binding to hemoglobin, 3:386 Carbutamide, 4 1 8 Carinamide, 3:139
2-Carbon chain elongation, Carcinogenesis, 5:3-5 Carisoprodol 'Z
2~460-461 and apoptosis, 5:16-19 CYP2C19 substrate, 4:627
I Carbon dioxide and diet, 5:7 Carmustine
i binding to hemoglobin, and DNA repair, 5:4,5:5 alkylating agent, 5:54, -
i 3:386-387 epigenetic changes, 5:7-10 564-65
Carbonic acid and ionizing radiation, 5 4 , L-Carnitine esters, 2562
533 a-Carotene, 4:369
production in kidneys, 3:58,
and transforming growth fac- p-Carotene, 4320-321,
3:68
tor p, 530-32 4368-370
Carbonic anhydrase, 3 6 8
UV radiation effects, asymmetrical cleavage,
action in kidneys, 3:58,3:59, 4465-466 4322-323
3:61 Cardiac arrhythmias, 2:76,3:3. symmetrical cleavage,
Carbonic anhydrase inhibitors, See also Antiarrhythmic 4321-322
1:718 agents; Long QT syndrome y-Carotene, 4369
diuretic agents, 3:66-70 and calcium, 3:159-162 Carotenes, 4 3 6 8 3 7 1
4-fluorobenzenesulfonamide disorders in electrical signal Carotenoids, 4:320321
binding to, 1:538 generation and conduction, Carpetimycin A, 5:670,5:671
molecular modeling, 1:120 329 CAR receptor, 2:79
selective toxicity, 5264 heart block, 329-30 Carrier-mediated transport,
virtual screening for, 1:316 malignant, 3:157-164 2:254,2:257-258,2:658
Carbonic anhydrase I1 inhibi- and myocardial oxygen de- Carrier moiety, prodrugs, 2:500,
tors, 1:718 mand, 3:9 2:503,2:525
novel lead identification by re-entrant, 3:30-31,3161 CART, 1:67
virtual screening, 1:320 role of extracellular potas- Carte0101
X-ray crystallographic studies, sium, 3:163-164 P-antagonist, 6:5
1~483-484 thyromimetics for, 3676 applications, 6:16
Carbon monoxide treatment perspective, Cartrol, 6:5
binding to hemoglobin, 3:386 333-34 Carumonam, 5:666
Carbon oxidation and reduction types of, 3:31 in vitro activity, 5:667
in metabolism, 2:441-445 Cardiac Arrhythmia Suppres- Carvedilol
Carboplatin sion Trial, 3:45 alp-antagonist, 6:5
alkylating agent, 5:54,5:59 Cardiac contractility, 3:9 applications, 6:16
with RSR13,5:190 Cardiac glycoside calcium chan- structure-activity relationship,
selective toxicity, 5257-258 nel blockers, 3 2 8 6:32
Carboprost, 4305 Cardiac physiology, 3:2-6 Carzelesin
p-Carboxybenzenesulfonamide, electrophysiology, 3:3-4 alkylating agent, 554,
3:70,3:71 excitation and contraction 561-64
Carboxylesterase, 2:538 coupling, 3:4-6 Cascade clustering
Carboxylic acids and ion channels, 3:6-8 with molecular similaritytdi-
pharmacophore points, 1249 Cardioangiography versity methods, 1:205
privileged structures, 1252 CT application, 4:568 CASE, 2:621
Carboxylic 3-deazaadenosine Cardiotoxicity screening, CASETox, 1246
activity against orthopoxvi- 2:627-628 CAS Numbers, 1:378, 1:400
ruses, 5439,5:440 assays in high-throughput Casodex, 3:717,3:720
Carboxypeptidase A screening, 2:62 P-Casomorphin analogs,
effect on urotensin-II,3:212 and long QT syndrome, 2:76 6:422-425
similarity of active site to Cardiotropin-1, 3269 CAS ONLINE, 1:361
ACE, 1:433, 1:746-747 Cardiovascular disease, 3:2 similarity searching, 1:383
Carboxypeptidase inhibitors Cardiovascular drugs Caspase-3
conformational changes on natural products as leads, and apoptosis, 5 1 8
binding, 1261 1:878-883 and cellular energetics, 6:680
Cumulative Index
i
I
Cumulative Index
side effects, adverse effects, Ethacrynic acid, 3:142 promotion of Fenton reaction,
drug interactions, and con- animal studies, 3:62 3503
traindications, 3:355, antisickling agent, 1:421 radiopaque chelating agent,
3:631-633 diurectic, 3:88, 3:89-92 4489-490
Estrogen response elements, sickle hemoglobin modifier, radioprotective agent, 5:163
3648, 3:653 3:461 N-Ethylmaleimide
P
Estrogens, 3:595,3:598-599 Ethambutol, 5:839-840 radiosensitizer, 5:177
for Alzheimer's disease, drug interactions, 5:864 Ethylpropion, 6:863-864
6:765-766,6:768 efficacy and dosage, 5:810, pharmacological activities,
5:863 6:865 -
androgen conversion to,
3690-692 side effects, 5:840 Ethyl triiodostearate
biosynthesis, 3:617, Ethamide, 3:7O radiopaque material, 4493
3:618-620 Ethanol Ethyndiol diacetate, 3:632,
adaptive responses caused by, 3:666
currently marketed, 3:632
2:618 Ethynyl estradiol, 3634
for eating disorders, 6:853,
Ethclorvynol, 6:208, 6:209 applications, 3:631,3:632
6:858 ADME, 6:221 in contraceptives, 3:654
environmental and dietary, Ethidium bromide dose for eating disorders,
3:651-652 thermodynamics of binding to 6:853
fate of conjugated equine, DNA, 1:183 for eating disorders, 6:858
3~635-636 Ethiodol enterohepatic recycling, 3:640
for female unwanted hair emulsified, 4:575 steroidal analogs, 3:656,3:657
growth, 4:434 liposome encapsulated, 4575 5-Ethynylnicotine, 6:52
first antiandrogens, 3:718 radiopaque material, Etiocholane
genesis of equine, 3:644-645 4:493-494 testosterone metabolite, a687
metabolism, 3:620-622 Ethionamide, 5:835-836 Etizolam, a554
oxidation and reduction, efficacy and dosage, 5:810, Etodolac, 4242-243
3:633- 634 5:863 classical resolution, 1:796-797
radioprotective agent, 5:165 side effects, 5:836 Etodroxizine, 6:212,6:214
side effects, 3:631-633 Ethnic differences Etomidate, 2538
soft drugs, 2553-554 in metabolism, 2:472-473 Etonitazene
temperature-dependent up- Ethnomedicine, 239 analgesic, 6:308
take, 3:686 Ethopropazine Etopophos, 5:135
tissue-selective, 3652-654 for Parkinson's disease, 6:731, Etoposide, 1:717,1:867
Estrone, 3594 6:732 with amsacrine, 5:71
biosynthesis, 3:633-634 Ethosuximide antitumor natural product,
dose for eating disorders, ADME, 6:282 5:110,5:134-135
6:853 anticonvulsant, 6:265, 6:267, gene expression profling
for eating disorders, 6:858 6:297,6:298 study of drug response,
enterohepatic recycling, 3:640 clinical efficacy, 6:273 4:609
equilin compared, 3658 dose, 6:270 P-glycoprotein substrate,
estrogen receptor affinity, side effects, 6:277 4631
3635 Ethotoin resistance to, 5:288
with medroxyprogesterone, ADME, 6:273 with tirapammine, 5:85
3650 anticonvulsant, 6:266 tumor-activated prodrug, 5:87
metabolism, 3:621,3:622 dose, 6:270 Etoricoxib, 4235, 4236
Estrone sulfate, a 6 2 1 Ethoxzolamide, 3:69,3:70 Etorphine, 1:851
Estropipate Ethyldienolone, 3708 e-Tox, 2:620
applications, 3:630,3:632 Ethyl diiodostearate Etozolin, 3:109-110
Etanercept, 4:182,4:183,5:229, radiopaque material, 4493 Etrinate, 4373474
5:234-235 Ethylenediaminetetraaceticacid Eucatropine
Etanidazole (EDTA) anticholinergic, 6:136
radiosensitizer, 5: 178 formation constants with iron mydriatic and cycloplegic ac-
ETC-216,3:374 and other metals, 3506 tivity, e l 5 3
ETC-588,3374 history of use for iron chela- relative anticholinergic activ-
Eterobarb tion, 3:514 ity, 6:157
anticonvulsant, 6:306-307 pM values, 3:484 Euclidean distance, 1:68, 1:202
Cumulative Index
Factor Xa, 3:285 Fansimef, kg34 Federal Food, Drug, and Cos-
in clotting, 3:299-308 Fareston, 3:343,3:632 metics Act of 1938,2:684,
hemostatic preparations, Farglitazar, 429-30 2:685-686,2:689,2:697,
3:292 Faropenem, 5:659,5:660 2:698
Factor Xa inhibitors, 1:103, Fas-associated death domain, and over-the-counter drugs,
1:738, a 3 2 4 3 2 5 5:17 4424
3D pharmacophores, 1:199 Fas ligand Fedotozine P
non-peptide peptidomimetics, and adaptive immunity, 5:225 analgesic, 6:394,6:395
1:662, 1:665 plasmid DNA-mediated gene Feiba
site-based pharmacophores, therapy study, 4654 hemostatic preparation, 3:292 -
1:235-236 Faslodex, 3:6O5- 606 Felbamate
target of structure-based drug FASTA, 1:347 ADME, 6:284-285
design, 1:442
Fastin, e l 7 1 anticonvulsant, 6:265, 6:268,
Factor XI
Fast ion bombardment, 1:586, (3269, 6:295,6:298
in clotting, 3:301-308
1:587 clinical efficacy, 6:273
plasma concentrations and
half-life, a296 Fastsearch index, 1:376-377, development, 6:302
Factor XIa inhibitors, a308 1:399, 1:404 dose, 6:270
Factor XIII, a285 Fast Track programs, FDA, pediatric indications, 6:274
in clotting, 3:303 2:689,2:697 removedlrestricted by FDA,
Factor XIIIa, 3:284, a285 Fat-absorption inhibitors, a844 4:642
in clotting, 3:303 clinical applications, 6:843 side effects, a278
Factor XIIIa inhibitors, Fat soluble vitamins, 4368 Felodipine, 2:567,3:21
3329-331 Fattiviracin A1 bioavailability and half-life,
Factor XIIa influenza antiviral agent, 3:23
and bradykinin production, 5:390 cardiovascular effects, 3:25
3:225 Fatty acids vascular-to-cardiac selectivity
in clotting, 3:299-308 as absorption enhancers in ratio, 3:172
Factorial designs, 1:65-66 lipid formulations, Female-pattern baldness, 4433,
bioassays, 2:46 2:673-674 4435
Factory Inspection Amendment and insulin resistance, 4 2 8 Female sex hormones, 3:598,
of 1953,2:686 in lipid formulations, 2:671 3:629-630. See also Contra-
Fact tables, 1:390, 1:401, 1:404 permeation enhancers, 2:675, ceptives; l7p-Estradiol; Es-
FAD, 4393,4394,4397 trogens; Progesterone
2:676
nitric oxide synthase binding aromatase inhibitors,
role of insulin in metabolism,
site, 6:698 3:642- 645
42-3
FADH, 4394 biosynthesis, 3:617,
FADH,, 4394 target of toxicity, 2:616 3642-645
Fadrozole, 3:643,3:668 Faverin, 6:485 clinical applications,
Failed Reactions database, 1:385 FBSS, 1:202 3:630-633
Famciclovir FCE 22101,5:659,6:660 discovery of SERMS,
for hepatitis B virus, FCE 22891,5:659 3:655-656
6:330-331 FCE 29464,5:660 drug metabolism, 3:633-642
for herpes virus, 5:309,5:310 FDA, See Food and Drug Admin- nonsteroidal analogs of estra-
selective toxicity, 5:267 istration diol, 3:659-666
Famiciclovir, 2:508 Fd4C nonsteroidal analogs of pro-
Familial hypercholesterolemia, for hepatitis B virus, 6:327, gesterone, 3:667-668
3:11 5:329,5:333 physiology and pharmacology,
Families, 1:93 FeatureTrees, 1:316, 1:321 3:645-655
Family competition evolutionary Febrifugine recent and future develop-
algorithm, 1:299 antimalarial, 5:996,5:997 ments, 3:668-669
Famotidine Federal Anti-Tampering A d of steroidal analogs of estradiol,
classification in various coun- 1982,2:687 3:656-659
tries, 4:430 Federal Courts Improvement steroidal analogs of progester-
Fananserin, 6:651 Act of 1982,2:741 one, 3666-667
Fanconi's anemia, 6:32 Federal Declaratory Judgments structure-activity relationship,
Fansidar, kg34 Act of 1934,2:741 3:656-668
Cumulative Index
and proteoglycans, 2:238 Hepatitis C virus NS3 protein, Herpes viruses, 5:294-295,
structure, 3:288 5:417-418 5297-299
synthesis, 2:235 Hepatocyte growth factor adiva- a-Herpesviruses, 5298
for thrombolytic therapy, tor inhibitor 1 P-Herpesviruses, 5298
3:166 mariptase inhibitor, 1:269, y-Herpesviruses, 5:298,5:299
trend to lower molecular 1:271 Herpes virus protease inhibitors,
7
weight, 2:204,2:210 Hepatography, 4567. See also 5324-325
Heparinase I, 2:215-216,2:220 Radiopaques Herpes zoster, 5:298
Heparin cofactor II,2:302 Hepatolienography, 4567. See Hesperidin
rotavirus antiviral, 5431 -
Heparin-induced thrombocyto- also Radiopaques
penia, 3:294 HEPES ligand HE-State index, 1:26
Heparin-induced thrombocyto- nicotinic acetylcholine recep- Heterochiral molecules, 1:782
penia with thrombosis, Heterocyclic antidepremmts, 6:486
tors, 2:363,2:364,
Heterocyclic compounds
3:294 2:375-376
radioprotective agents,
Heparin-linked fibroblast Hephaestin, 3:501
5163-165
growth factor (FGF), HEPT Heterocyclic sedative-hypnotics
2:235-236 allosteric effector, 2:314, ADME, 6:221-223
Heparin rebound, 3:308 2:315 clinical pharmacology,
Heparin sulfate proteoglycans, Heptylphysostigmine 6:234-235
521, 5:216 anticholinesterases related to, currently used drugs,
Hepatic clearance, 2:637 6:89 6:210-211
Hepatic extraction ratio, Herbal remedies, 2:39 discovery, 6:235
2~637-638 Herceptin, 2:87-88,5228,5:230 pharmacologic effects, 6:230
Hepatitis B virus, 5:297, selective toxicity, 5:257 side effects and drug interac-
5299-301 Hereditary persistence of fetal tions, 6:210-211
Hepatitis B virus antiviral hemoglobin, a457 6-Heterocyclylmethylenepen-
agents, 5326 HERG protein cardiotoxicity ems, 5:698-699
antisense oligonucleotides, screening, 2:76,2:628 Heteronuclear multiple bond
5:337 Heroin, 1:849-850 correlation spectroscopy
combination treatments, as "accidental" prodrug of natural products, 1518
5334-335 morphine, 2500 Heteronuclear single quantum
purines, 5330-333 synthesis, 6:360,6:362 correlation spectroscopy,
pyrimidines, 5:326-330 Herpes simplex virus 1512
resistance, 5:333-334 chemokine receptors, Hexahydrosiladifenidol
targeting drugs to liver, 4170-171 anticholinergic, 6:132
5335337 in suicidal gene cancer ther- Hexamethonium
Hepatitis C virus, 5:361362 apy, 5 4 4 nicotinic receptor antagonist,
and HBV infection, 5300 Herpes simplex virus-l,5:297, 2:397
model systems to study repli- 5298 Hexestrol, 1:707
cation, 5:427 Herpes simplex virus-2, 5298 electrophilic metabolites, a638
Hepatitis C virus antiviral Herpes virus antiviral agents, estrogen receptor affinity,
agents 5:305 3:659
inhibitors of HCV 5' untrans- compounds under develop- Hexobarbital, a204
lated region and core gene, ment, 5:310326 CYP2C19 substrate, 4:627
5415-417 currently approved drugs, Hexocyclium, 6:139
inhibitors of NS5B RNA-de- 5:305-310 relative anticholinergic activ-
pendent RNA polymerase, immune modulators, ity, 6:157
5:425-426 5325-326 HHS-8 virus, 4:171
inhibitors of NS3 helicase, nonnucleoside inhibitors and HHV-6,5298-299
5425 other targets, 5321324 HHV-7,5:298-299
inhibitors of NS3 protease, prodrugs increasing oral avail- HHV-8, 5299
5418-425 ability, 5:309 Hib vaccines, 2:209,2:239
interferon-a applications, prodrugs under development, Hierarchical clustering, 1:220,
5:361364 5318-321 1:401, 1:405
Hepatitis C virus inhibitor resistance to, 5:309310 with molecular similaritytdi-
antisense drug, 2:120 selective toxicity, 5:267 versity methods, 1:205
Cumulative Index
High ceiling diuretics, 3:88, and combinatorial chemistry, Histamine receptor antagonists
3:89-111, a142 2:32 molecular modeling, 1:143
High-content metabolic assays, compound deck construction Histamine1 receptors, e l 1 2 -
2~72-79 and screening for hits, reporter gene assays studies, .
High-content toxicity screening, 2:54-59 2:51
2:626 following the competition, Histamine2 receptors, 6:112
High density lipoproteins 2:54 Histamine3 receptors, &I12 *
(HDL),2:616,3:340-341 lead discovery process, Histidine
and Alzheimer's disease, 2~41-62 chemical modification re-
6:748-749 mass spectrometry applica- agents, 1:755 -
and coronary artery disease, tions, 1:591, 1:592-596 gating of anion-selectivechan-
3:lO miniaturization, 2:66-67 nels, 2:360
current drugs to raise,
and molecular modeling, 1:155 Histone deacetylases, 5:8
3:342-352
molecular similarity/diversity Histoplasma capsulatum, 6:883
new treatments for raising
methods, 1:191 Histoplasmosis, 5:883
while lowering TG,
3:370-372 profiling hits, 2:59-62 Hit list, 1:380, 1:405, 1:411
transport, 3:359-361 raw data points obtained by HIV-DNA polymerase (reverse
High density oligonucleotide ar- companies, 1:50 transcriptase), 5462-463
rays, 1:344 for receptor target lead discov- HIV fusion proteins
Higher osmolality intravascular ery, 2:344 allosteric protein target,
contrast agents, 4495, screening hits analysis, 2:59 2:314,2:315
4538 screen validation and reagent and CXCFM, 4166-170
Highly active antiretroviral scale up, 2:53-54 HIV integrase inhibitors, 5:464
therapy (HAART), 6:884 target discovery and valida- HIV protease, 6:464
HIVIAIDS, k479 tion, 2:42-43 HIV protease inhibitors, 1:717,
High performance liquid chro- and toxicology, 2:610 2:610,6:475-477
matography (HPLC), ultra-high-throughput, 2:40 binding-site molecular models,
1:586-589 with virtual screening, 1:316 1:130
for combinatorial library X-ray crystallography applica- comparative molecular field
screening, 1:592-596, 1:598, tion, 1:472 analysis, 1:153
1:599, 1:607 Himbacine consensus scoring study,
denaturing, 4:622 anticholinergic, 6:116, a133 1:266
fast, 1:596 HIN file format, 1:369 3D CoMFA, 1:59
for hydrophobicity determina- HINT descriptors, 1:56 de novo design, 1:113
tion, 1:16-17, 1:23 for studying allostery, fluorescence polarization stud-
for separation of chiral mole- 2:309-312 ies, 2:48
cules, 1:783, 1:788-792 Hint!-LogP, 1:389 force field-based scoring study,
High pressure liquid chromatog- HipHop, 1:60, 1:256 1:307
raphy (HPLC),4 4 8 Hippocampus homology modeling, 1:123
High-throughput chemistry, nicotinic acetylcholine recep- knowledge-based scoring
1:358, 1:405 tors, 2:389, 2:391 study, 1:311
chemical libraries for, 1:367 Hirsutism, 4:433,4:440 molecular modeling,
and natural product screen- Hirudin and analogs, 3:29, 1:103-104, 1:105, 1:108,
ing, 1:848 3:285,3:316-318 1:109, l:lll, 1:117, 1:120,
High Throughput Crystallogra- marketed antithrombotics, 1:122
phy Consortium, 1:418 3:286 nevirapine synthesis,
High-throughput pharmacology, Histamine 2:420-429
25'2-79 role in gastric secretion, NMR binding studies, 1:533,
High-throughput pharmacology 6:112-113 1:559-562
system, 25'3-74,2:78-79 role in spasms, 6:111 non-peptide peptidomimetics,
High-throughput screening, and ulcers, 6:119 1:659-660
1:283,2:37-41 Histamine H2 receptor antago- novel lead identification by
automation, 2:40, 2:63-66 nists, 6:112-113 virtual screening, 1:320
bioassay design and screen antiulcer application, 6:155, reagents for screening, 2:91
construction, 2:43-54 6:158 recombinant DNA technology
candidate selection, 2:59-62 discovery, 2:40 applications, 2:97-99
Cumulative Index
Insulin dependent diabetes mel- Interatomic distance variant an- modulator studies, 4174-175
litus, See Diabetes mellitus, alog~,12710-712 role in blood cell development,
type 1 Intercalated disk, 3:3 3253
Insulin detemir, 411 Intercalation, $67 X-ray crystallographic studies,
Insulin glargine, 4:6,47,49 Intercellular adhesion molecule 1:490
development of, 411 1 Interleukin-3,3:268-270,
receptor binding affinities, antisense inhibitors, 2:126, 3~271-272 V
410 2:127,2:138 role in blood cell development,
Insulin lente, 4 6 , 4 7 recombinant DNA studies, 3:253,3:254-255
Insulin-like growth factor 1 2:105 with stem cell factor, a268 -
insulin binding, 49-10 X-ray crystallographic studies, X-ray crystallographic studies,
radiosensitizer, 5:194 1:489 1:490
X-ray crystallographic studies, Interdigitation-fusion vesicles, Interleukin-4
1:489 4573 and adaptive immunity, 5:225
Insulin-like growth factor 2 Interference, with patents, modulator studies, 4:175-176
X-ray crystallographic studies, 2:735-738 plasmid DNA-mediated gene
1:489 Interferon therapy study, 4656
Insulin lispro, 4:6, 4 7 , 4 9 dosing interval, 2:643-644 radioprotective agent, $171
development of, 411 induction by polyribosine: role in blood cell development,
receptor binding affinities, polyribonocytidine, 2:117 3:253
410 radioprotective agents, 5:171 X-ray crystallographic studies,
Insulinotropic agents, 411-20 Interferon-a 12490-491
Insulin overdosing, 2:618 dosing interval, 2:644 Interleukin-5
Insulin receptor, 4 9 for hepatitis B virus, 5:326, and adaptive immunity, 5:225
Insulin resistance, 4 3 5:334 glucocorticoid downregulation,
and free fatty acids, 4 2 8 for hepatitis C virus, 5:361, 4:172
Insulin-sensitizing agents, 5:362-364 modulator studies, 4176-178
420-31 for papillomavirus, $337-338 role in blood cell development,
Insulin ultralente, 46,47,4:9 volume of distribution, 2:640 3:253
Integrase, 5:616 Interferon a1 X-ray crystallographic studies,
Integrilin, 3:29, a287 X-ray crystallographic studies, 1:491
Integrin a4pl 1:490 Interleukin-6,3:268-270
interaction with sickled cells, Interferon y and adaptive immunity, 5:225
a458 and adaptive immunity, 5:225 with IL-11,3:265
Integrin aM IL-5 modulator, 4177 modulator studies, 4178-180
X-ray crystallographic studies, modulator studies, 4183-184 plasmid DNA-mediated gene
1:489 plasmid DNA-mediated gene therapy study, 4657
Integrins, 5:13 therapy study, 4656, radioprotective agent, Ex171
recombinant DNA studies, 4669-670 role in blood cell development,
2:105 X-ray crystallographic studies, 3:253,3:254-255
Integrin aVp3 1:490 with stem cell factor, 3:268
interaction with sickles cells, Interleukin-1,4656 X-ray crystallographic studies,
3:458 enhances IL-11,3:264 1:491
Integrons modulator studies, 4:173-174 Interleukin-7
and p-lactam resistance, radioprotective agent, 5:171 role in blood cell development,
5:614-616 role in blood cell development, 3:253
Intellectual property, 2:703-708. 3:253,3:254-255 Interleukin-8
See also Patents; Trade- and stem cell factor, 3:267 and CXCRl/CXCR2,
marks; Trade secrets X-ray crystallographic studies, 4161-164
and gene hnction, 2:43 1:490 mutation and chimera studies,
other forms of protection (be- X-ray crystallographic studies 4137
sides patents, trademarks, of receptor, 1:490 X-ray crystallographic studies,
and trade secrets), Interleukin-2 1:491
2:770-771 and adaptive immunity, 5:225 Interleukin-10
Intent-to-use applications, trade- glucocorticoid downregulation, and adaptive immunity, 5:225,
marks, 2:760 4:172 a226
Cumulative index
plasmid DNA-mediated gene Interrogatories, in patent in- review flow chart, 2:694
therapy study, 4656 fringement cases, 2:749 types of, 2:689-690
X-ray crystallographic studies, Interstitial collagenase, 5:13 Investigator Investigational New -
1:490 Intestinal metabolism, Drugs application,
Interleukin-ll,3:264-265, 2:677-678 2:689-690
3:269,3:272 Intestinal peptide transporter, In vitro assays, 2:72
bioactivity, 3:265 2274-275 gastric acid secretion inhibi- r
preparations, 3:266 Intestinal permeability, See Per- tors, 495-96
role in blood cell development, meability In vitro cell-based toxicity test-
3253 Intestine transplantation, 5:487 ing, 2:611,2:624-628 -
therapeutic indications, side Int-2 growth factor, 5:22 In vitro evolution (SELEX),
effects, and pharmacokinet- Intoplicine 2:92-94
dual topoisomerase ID1 inhibi-
In vitro studies
ics, 3:265-266
shortcomings of, 3:62
Interleukin-12, 4656 tor, 5:68,5:73,5:74
In vitro toxicology
glucocorticoid downregulation, Intracellular adhesion molecule
phosphorothioate oligonucleo-
4172 1 (ICAM-1) tides, 2:139
modulator studies, 4180-181 target of NMR screening stud- In vitro uptake
role in blood cell development, ies using SAR-by-NMR, phosphorothioate oligonucleo-
3253 1:566-567 tides, 2:121,2:132-133
X-ray crystallographic studies, Intracellular receptors, 2:334 In vivo assays
1:490 Intraepithelial neoplasia, 5:35 gastric acid secretion inhibi-
Interleukin-13 Intrahepatic anion-binding Y tors, 496-97
modulator studies, 4181-182 protein In vivo pharmacokinetics
X-ray crystallographic studies, binding to contrast agents, phosphorothioate oligonucleo-
1:490 4557 tides, 2:133-135
Interleukin-17 Intratransguanylation, 5:158 In vivo pharmacological activi-
role in blood cell development, Intrinsic tenad complex, ties
a253 35299-300 phosphorothioate oligonucleo-
Interleukin-lp-convertingen- Invader assay, 4625 tides, 2:136-138
zyme (ICE) Invadopodia, 5: 13 In vivo toxicity testing, 2:611,
transition state analog inhibi- Invasins, 2:262-263 2:619-620
tors, 1:655 Invasive cleavage assays, 4625 In vivo toxicology
Interleukins, 4130 Invention disclosure, 2:711-712 phosphorothioate oligonucleo-
role in blood cell development, Inventory data, 1:405 tides, 2:139-142
3:252-255 Inverse folding, 1:123-125 Inward rectification
Intermediate density lipopro- Inverse QSAR, 1:4 nicotinic acetylcholine recep-
teins (IDL), 3:340-341 Inverted keys, 1:405 tors (neuronal), 2:388
Intermolecular forces, 1:6 Investigational New Drugs Ap- Inward-rectifying K+ current,
Internal ribosome entry signal, plication Process, 2:72 3:7
5:41 guidance documents, Iobenzamate
International cell depository au- 2:690-691 biotransforrnation, 4:566
thority, 2:723 information submitted with, Iobenzamic acid, 4497
International Conference on 2:691 properties, 4506
Harmonization of Technical meetings with FDA, Iobitridol, 4497
Requirements for Registra- 2~695-696 excretion, 4564
tion of Pharmaceuticals for over-the-counter drugs, 4428 nephrotoxicity, 4552
Human Use, 2:701 parallel track, 2:689-690 properties, 4506
International patent agree- pharmacokinetic data submis- Iobutoic acid, 4497
ments, 2:751-752 sion, 2:645 properties, 4:506
Internet Phase I, 2:691-693 Iocarmate
increased availability of medi- Phase II,2:693-695 excretion, 4565
cal information on, 4423 Phase III,2:695 pharmacokinetics, 4562
Inter partes patent proceeding, Phase IV, 2:695 Iocarmic acid, 4497
2:739 pre-IND meeting, 2:690 angiography application,
Interpro, 1:349 resources for application prep- 4:569
InterProScan, 1:349 aration, 2:690-691 hyperosmolality, 4543
Cumulative Index
Mercury search program, 1:387 carbon oxidation and reduc- sulfur oxidation and reduc-
Merged Markush Service, 1:386 tion, 2:441-445 tion, 2:447-448
Meridia, 6:844 and chirality, 2:478-479 3D quantitative structure-
Merimepodip conjugation reactions, metabolism relationship
structure-based design, 1:447 2:450-475 (3D-QSMRs),2:481-483
MERLIN, 1:39, 1:386 conjugation with CoA, thyroid hormones, 3569-570
Meropenem 2:459-461 Metabolism databases, 1:385,1:386 r
antimycobacterial application, and disease, 2:474-475 Metabolism screening, 1:591
5:858 electronic factors, 2:480-481 pulsed ultrafiltration applica-
discovery, 5:668-669, enantiomers, 1:786-787 tion, 1:605 -
5:680-681 enzyme induction, 2:476-478 Metabolite database, 1:386
pharmacokinetics, 5:623-624 enzyme inhibition, 2:475-476 Metabolite toxicity, 2:621-624
side effects and interactions, Metabonome, 2:621
ethnic differences, 2:472-473
5:625 Metabonomics
functionalization reaction en-
use recommendations, 5:626 toxicology implications, 2:623
in vitro activity, 5:692 zymes, 2:437-441
Metadata, 1:375, 1:376, 1:406
Merozoites, 5:920,5:922 functionalization reactions, Metadate ER, 6:170
Merrifield resin, 2:30 2:431-4352~437-465 Meta-layer searching, 1:395
Mersalyl, 3 6 5 gender differences, 2:472-473 Metal chelates
Mersoben, 3:66 genetic factors, 2:467-472 radiopaque, 4:489-490
Mesna global expert systems to pre- Metal ion complexes
radioprotective agent, &I72 dict biotransformation, radioprotective agents, 5963
Mesopin, a117 2:481-483 radiosensitizers, 5:196
Mesoridazine glucuronidation and glucosi- Metalloelastase, 5:13
dose, a 6 0 1 dation, 2:453-456 Metallopeptidase inhibitors
receptor binding and side ef- glutathione conjugation and transition state analogs,
fects, a606 redox reactions, 2:461-464 1:649-652
Mespirenone, 3:118, 3119 hydration/hydrolysis, Metarnitron, 1:42
Messenger RNA, See mRNA 2:449-450 Metaprel, 6:4
Mesterolone, 3:709 interindivual factors, Metaproterenol
Mestranol, 3:634,3:635 2:466-473 p-agonist, 6:4
applications, 3631, 3:632 intraindivual factors, applications, 6:14
oxidative metabolism, 3:636 2:473-478 structure-activity relationship,
Metabolic assays inversion of arylpropionic ac- 6:29
high-content, 2:72-79 ids, 2:460 Metaproterenol sulfate inhaler
Metabolic inhibitors and lipophilicity, 2:479-480 reversed from OTC back to
radioprotective agents, methylation, 2:450-452 prescription status, 4426
5:162-163 modulation by structural vari- Metaraminol
radiosensitizers, 5:193-196 ations, 2:483-490 a,-agonist, 6:3
Metabolic profile molecular modeling, applications, 6:12
in vivo toxicity testing, 2:619 2:48l- 483 structure-activity relationship,
Metabolic rate nitrogen oxidation and reduc- 6:30
thyroid hormone effect, tion, 2:445-447 Metastable forms, 2:669
3:570-571 @oxidationand 2-carbon Metastasis, 5:12
Metabolism, 2:431-435. See also chain elongation, 2:460-461 Metazocine, 1:708, &375
Absorption, distribution, oxidative cleavage, 2:448-449 Met-chemokine P-7,4150
metabolism, and excretion presystemic elimination, Metconazole, 1:41, 1:42
(ADME);Retrometabolic 2:677-678 Met-enkephalin, 6:331,
drug design; Toxicophores; and prodrug design, 6:356-358
Xenobiotic metabolism 2:484-487,2:499-526 Metformin, 421-23
acetylation and acylation, questions related to answered a-Methadol, 6:385
2:456-459 in lead discovery and opti- p-Methadol, a 3 8 5
and age, 2:474 mization, 2:435 Methadone, 1:708,6:332, &333
antidepressants, 6510-516 questions related to answered analogs, 6:385-387
biological factors influencing, in preclinical and clinical cytochrome P4S0isozyme sub-
2:466-478 phases, 2:435 stratelinhibitor, 6:339
and biological rhythms, 2:474 sulfation, 2:452-453 dose, &334
Cumulative Index
Mu-selective opioid receptor an- Mycobacterium chelonae, 5:815 Myeloid progenitors, 3:253,
tagonists, 6:333,6:348 Mycobacterium fortuitum, 5:815 3254
somatostatin derived, regimen for treatment, k867 Mylotarg, 5:228,5:231
6:437-438 Mycobacterium haemophilum, tumor-activated prodrug,
Musettamycin 5:815 5:83,5:92
antitumor natural product, Mycobacterium kansasii, 5:815 Myocardial cells, 3:3-4
5:124,5:126 regimen for treatment, 5866 Myocardial diseases T
Mustards, See Nitrogen mus- Mycobacterium leprae, 5:809, plasmid DNA-mediated gene
tards 5:825-826 therapy, 4655-656
Mustargen susceptibility testing, k827 Myocardial infarction, 3:3,3:289
alkylating agent, 5:54 Mycobacterium malmoense, 5:815 coronary occlusion, 3156-157 -
Mutarnycin regimen for treatment, 5:866 and lipids, 3:340
Mycobacterium marinum, 55315 malignant arrhythmias,
antitumor natural product,
regimen for treatment, 5:866 3157-164
5:120-122
Mycobacterium microti, 5:814 pathophysiology, 3:156-164
Mutated in multiple advanced
Mycobacterium scrofilaceum, preconditioning, 3:157
cancers (MMAC) tumor sup- 5:815 AT, receptor expression in,
pressor gene, 5:30 regimen for treatment, 5:866 3:197
Mutation Mycobacterium simiae in stroke patients, 6:675-676
in genetic algorithms, 1:87, regimen for treatment, 5:866 treatment regimens, 3298
1:88 Mycobacterium tuberculosis, ventricular remodeling, 3:164
ionizing radiation, 5:153-154 5:809,5:813-825 Myocardial infarction agents,
in nicotinic acetylcholine re- susceptibility testing, 5826-829 3:155-156. See also Antiar-
ceptors (muscle-type), Mycobacterium ulcerans, 55315 rhythmic agents
2:375-382 Mycobacterium xenopi, 5815 for pain relief, 3:164-165
and tumorigenesis, 5:2 Mycobadin M, 5:818 for remodeling prevention,
Mutation hot spots, 5:5 Mycobadin P, 5:818 3:180-181
Mutual Recognition Procedure Mycobactin S, 3:486,3:487, for treatment of arrhythmias,
(European over-the-counter 3:493,5:818 3167-180
drugs), 4428-429 synthesis, 3:524 for treatment of thrombolysis,
Muzolimine, 3:108 Mycobutin, 5:811 3:165-167
MVIIA (Ziconotide) Mycolic acid, 5:816 Myocardial ischemia, 3:3,3:157,
NMR spectroscopy, Mycophenolate mofetil, 1:849, 3:289,6:675-676
5:489, 5:490 extracellular potassium accu-
1:518-523, 1:526, 1:534
clinical use for organ trans- mulation during, 3:162-163
MVT-101, 1~103-104,1:105,
plants, 5:518-521 and oxygen supply, 3:8,3:9
1:117
for herpes, 5:321-322 plasmid DNA-mediated gene
Myambutol, 5:810 with leflunomide, 5:523 therapy study, 4655
Myasthenia gravis side effects, 5:521 treatment of arrhythmias in-
cholinergics for, 6:41 structure-activity relationship, duced by, 3:167-180
Mycobacteria, 5:813-821. See 5:520-521 Myocardial oxygen demand, 3:9
also Antimycobacterial Mycophenolic acid, 5:518 Myocardial oxygen supply, 3:8-9
agents flavivirus antiviral, 5:428 Myocardial pacemaker cells, 3 4 ,
pathogenesis and epidemiol- for herpes virus, k321 329
ogy, 5821-826 structure-based design, Myocardium, 3:3,3:4
Mycobacteria other than tuber- 1:446-447 Myoglobin, 1:419
culosis, 5:814, 5:824 Myc protein, 5:22,5:25-26 N-Myristoyl transferase inhibi-
Mycobacterium, 5:813-814 Mydriacyl, a 1 3 8 tors, 5:908-909,5:910
Mycobacterium abscessus, 5:815 Mydriasine, a117 M I zinc finger peptides
regimen for treatment, 5867 Mydriatics influenza antiviral agents,
Mycobacterium africanum, 5:814 anticholinergic activity assay, 5:387
Mycobacterium avium, 5:809, 6:119-120,6:153
5:814,5:815 anticholinergics, 6:114-115 Nabilone, 1:853
regimen for treatment, 5:866 Myelography, 4:567. See also Nabi-NicVax
Mycobacterium bovis, 5:813, Radiopaques experimental smoking cessa-
5:814,5:815,5:820-821, radiopaques applications, tion agent, 4459
56323 4:570-571 Nacton, a138
Cumulative Index