ANNUAL REPORTS IN MEDICINAL
CHEMISTRY
Volume 19
Academic Press Rapid Manuscript Reproduction
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ANNUAL REPORTS IN MEDICINAL
CHEMISTRY
Volume 19
Academic Press Rapid Manuscript Reproduction
ANNUAL REPORTS IN MEDICINAL CHEMISTRY Volume 19 Sponsored by the Division of Medicinal Chemistry of the American Chemical Society Editor-in-Chief: DENIS M. BAILEY STERLING-WINTHROP RESEARCH INSTITUTE RENSSELAER, NEW YORK
SECTION EDITORS BARRIE HESP WILLIAM T. COMER FRANK C. SCIAVOLINO BEVERLY A. PAWSON ROBERT W. EGAN RICHARD C. ALLEN
ACADEMIC PRESS, INC.
1984
(Harcouft Brace Jovanovich, Publishers)
ORLANDO SAN DIEGO NEW YORK LONDON TORONTO MONTREAL SYDNEY TOKYO
COPYRIGHT 0 1984, BY ACADEMIC PRESS,INC. ALL RIGHTS RESERVED. NO PART OF THIS PUBLICATION MAY BE REPRODUCED OR TRANSMITTED IN ANY FORM OR BY ANY MEANS, ELECTRONIC OR MECHANICAL, INCLUDING PHOTOCOPY, RECORDING, OR A N Y INFORMATION STORAGE AND RETRIEVAL SYSTEM,WITHOUT PERMISSION IN WRITINQ FROM THE PUBLISHBB.
ACADEMIC PRESS,INC.
Orlando, Florida 32001
United Kingdom Edition published by ACADEMIC PRESS. INC. ( L O N D O N ) LTD.
24/20 Oval Road, London N W l 7DX
LIBRARY OF CONQRESS
CATALOG CARD NUMBER:
I SBN 0- 12-0405 19-9 PRINTED IN THE U N m STATES OF AMERICA 84 85 86 81
9 8 7 6 5 4 3 2 1
6 6 - 26 84 3
CONTRIBUTORS PREFACE
xi
...
Xlll
I.
CNS AGENTS
Section Editor: Barrie Hesp, Stuart Pharmaceuticals, Division of ICI Americas, Wilmington, Delaware 1.
Analgesics 0. William Lever, Jr., Kwen-Jen Chang, and John D. McDermed, Wellcome Research Laboratories, Burroughs Wellcome Co., Research Triangle Park, North Carolina
1
2 . Antianxiety Agents, Anticonvulsants, and Sedative-Hypnotics Joseph P. Yevich, James S. New, and Michael S. Eison, Bristol-Myers Research and Development, Evansville, Indiana
11
3. Antipsychotic Agents Tomas de Paulis and Sten Ramsby, Astra Lakemedal AB, Sweden
21
4. Cognitive Disorders Fred M. Hershenson and John G. Marriott, Warner Lambertl Parke-Davis, Ann Arbor, Michigan
31
5.
Adaptive Changes in Central Nervous System Receptor Systems B. Kenneth Koe and Fredric J . Vinick, Pfzer Central Research, Groton, Connecticut
6 . CNS Autoreceptors as Targets for Drug Design Pieter B. M. W. M. Timmermans, Department of Pharmacy, Division of Pharmacotherapy, University of Amsterdam, Plantage Muidergracht 24, 1018 TV Amsterdam, The Netherlands V
41
51
vi
Contents
11. PHARMACODYNAMIC AGENTS Section Editor: William T. Comer, Bristol-Myers Research and Development, 345 Park Avenue, New York, New York 7. Antihypertensive Agents Peter Sprague and James R. Powell, The Squibb Institute for Medical Research, Princeton, New Jersey
61
8. Cardiotonic Agents Bernd Wetzel and Norbert Hauel, Dr. Karl Thomae GmbH, 0-7950 Biberach, Federal Republic of Germany
71
9. Agents for the Treatment of Peptic Ulcer Disease David E. Bays and Roger Stables, Glaxo Group Research Ltd., Ware, Hertfordshire, England
81
10. Pulmonary and Antiallergy Agents John H. Musser, Anthony F. Kref, and Alan J . Lewis, Wyeth Laboratories, Inc., Philadelphia, Pennsylvania
93
111. CHEMOTHERAPEUTIC AGENTS
Section Editor: Frank C. Sciavolino, Pfizer Central Research, Groton, Connecticut 11, Antibacterial Agents E. S. Hamanaka and M. S. Kellogg, Pfizer Central Research, Groton, Connecticut
107
12. Antiviral Agents James L. Kelley, Wellcome Research Laboratories, Burroughs Wellcome Co., Research Triangle Park, North Carolina
117
13. Antifungal Chemotherapy Michael B. Gravestock and John F. Ryley, ICI Pharmaceuticals Division, Mereside, Alderley Park, Macclesfield, Cheshire, England
127
14. Antineoplastic Agents Terrence W. Doyle, Bristol-Meyers Pharmaceutical R & D Division, P.O. Box 4755, Syracuse, New York
137
15. Antiparasitic Agents Bernard J . Banks, Pfizer Central Research, Sandwich, Kent, England
147
Contents
vii
IV. METABOLIC DISEASES AND ENDOCRINE FUNCTION Section Editor: Beverly A. Pawson, Roche Research Center, Hoffmann-La Roche Inc., Nutley, New Jersey 16. Progress in the Development of Antiobesity Drugs Ann C. Sullivan, Susan Hogan, and Joseph Triscari, Roche Research Center, Hoffmann-La Roche Inc., Nutley, New Jersey 17. Aldose Reductase Inhibitors as a New Approach to the Treatment of Diabetic Complications Christopher A. Lipinski and Nancy J . Hutson, Pfizer Central Research, Groton, Connecticut 18.
Vitamin D: Metabolism and Mechanism of Action Hector F. DeLuca and Heinrich K . Schnoes, Department of Biochemistry, University of Wisconsin-Madison, College of Agricultural and Life Sciences, Madison, Wisconsin
19. Interleukin 2 John J . Farrar, William R. Benjamin, Lorraine Cheng, and Beverly A. Pawson, Roche Research Center, Hoffmann-La Roche Inc., Nutley, New Jersey
157
169
179
191
V. TOPICS IN BIOLOGY Editor: Robert W. Egan, Merck Institute for Therapeutic Research, Rahway, New Jersey 20. The Inactivation of Cytochrome P-450 Paul R. Ortiz de Montellano, Department of Pharmaceutical Chemistry, School of Pharmacy, University of California, San Francisco, California
20 1
2 1. Phospholipases Eduardo G. Lapetina, Department of Molecular Biology, Wellcome Research Laboratories, Burroughs Wellcome Co., Research Triangle Park, North Carolina
213
22. Vaccine Synthesis by Recombinant DNA Technology Dennis G. Kleid, Department of Vaccine Development, Genentech Inc., 460 Point San Bruno Blvd., South San Francisco, California
223
23. Collagenases in Tumor Cell Extravasation T. Turpeenniemi-Hujanen, U . P. Thorgeirsson, and L. A . Liotta, Laboratory of Pathology, NCI, NIH, Bethesda, Maryland
23 1
viii
Contents
24. Biology of Leukotrienes William Kreutner and Marvin I . Siegel, Schering-Plough Corporation, Bloomfield, New Jersey
24 1
25. Endogenous Natriuretic Agents Mary Anna Napier and Edward H . Blaine, Merck Sharp & Dohme Research Laboratories, Rahway, New Jersey and West Point, Pennsylvania
25 3
VI. TOPICS IN CHEMISTRY AND DRUG DESIGN
Section Editor: Richard C. Allen, Hoechst-Roussel Pharmaceuticals Inc., Somerville, New Jersey 26. Enzymic Methods in Organic Synthesis Mark A. Findeis and George M . Whitesides, Department of Chemistry, Harvard University, Cambridge, Massachusetts
263
27. Stable Isotopes in Drug Metabolism and Disposition Thomas A. Baillie and Albert W, Rettenmeier, Department of Medicinal Chemistry, University of Washington, Seattle, Washington Lisa A. Peterson and Neal Castagnoli, Jr., Department of Pharmaceutical Chemistry, University of California, San Francisco, California
273
28. Drug Discovery at the Molecular Level: A Decade of Radioligand Binding in Retrospect Michael Williams and David C. U'Prichard, Nova Pharmaceutical Corporation, P,0. Box 21204, Wade Avenue, Baltimore, Maryland 29. Forskolin and Adenylate Cyclase: New Opportunities in Drug Design Kenneth B. Seamon, National Center for Drugs and Biologics, FDA, Bethesda, Maryland
30. Recent Progress in the Rational Design of Peptide Hormones and Neurotransmitters Victor J . Hruby, John L. Krstenansky, and Wayne L. Cody, Department of Chemistry, University of Arizona, Tucson, Arizona
VII.
WORLDWIDE MARKET INTRODUCTIONS
Section Editor: Richard C. Allen, Hoechst-Roussel Pharmaceuticals Inc., Sornerville, New Jersey
283
293
303
Contents
31.
To Market, to Market-1983 Richard C . Allen, Hoechst-Roussel Pharmaceuticals Inc., Somerville, New Jersey
COMPOUND NAME AND CODE NUMBER INDEX CUMULATIVE CHAPTER TITLES KEYWORD INDEX
ix
313
327 335
This Page Intentionally Left Blank
Allen. Richard C . . . . Baillie. Thomas A . . . Banks. Bernard J . . . . Bays. David E . . . . . Benjamin. William R . . . Blaine. Edward H . . . . Castagnoli. Neal. Jr . . . Chang. Kwen-Jen . . . . . . Cheng. Lorraine Cody. Wayne L . . . . DeLuca. Hector F. . . . de Paulis. Tomas . . . Doyle. Terrence W . . . Eison. Michael S . . . . Farrar. John J . . . . . Findeis. Mark A . . . . Gravestock. Michael B . . Hamanaka. E . S . . . . Hauel. Norbert . . . . Hershenson. Fred M . . . Hogan. Susan . . . . Hruby. Victor J . . . . Hutson. Nancy J . . . . Kelley. James L . . . . Kellogg. M . S . . . . . Kleid. Dennis G . . . . Koe. B . Kenneth . . . Kreft. Anthony F. . . . Kreutner. William . . . Krstenansky. John L . . . Lapetina. Eduardo G . . . Lever. 0. William. Jr. . .
. . . 313 . . . 273
Lewis. Alan J . . . . . . Liotta. L . A . . . . . . Lipinski. Christopher A . . . Marriott. John G . . . . . McDermed. John D . . . . Musser. John H . . . . . Napier. Mary Anna . . . New. James S . . . . . . Ortiz de Montellano. Paul R . . Pawson. Beverly A . . . . Peterson. Lisa A . . . . . Powell. James R . . . . . Ramsby. Sten . . . . . Rettenmeier. Albert W. . . Ryley. John F. . . . . . Schnoes. Heinrich K . . . . Seamon. Kenneth B . . . . Siegel. Marvin I . . . . . Sprague. Peter . . . . . Stables. Roger . . . . . Sullivan. Ann C . . . . . Thorgeirsson. U . P. . . . Timmermans. Pieter B.M. W.M. Triscari. Joseph . . . . . Turpeenniemi.Hujanen. T. . U’Prichard. David C . . . . Vinick. Fredric J . . . . . Wetzel. Bernd . . . . . . Whitesides. George M . . . Williams. Michael . . . . Yevich. Joseph P. . . . .
. . . 147 . . . 81 . . . 191
. . . 253 . . . 273 . . . 1 . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . .
. 191 . 303 . 179
. 21 . 137 .
11
. 191 . 263 . . . . . . .
. . . .
. . . .
. . .
127 107 71 31 157 303 169 117 107 223 41 93 241 303 21 3 1
xi
. .
93
. . 231 . . 169 . . 31 . .
1
. .
93 253 11 201 191 273 61 21 273 127 179 293 241 61 81 157 231 51 157 231 283 41 71 263 283 11
. . . .
. . . .
. . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . .
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PREFACE
Volume 19 of Annual Reports in Medicinal Chemistry contains 30 chapters organized in the traditional format of previous volumes-in sections titled CNS Agents, Pharmacodynamic Agents, Chemotherapeutic Agents, Metabolic Diseases and Endocrine Function, Topics in Biology, and Topics in Chemistry and Drug Design. In addition, this year the editors experiment with a new section that summarizes worldwide first market introductions of therapeutic agents during the previous calendar year. In the first six sections are included a mix of literature updates on a broad sweep of drug research areas, as well as reviews of highly specialized and newly emerging technologies. Topics covered for the first time include CNS autoreceptors, interleukin 2, endogenous natriuretic factors, and enzymic methods in organic synthesis. Phospholipases and collagenases, touched on in earlier Annual Reports, receive in-depth coverage in separate chapters in this volume. Reviews on recombinant DNA technology and the biology of leukotrienes bring the reader up to date in these highly important and active areas. In the new section, Worldwide Market Introductions, the chapter “To Market, to Market” presents an aspect lacking in the previous 18 volumes of Annual Reports-an organized follow-up on market introduction, the ultimate endpoint of the many agents reviewed. The accumulation of data for this chapter proved formidable and while a number of sources were used, it is inevitable that we missed some drugs. We hope we have not made a major omission! Finally, I want to extend my gratitude to the section editors and contributors whose time and effort made Volume 19 possible, and in particular to Martha Johnson, whose assistance was invaluable in preparing the final copy. Denis M . Bailey Rensselaer, New York M a y I984
...
XI11
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Section I
-
CNS Agents
Editor: Barrie Hesp, Stuart Pharmaceuticals, Division of ICI Americas, Wilmington, DE 19897 Chapter 1.
Analgesics
0. William Lever, Jr., Kwen-Jen Chang and John D. McDermed Wellcome Research Laboratories, Burroughs Wellcome Co. Research Triangle Park, NC 27709 Efforts toward alleviation of pain continue at an intensive pace at the clinical, pre-clinical and basic research levels. In this review, recent research developments in opioid and non-opioid analgesia are presented, along with selected clinical highlights. Opioid Receptors and Ligands Endogenous opioids and their multiple receptors continue as the focus of vigorous research. The proceedings of the 1983 International Narcotic Research Conferenc were published,’ and reviews were recently p o ided on opioid peptides,2,$ opioid receptors and their functional roles, macology and therapeutic implications of enke halins and endorphins,Shycardiovascular effects of endogenous opioids, opioids and the adrenalpituit ry axis, opioids and the hippocampus,l2 substance in nociception,’$ potential therapeutic roles for opioid antagonists’‘, roles of opioid pept‘des in appetite control,l 5 and autoradiography of opioid receptors. 16
6-8
’’
Biosvnthesis and Metabolism of Opioid Peptideg - Opioid peptide biosynzhesis was summariz in our previous review nd additional overviews of the biosynthesis”-22 and distribution2o 9 7 2%25 of endogeneous opioids have appeared. Several inhibitors of enkephalin convertase, a carboxypeptidase which may liberate enkephalins from immediate precursors, Degradation of enkephalins by peptidases continues were described 2 6 9 2 7 to be s t ~ d i e d , ~ ~ , ~ the ~ analgesic properties of inhibitors of as28z54 Thiorphan, N-[(R,S)-3-mercapto-2-benzylproenkephalin metabolism. panoyllglycine, is an enkephalinase inhibitor (Ki = 3.5 nM) which also inhibits (Ki = 140 nM) angiotensin-converting enzyme (ACE).32 By contrast, the retro-inuerso isomer, retro-thiorphan [(R,S)-HS-CH2-CH(CH2Ph)- N H CO-CH2C02H], does not inhibit ACE but retains potent enkephalinase inhibition (Ki = 6 nM) and has an inuiuo analgesic potencs2(i.c.v.; mouse hot plate, mouse writhing) similar to that of thiorphan. Thiorphan was reported to reduce postmyelographic side effects (headache, nausea, sciatica) when administered (i.v.) to patients prior to m y e l ~ g r a p h y . ~ ~ Oral administration of D-phenylalanine, a carboxypeptidase inhibitor, was found to be of benefit in patients with certain chronic ain complaints; the drug was most effective after 3-5 weeks of therapy.38
’’
Opioid Receptors and Pharmacology - The previously defined opioid receptor sub-types ( P , ~ , K , E , U ) continue to be studied, and an additional binding site, designated the A site, was recently described in rat brain.35 The A
ANNUAL REPORTS IN MEDICINAL CHEMISTRY-I9
Copyright 0 1984 by Academic Press, Inc. All nghts of reproduction in any form reserved. ISBN 0-12-040519-9
-2
Sect. I
- CNS Agents
Hesp, Ed.
sites have a regional distribution in brain different from that of p receptors and have a high affinity for 4,5-epoxymorphinans (u., morphine, naloxone) and low affinity for non-epoxymorph‘nan p ligands, K ligands, 6 ligands, and “universal” ( p , 6 , ~ )ligands.j5 Evidence that u receptors may differ from phencyclidine receptors was provided by the report of a naloxone-inaccessible u receptor in rat brain and spinal cord which stereoselectively binds (+)-ethylketocyclazocine and (+)-N-allylnormetazocine [(+)-SKF 10,0471 and has drug selectivity and regiona13$istribution different from those of p, 6 and phencyclidine receptors. Additional studies on u agon t and on their phencyclidine-like behavioral effects were reported.jf-81The benzomorphan Mr 2034 [ ( - ) - N (2-tetrahydrofurfuryl)normetazocinel, originally proposed as a K liga d is more accurately described as a universal ( p , 6 , ~ , 0 ) opioid ligand.42 The nature of opioid binding continues to be e ~ p l o r e d . ~ ~In ’ ~rat ~ brain and spinal cord, p a@jl 6 sites predominate, and K sites represent Both 6 and p receptors mediate antinocionly 10% of total binding. pro il s of i.~and K agonists were ~ e p t i o n . ~ The ~ - ~antinociceptive ~ reported to be distinguishable inuiuo, 55,5‘ and the affinities of K agonists for p and 6 receptors invivo in the rat were assessed.55 Respiratory depression is caused by opioid agonists but may be mediat b a receptor population different from that producing antinociception.5‘-521 Evidence suggesting th t the p sites in mouse vas deferens and guinea pig ileum are different,60,g1 and support for the possibility that opio agonists and antagonists interact with different sites on )1 receptors? were also presented. The antitussive effects of opioids appear to arise from interaction with pharmacological receptors which are less st eoselective and less sensitive to naloxone than the analgesic c tors.g5 Attempts Additional data to purify opioid receptors have progressed slowly. were cited for the coexistence of allosterically coupled morphine an enkephalin receptors within an opioid receptor complex in rat brain.26,67
sQ*gFs
-
Newer Endonenous ODioid Peptides The number of endogenous opioid peptides continues to grow rapidly. Several fragments of proenkephalin A have received recent attention. A u-selective octapeptide fragment, Gly-Gly-Phe-Met-Arg-Arg-Val-NH2, has been isolated from bovine brain6”;nd from h y g n pheochromo~ytoma~~ and has been designated both as metorE, a 25-amino acid fragment, phamide and as a d r e n ~ r p h i n . ~Peptide ~ binds preferentially to 1.1 and K receptors but has minimal analgesic tail flick) in potency (i.c.v., The C-terminal heptapeptide fragmen mous: Met -enke h lin-Argg-Phe7, has e n found in human and rabbit pl sma,72, rat brain 75-iJ5and spinal c~rd,~‘,?~ and lung of several species.78 A unique high affinity binding site was described in rat lung membranes.78 The role of this peptide as a Met-enkephalin pr cursor or a transmitter is speculative. The fragment Met5-enkephalin-Arg&-Gly7-Leu8 is a p- and &-selective ligand which binds poorly to K receptors; synthetic extension to incorporate a Lys9 residue improved K affinity 10-fold but did not alter p or 6 binding or the p,6-~electivity.~~
-
Svnthetic ODioid PeDtide Analonues Structure-activity studies on analogues of the exceptionally selective ligand morphiceptin (TyrPro-Phe-Pro-NH2) were reported.g’ One of the most potent analgesics was PL017 ( Tyr-Pro-NMePhe-D-Pro-NH2). 6o A peptide containing the PL017 sequence at the N-terminus and the dyn-A -17) sequence at the C-terminus had high affinity for both p and K s i t e s 3 Analogues of 8-endorphin containing the dynorphin (5-13) or (6-13) sequences showed excitatory behavioral effects in the mouse t had no analgesic properties in the By contrast, a 8-endorphin analogue mouse tail flick assay (i.c.v. 1 with a dermorphin sequence (Tyr-D-Ala-Phe-Gly-Tyr-Pro-Ser) in residues
.”
Chap. 1
Analgesics
Lever, Chang, McDermed
3
(1-7)wag2several-fold more potent than B-endorphin (mouse tail flick, A number of additional studies on the dermorphins, potent i.c.v.). Tyr-D-Ala-Phe-Gly-Tyr-Pro-Ser-NH2) originally opioid heptapeptides (u., isolated from the skin o S th American frogs, and their synthetic analogues were reported.83-gH A heptapeptide is not required for high activity. The dermorphin (1-4) analogue H2N-C(NH)-Tyr-D-Ala-Phe-Gly-NHadamantyl, which contains a guanidino N-terminus and an N-adamantyl amide moiety, is a powerful p-agonist (mouse tail flick, i.c.v.) with potency 30,000 and 1000 times that of Met-enkephalin and morphine, r e ~ p e c t i v e l y . ~ ~ New &-selective li ands have emerged, including deltakephalin (TyrD-Thr-Gly-Phe-Leu-Thr),8g certain enkephalin dimers,l7vgo and cyclic enkephalin analogues containing disulfide-linked penicillamine and cystine residues.91 Possibly the most highly 6-selective ligands reported to date are the bis-penicillamine 14-membered cyclic disulfide-containing en~ kephalin analogues DPDPE (la) and DPLPE ( & ) , which gave 6 4 selectivity By contrast, ratios in isolated tissues of 3164 and 1088, respectively. the 14-membered cyclic enkephalin analogue 2, as well as a number of partial retro-inuerso modifications of this cyclic structure, are p-selective ligands 93 Other approaches to conformational restriction were reported 941 95 as were enkephalin analogues incorggrating dehydroamino cyclopropyl-containing amino acids, y g 9 p-nitro substitution in Phe4,100 ketomethylene replacement of the T r-Gly and Gly-Gly amide linka e lo' modifications at the C-terminus,182 S-oxidized m t io nine,'Oj'and phosphonate analogues of the C-terminal residues.7ob -
acid^,^^,^^
!Q
R1 = C02H, R 2 = H
lb
R1 = H, R 2 = C 0 2 H (DPLPE)
(DPDPE)
T y r -D -Ala-G Iy -P he- Leu - NH-C H, C H,-X
QN3
3 X=NH X = NHCO(CH2)2NH
4
NO z
New Ligands, SAR, Structural Studies - Irreversible opioid ligands are of interest as receptor probes. The photolabile enkephalin d r'vatives 3 and and the 2, lo5 the chloromethyl ketone Tyr-D-Ala-Gly-Phe-Leu-CH$l, fentanyl isothiocyanate 5Io7 all appear to label 6 receptors selectively, whereas the etonitazene isothiocyanate 61°7 appears to inactivate only p receptors. A variety of epoxymorphinans with alkylatin groups attached Both B-CNA (1) through amino groups at C-6 are opioid affinity labels.'ol and a-CNA (8) produce irreversible antagonism at p, K and 6 receptors, and a-CNA appears to have an additional irreversible agonist property in the guinea pig ileum assay.lo9 Further studies in the ileum with B-FNA ( 9 ) and B-FOA (lo) led to the suggestigs that 1.1 agonists and antagonists interact with distinct receptor sites. Comparisons of isolated tissue data for various epimeric antagonist affinity labels indicated that the chiralit at C-6 is an important determinant of receptor alkylating The configuration at C-6 was shown to determine the ring C ability.'lo solution conformations of the oxymorphamines: rinh C is a chair in the B-epimer 11 and a twist boat in the a-epimer 12.
4 -
Sect. I
- CNS Agents
Hesp, Ed.
7 -
R1= N(CH2CH2C11 R2=H, R 3 = C H 2 d 2‘
(P-CNA)
8
R’= H, R2= N(CH2CH2C1)2, R3= C H 2 U
(a-CNA)
9
R1= NHCOCH= CHC02Me, R2=H, R3= C H 2 - a
10 R 1 = NHCOCH= CHC02Me, R2: 11 R1= NH2, R2= H, R3= Me 12 R1= HI R2= NH2, R3=Me
H, R3= Me
(P-FNA)
( 0 ~ 0 ~ )
Of four pentazocine analogues in which the N-alkyl group was folded was the most potent ( . 5 X back into 6- or 7-membered riyj$,lFgmpound pentazocine, mouse writhing). . Conjugate addition of alkyl groups to the 8-position of codeinone gave a series of analgesics in which the 8 - a - e p i m ~were ~ ~ generally more potent (mouse tail flick) than the 8-BSyntheses of two isomeric groups of thiazolomorphans were epimers. reportjTd o which 14 was the most potent (10 X codeine, mouse writhing) 5:11g Compound 14 is a 9-B-epimer and is somewhat more potent than the 9-a-epimer, in contrast with precedents from the benzomorphans,
a
.
u
14
15
There wer r rts of further modifications of ring C of morphine and From a large series of active compounds oxet n codeine . 1’i7-ci88 was about 700 X more potent (mouse writhing, s.c.) than morphine. Translocation of the hydroxyl group from the 4- to the 1-position destroyed activity in N-meth lmorphinan-6-ones.120 S ntheses of r’ng-C-contracted 6,14-exo-ethen m rphinans,12’ 2‘-thiobenzomorphans,12’ morphinanones,82j and more exotic heterocyclic analogs12‘ were described without indications of biological activity. A series of fentanylenkephalin hybrids were essentially inactive in isolated tissue assays. 125
?18
The roles of hydrophobic and hydrophilic (amphiphilic) domains of the &endorphin surface in relation to ph s’cochemical,biochemical, analgesic and binding activities were explored. ‘2‘,127 Studies on four large synthetic peptides designed to simulate or modify the surface properties of &endorphin led to the conclusion that exact residue homology may be less important to bioactivity than the gross physicochemical properties of the binding surface. In a complementary approach, a 40-residue peptide designed to mimic the binding site of opioid receptors was synthesized. 128 It displayed modest but significant affinity for several opioid peptides and discriminated them from non-opioids. (Leu-enkephalin, KD = 5.8 x
Chap.
Analgesics
1
Lever, Chang, McDermed
2
Non-Opioid Analgesia Direct modulation of opioid pathways has been thetmajor focus of modern analgesic research, but other modalities for antinociception appear to involve such pathways only indirectly or not at all. Stress, of which traditional Chinese acupuncture and modern electroacupuncture may be considered special cases, is one such mode. Early reports of naloxone blockade of acupuncture analgesia suggested mediation by endogenous opioids.129 However, more recent studies contradict this hypothesis. 130 A superficially related clinical procedure for control of chronic pain is electrical transcutaneous or spinal cord nerve stimulation. A doubieblind placebo-control d study indicated that such analgesia is not The neurophysiological mechanisms mediating naloxone-reversible. stress-induced autoanalgesia complex and depend subtly on the paraElectrical stress-induced analge meters of the applied stress. tg3 animals apparently involves both opioid and non-opioid substrates. Front foot shocks in rats produced naloxone-blocked morphine-tolerant analgesia (tail flick) unaffected by h pophysectomy, and therefore describable as opioid, non-hormonal.13y However, similar shocks to the hind feet gave analgesia unaffected by naloxone, tolerance, or morphtS3 Moreover, the hypophysectomy and therefore non-opioid, non-hormonal. footshock regimen influenced the nature of antinociception: prolonged intermittent shocks produced opioid-dependent analgesia, whereas br'ef continuous shocks produced non-opioid analgesia (rat tail flick). 134 esions of the raphe nucleus attenuated the latter but not the ~ ~ ~ ~ though ~ ~ other ~ ' studies i 3 h have i licated the raphe nucleus in mediation of both types of analgesia. 1%
'3'
's5
79,
GABA-ergic mechanisms have been implicated in antinociception. In normal volunteers the GABA agonist THIP (16)produced analgesia against dental pain without tolerance, atory depression, or other characteristics of opiate therapy. resg3S In an open study of cancer patients with chronic pain refractory to mild analgesics, THIP (20 mg i.m.) gave subjective pain control, but side effects (blurred vision, sedation) were dose-limiting.137 The GABA;ergic benzodiazepine midazolam (IJ) given intrathecally to anaesthetized dogs strongly att n ated nociceptive sympathetic reflexes to electrical nerve stimulation,'38 Baclofen and other GABA agonists have a so en found to be central antinociceptives in exThese effects were generally naloxone-in perimental animals. sitive, though indi ct effects o opioid substrates may be present.785GABA analgesia may"? o r may not144 be cross-tolerant to morphine.
139-1''
*N \
16
17
HO
18 -
Intrathecal administration of the -agonist clonidine attenuated nociceptive responses in rats and cats.9L45 Attempts to characterize further the receptors involved as a l or a2 gave inconclusive results.146 Studies with monoamine depletors indicated that intact spinal adrenergic and serotonergic fibers are 'nvolved in a complex way in expression of morphine analgesia in rats. 147 Adrenergic systems may mediate foot shock analgesia (rats, tail flick).148 A study in terminal cancer patients
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Sect. I
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Hesp, Ed.
indicated effective control of chronic pain by epidural labetalol, a mixed a- and 8-antagonist though the result might have been due to a local anaesthetic effect. Several studies indicate that opioids acti ate erotonergic neurons and increase 5-HT turnover in the spinal cord. 130-155 Intrathecal 5-HT produced antinociception in mice (tail flick).153 The role of serotonergic neurons in opioid analgesia may be modek-dependent.152. Central cholinergic fib rs may also mediate analgesia,l5 and both m ~ s c a r i n i c l ~ ~ and nicotinicl5' agonists produce analgesia in rodents by central mechanisms. There is evidence that cholinergic connections m d'ate the expression of both endogenous and exogenous opioid analgesia.754,157 Meptazinol is one drug in clinical use (videinfra) which appears to take fortuitous advantage of the opioid-cholinergic connection. In rodent antinociceptive models it displays ph r acology appropriate for an opiate partial agonist and a cholinomimetic.?58 The analgesic properties of cannabinoid derivatives are well known, though no such drugs have yet been demonstrated to be substantially free from the CNS side eff ct to which cannabissativa owes its popularity as a substance of abuse. 15% 7 Igo However, cannabinoid analgesia has appeal due t failure to induce to its opioi independent mechanism and its con dependence.1 g i . A series of preliminary reports'ig'"985 have described the syntheses of analgesics modelled on 9-nor-9-8-hydroxyhexa drocannabinol; compound 18 was the most potent member (>60 X morphine). %1 Clinical Hinhliahts ODioid Analgesics - Clinical evaluation of the newer opioi agonistantagonist ugs continues, and theirl@algesic efficacy,lg6 phar aand dependence profileslgl9 were cokinetics,%7 receptor pharmacology, recently revi w d. A review on the pharmacology and therapeutic efficacy of nalbuphin 778 and a compilation of clinical experiences with injectable meptazinol'71 were ovided. Human pharmacokinetic data for meptazi 01 were reported. 1729 lSi5 Efficacy studies with epidura pentazocine,174 epidural buprenorphine,175 and parenteral d e ~ o c i n e 'for ~ ~ postoperative pain relief were reported. Oral cirayadol was superior to codein relief of advanced cancer ain. 177 The clinical pharmacokinetics77p;nd applications in anesthesiaq7g of fentanyl and its derivatives were a1 additional reports on enkephalin analogues reviewed. 8 ' e % ' For relief of severe episiotomy pain, metkephamid appeared. 1 acetate (140 mg, i.m.) was superior to mepiridine (100 mg, i.m.) but minor side effects wer more frequent; a lower dose (70 mg, i.m.) of metkephamid was ineffegbive.'80 The preclinical pharmacology of metkephamid was Intrathecal administration of D-Ala2-D-Leu5-enkephalin described. (DADL) to a single patient showed that DADL could provide a powerful, long-lasting analgesic effect, and also is ?@able of producing profound, naloxone-reversible respiratory depression.
'
- Therapeutic appliNon-Ster%dal Antiinflammatory Drugs (NSA ' O f clinical pharmacokineticsq and mechanism of action189 cations, of the peri herally-acting N AIDS were reviewed, and the pharmacology of and suprofen19s were discussed in detail. Ibuprofen diflunisal (400 mg, p.0.) was more effective than z mepirac (100 mg, p . 0 . ) or aspirin (600 mg, p.0.) against episiotomy pain,182 and rectal indomethacin (100 mglgfight hourly) provided effective analgesia following thoraA dose-r sponse study with i.v. indoprofen for postoperative cotomy pain was reported,19f and in women with moderate to severe cancer pain, indoprofen (400mg, i.v.) appeared equivalent to morphine (10 mg,
-
.
Chap. 1
Analgesics
Lever, Chang, McDermed
1
i.m.). lg5 Additional studies with diflunisal,l g 6 9 l g 7 flurbiprofen,lg8 fendosal, Ig9 and diclofenac2” were reported. Therapeutic applications of 2@ were rsJ&ewed.201 f202 In preclinical NSAIDs in primary dysmenorrh were described as effective studies, propionic acids and 3 NSAIDs with reduced ulcerogenic properties.
Migraine - Headache is the most common pain syndrome for which analgesic medication is applied and is associated with a large number of disorders with different etiologies. Comments here will be restricted to vascular headache, particularly migraine, which is probably a symptom of a reactive compensatory mechanism occurring late in a poorly understoojofathogenetic A classisequence. A provocative review of this topic is available. cal migraine attack begins with a prodromal phase which i vol es regional cerebral vasoconstriction leading to hypoxi ischemia,209-208 and possibly reduced neuronal 5-HT synthesis.208’ Onset of the painful headache phase is marked by vasodilation,206 which may reflect a defective vascular homeostatic mechanism.209 Partly because of a dearth of animal models, treatment has evolved empirically from clinical experience and has traditionally consisted of symptom management.210,21 Most drug treatment in the past focused acutely on the painful vasodilation phase. The efficacy of dihydroergotamine212 apparently results from its vasoconstrictor effects. NSAIDs inhibit prostacyclin biosynthes’s a d reduce the intensity, but not the frequency, of attacks.213,214 Newer therapies have addressed prevention of attacks. The prophylactic effects of B-blockers have been established, though their mechanism of action remains speculative. In a long-term placebocontrolled study of propranolol in 245 migraineurs, more than 70% of the subjects had fewer and less severe attacks, and 46% reported th t their improvement was maintained after discontinuation of medication.$15 Nadolol was shown to similarly prophylactic, whereas pindolol and alprenolol were not.28g Calcium channel blockers are the most recent additions to migraine prophylaxis. Clinical studies with flunarizine, a Ca++ blocker with anti-vasoconstrictive and anti-hypoxic effects in animals, indicate that it is clearly ophylactic, though its effect on severity of attacks is marginal.217,28‘ Small clinical studies with nimodipine, nifedipine, and verapamil indicate efficacy for these also.219 Nimodipine appears to produce the fewest side effects, probably because of its high selectivity for cerebral vasculature. These agents appear to prevent migraine attacks by attenuating Ca++-dependent cerebrai vasoconstriction irrespective of the nature of the triggering constrictive stimulus.220’ References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13.
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-8 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38. 39. 40. 41. 42. 43. 44. 45. 46. 47. 48. 49. 50. 51. 52. 53. 54. 55. 56. 57. 58. 59: 60. 61. 62. 63. 64. 65. 66. 67. 68. 69.
70.
71. 72. 73. 74. 75. 76. 77. 78. 79. 80. 81. 82. 83. 84. 85. 86. 87.
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CNS Agents
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s,
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G.8,
a,
C.d
Chap. 88.
89 90. 91.
1
Analgesics
Lever, C h a n g , McDermed
2
E.C. Degli-Uberti, G. Trasforini. S. Salvadori, R. Tomatis, A. Margutti, M. Bianconi, C. Rotola and R. Pansini, J Clin. Endocrinol. Metab. 56 1032 (1983). J.-M. Zajac, G. G a d , F. k&t, P. Dodey, P. Rossignol and B P. Roques, Biochem. Biophys. Res. Commun., 111, 390 (1983). D. Rodbard, T. Costa, Y Shimohigashi and S. Krumins, J. Rece t Res 3 21 (1983). H.I. Mosberg, R. Hurst, V.J. Hruby, J.J.Galligan, T.F. Burks, 8 . G e e H’dH.I. Yamamura, Life Sci., 32,2565 1l l1 l 0 ~ 9 ) ” ” , .
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z,
10 157. 158. 159. 160. 161. 162. 163. 164. 165. 166. 167. 168. 169. 170. 171. 172. 173. 174.
175. 176. 177. 178. 179. 180. 181. 182. 183. 184. 185. 186. 187. 188.
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Sect. I
- CNS Agents
Hesp, Ed.
J.W. Lewis, J.T. Cannon and J.C. Liebeskind, Brain Res., 270,289 (1983). D.J. Bill, J.E. Hartley, R.J. Ste hensand A.M. Thompson,=. J. Pharmacol., 79,191 (1983). R.K. Razdan and J.F. Howes, d e d . Res. Rev., 2,119(1983). L. Lemberger, Ann. Rev. Pharmacol. Toxicol., 20,151 (1980). L.S. Harris, in "The Therapeutic Potential of X r i h u a n a , " S. Cohen and R.C. Stillman, eds., Plenum, New York, 1976, p. 299. L. Melvin, M. Johnson and G. Milne, Am. Chem. Soc. 186th Nat. Mt Abstr , Medi. 22,1983. M. Johnson, J. Eggler, L. Melvin and G. Milne, Am. Chem. Soc. 186% Nat. Gtg. Abstr., Medi. 1, 1983. L. Melvin, M.Johnson and G. Milne, Am. Chem. Soc. 186th Nat. Ytg. Abstr., Medi. 2,1983. L. Melvin, M. Johnson, C. Harbert, G . Milne and A. Weissman, J. Med. Chem., 2'7,67 (1984). E.M. Zola and D.C. McLeod, Drug Intell. Clin. Pharm. 17,411 (1983). R.E.S. Bullin ham, H.J. McQuay and R.A. Moore, ClinTharmacokinet., 8,332 (1983). S.R. Hameroa, Contemp. Anesth. Pract., 27 (1983). W.L. Woolverton and C.R. Schuster, Pharmacol. Rev., %,33 (1983). J.E. Errick and R.C. Heel, Drugs, 26,191 (1983). "Clinical Experience with Injectabx Meptazinol-a New Strong Analgesic. Proceedings of a Symposium," Postgrad. Yed. J., 59 (Su pl I). pp: 1-94 (1983). G. Davies, A.J. Sinxair, 8.J;Warrington, R. Franklin, T. Frost, A. Latham and P.J. Robson, Eur. J. Clin. Pharmacol., 3 , 5 3 5 (1982). H.M. Norbury, R.A. Franklinand D.F. Graham, Eur. J. Clin. Pharrnacol., 25,77 (1983). P.K. Kalia, R. Yadan, R. Saksena, R.K. Batra and G.R. Gode, Anesth. AnaE., 62,949 (1983). J. Cahill, D. Murphy, D.OBrien, J. Mulhall and G. Fitz atrick, Anaesthesia, 3% 760 (1983). B.T. Finucane, d.B. Floyd, D.J. Petro and R. Braswell, &n. Anaesth. SOC. J. 2 r 5 0 0 (1982). J.E. Stambau h J r J McAdamsand G. Freeland, Clin. Pharmacol. Ther., 33,i98(1983). L.E. Mather, 8lik ]iiharmacokinet., 8,422 (1983). J.D. Borel, Contem Anesth. Pract.? l(1983). S.S. Bloomfield, T.I?Barden and J. M%chell, Clin. Pharmacol. Ther., 34,240 (1983). R.C. Frederickson. C.J. Parli, G.W. DeVaneand M.D. Hynes, Natl. Inst. DrugAbuse Monogr. Ser., 43,150 (1983). B.M. Onofrio and T.L. Yaksh, Lancet, I, 1386 (1983). N. Gilchrist, J.E. Bolton, R.A. Donald, J.H. Livesey, H.K. R0udandM.G. Nicholls, Br. J. Clin. P h a r m a c o l . , s , 465 (1983). A. Roby, J.-C. Willer and B. Bussel, Neuropharmacol., 22,1121 (1983). G. Nuki, Brit. Med. J., 287,39 (1983). S.A. Cooper, Ann. Rev.xarmacol. Toxicol., 23,617 (1983). H.L. Crossley, S.A. Bergman and R.L. WynnJ. Am. Dent. Assoc. 106 61 (1983). R.K. Verbeeck, J.L. Blackburn and G.R. Loewen, Clin. PharmacokG'l., 8,297 (1983). R.J. Capetola, Y.E. Rosenthale, B. Dubinsky and J.L. McGuire, J. Clin. Pharmacol., 23,545 (19831. R.O. Davies, Pharmacother., 9(2, pt. 2). 9 s (1983). Pharmacol.. 27 (Su 1 ), p 1 96 (1983). uns ine,x.Z. 8Eon, b M : Laska, I. Zighelboim, A. decastroand C. desarrarin, Clin. Pharmacol. Ther., 34, :5:(198h). D.J.M. Keenan, K. Cave, L. Langdon and R.E. Lea, Brit. Wed. J., 287,1335 (1983). G. Rigamonti, E. Zanella, R. Lampugnani, D.Marrano, 0.Campione, 0.Bruni, V. Mandelli and G. Sacchetti, Br. J. Anaesth., 55,513 (1983). A. Pellegrini, B.Wassidda, P. Pelle ini, E. Defraia, A. Usai, L. Parrinello, G. Bruni, G. Rigamonti and C. Sacchetti, Int. J. Clin. Pharmacol. &er. Toxicol., 21,483 (1983). E.R. Umbenhauer, Pharmacother., 2,55S (1983). J.A. Forbes, A.L. Kolodny, W.T. Beaver, R.W. Shackelford and V.R. Scarlett, Pharmacother., 2,47S (1983). A. Sunshine, N.Z. Olson, E.M. Laska, I. Zighelboim, A. DeCastro and C. DeSarrazin, Pharmacother., 2,177 (1983). P.J. Desjardins, S.A. Cooper, C.M. Ruderman, L.T. Gallegos, D.C. Reynolds andG.0. Kruger, Pharmacother., 3, 52 (1983). E.C. Huskisson and R.Bryans, Curr. Med. Res. Opinion 8 350 (1983). N.J. Wenzloff and L. Shimp, Dru Intell Clin. Pharm., i6'2Z (1984). W.Y. Chan, Ann. Rev. Pharmacof. Toxicol., 23,131 (1983. A. Marzo, G. Quadro, G. Bruno, E. Treffner.F'L. Reali, G. Borrelli and R. Stradi, Arzneim.-Forsch.. 3 ( 1 ) , 198 (1983). K. Hino H. Nakamura, Y.Nagai H. Uno and H. Nishimura, J. Yed. Chem., 222 (1983). J.R. Gra'ham, Headache, 21,243 (1981). E. Skinhoj, Arch. Neurol.79,95 (1973). 83 (1982). W.K. Arner Cephalalgia M. Botney, keadache, i7b (1981). R.J. McCaffrey and W. Isaac, Headache, 23,152 (1983). R. Peatfield, Dru s 26 364 (1983). J.E. Sutherland, !m?am. Physician, 27,137 (1983). N. Martucci, V. Manna, P. Mattesi, G. n o i a n i , G . Manzoni, M. Lanfranchi, G . Bono and G . Micieli, Cephalalgia, 3(Su pl I), 151 (1983). B.H.$eters, C.J. Fraimand B.E. Masel, Am. J. Wed., 74,36(1983). R.C. Peatfield, R.G. Petty and F.C. Rose, Ce halalgia 3 129 (1983). S. Diamiond, L. Kudrow. J. Stevens and D.{hapiro. HGdache, 22 268 (1982). R.E. Ryan, Sr., R.E. Ryan, Jr., and A. Sudilovsky, Headache, &$6 (1983). P. Louis, Headache, 21.235 (1981). W.K. Amery, Headaxe, 23,70(1983). J.S. Meyer and J. HardenGrg, Headache, 23,266 (1983). S.J. Peroutka, Headache, 23,278 (1983).
z,
z,
a,
Chapter 2 .
Anti-Anxiety Agents, Anticonvulsants & Sedative-Hypnotics
Joseph P. Yevich, James S. New and Michael S. Eison Bristol-Myers Research and Development, Evansville, Indiana 47721
-
Introduction The prevalence of anxiety and the diverse utility of existing anxiolytic drugs continues to promote a broad spectrum of research in the anxiolytic area. The challenging basic science of anxiolytic drug action and the potential for compounds with profiles different from the traditional antianxiety drugs has focused attention upon more selective structure activity relationships. Several excellent review articles emphasizing medications currently used, approaches to new drug discovery, newer agents and theories, side-effects and hazards 4-8 and the use of benzodiazepines (BZ) as preoperative medications’ have been published. Insights into the GABAergic effects of these drugs and their anticonvulsant properties continue to grow. Animal Models - Conflict tests play a prominent role in anxiolytic research, and may be used to demonstrate proconflict, anticonflict, and anxiolytic anta onist actions.l5 A chronic conflict paradigm has been described. The effects of drugs upon deprivation-induced drinking should be considered to avoid false-positive indications.l 7 The susceptability of compounds’ anticonflict effects to blockade by BZ antagonists appears to distinguish BZ receptor binders from nondisplacers. The use of anticonvulsant activity and effects upon BZ binding as indices to dissociate pro-drug from directly acting BZ effects has been described,lg as has a chronic epilepsy model in rats.2 o Drug-discrimination techni ues have revealed that the diazepam stimulus complex is specific.” It has been proposed that drug discrimination techniques can detect anxiogenic BZ withdrawal effects.22 923 In other behavioral tests, the increase in response rates induced by BZ’s to rewarding electrical brain stimulation i s facilitated by GABA antagonist^,^^ and is observed even in rats trained on a DRL schedule (rewarding low rates of response).25’26 Conditioned startle,27 defensive burying,28 and social intera c t i o n ~ ~continue ~ ’ ~ ~ to be used to investigate anxiolytic and BZ anta onist activity. Quantitative EEG activity in animals31 and man3! complement cellular electrophysiological investigations of anxiol tic drug Effects of BZs upon memory,35 and electrically-induced head turning37 have been investisleep gated, as have effects upon feeding and d r i n I ~ i n g . ~ ~ -A~ protective O action of diazepam against gastric ulceration in rats induced by water-immersion or by immobilization stress has been described.41 Tolerance developing to anxiolytic drugs ,42 ’ 4 3 and models of physical dependence to BZs in rat44 and dog45 have been reported.
,I6
Benzodiazepine Receptor Neurochemistry and Pharmacology - The concept of heterogeneous benzodiazepine (BZ) receptor sites has stimulated considerable research in the development of drugs which may be more anxioselective than the BZ class. The functional linking of a
ANNUAL REPORTS IN MEDICINAL CHEMISTRY- 19
Copyright 0 1984 by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0-12-0405 19-9
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chloride ionophore, BZ and GABA receptor sites is substantiated by their isolation in a supramolecular complex in which GABA stimulation of [ 3H]flunitrazepam binding (FLU, &) remains intact.4 6 Affinity chromatography has also led to the successful isolation of a homogeneous GABA/BZ receptor complex from bovine cerebral cortex.4 7 Data continues to accrue indicating GABA, and its agonists, enhance the binding of the BZs to these receptors; diazepam (DZ, ) & l enhances ~O the binding of GABA and muscimol but has no effect on T € I I P . ~ ~ - The presence of a single class of saturable [3H] muscimol binding sites (KD = 23 nM) on a purified BZ receptor is evident.51 The potentiation of [ 3H]DZ binding is also elicited by ethylenediamine analogues in a process mediated predominantly by a change in receptor number and not receptor affinitye5* Both [3H]DZ and [3H]GABA binding are enhanced by the hypnotics (+)etomidate and pentobarbital, presumabl through a similar mechanism, which is blocked by GABA antagonists. 545 GABAergic ligands were also observed to protect soluble BZ receptors from heat inactivation.54 BZ Type 1 and Type 2 receptors, initially defined on the basis of their different affinities for CL 218,872, have been further characterized in rat cerebellum and cerebral cortex. CL 218,872 preferentially displaced [3H]FLU from Type 1 receptors; Type 1 R' R' R' Et'Y receptor binding is H F H O la NO. CHI stimulated in a Y lb CI CHI H H H O chloride H F H S Ra & CI CHzCFa manner byion ImMdependent sodium Id CI CHI H H C I O pentobarbital, while H H O le CI CHzCCFa H OH CI H 0 Type 2 sites remain If CI H relatively ~naffected.'~~ 1g CI H COOH H H 0 Distinction between l h CI (CH~)ZN(CZHI)Z H F H 0 R OH H H O 11 CI CHI Type 1 and Type 2 -1 H H H O 1j NO' H receptor populations is reported to be possible through their differential sensitivity to detergent solubilization techniques.56 The effects of GABA agonists on [3H]FLU binding to these sites in both cerebellar and hi pocampal membranes were found to be broadly similar in one study.5q Alternatively, the effect of GABA on FLU binding to [3H]FLU and [3H]f3-CCP (2a) labelled Type 1 and 2 receptors was found to involve different degrees of GABA regulation dependent on the specific brain region.58 SAR studies have examined the f3-carbolines as templates in the desi n of more specific, high affinity ligands for the BZ recep[3H)DMCM (b)interacts with high affinity BZ receptors with binding properties different. from those of [ 3H]FLU and $-CCP which may reflect a subclass selectivity within these receptors Intraventricular injections of $-CCE (g)increased the number of [3H]DZ binding sites in a variety of brain regions but, in a different study, decreased the total number of [3H]GABA binding sites in rats habituated to handling procedure^.^*'^^ f3-CCM (&) has been shown to antagonize the anxiolytic effects of the BZs, possess convulsant properties, and exhibit anxiogenic properties.6 4 [ 3H]$-CCE binds to a homogenous population of recognition sites in rat brain and is completely dissociable by low concentrations of B Z S . ~ The ~ $-carboline FG 7142 (2f) was found to induce severe anxiety in man.66 Pharmacokinetic factors were studied as a para-
Chap. 2
Anti-Anxiety Agents
Yevich, New, Eison
meter controlling the convulsant activity of fi-CCM, DMCM, and the n~nconvulsantproperties of fi-CCE in the rat; a slow rate of degradation of these compounds by rat plasma vitro) paralleled their potencies as convulsants.67
(s
BZ receptor heterogeneity has been further delineated from the differential affinities observed between pairs of ligands for these sites. Subpopulations of [3H]DZ receptor sites are defined by their interactions in bicuculline/GABA or barbiturate/pyrazolopyridine binding studies.6 8 Discrimination between these subtypes was also possible with RO 15-1788 but not with CGS 8216.68 A study of various BZ receptor ligands, with different biological activities, yielded no simple correlation between their biological activity and the observed temperature dependence of their binding.69
(z),
The non-homogeneity of BZ-receptors has generated several models which attempt to rationalize their binding characteristics with a structurally diverse group of ligands. Kinetic studies suggest these receptors exist as a single population in two distinct conformational states whose interconversion is affected by BZ agonists, GABA, chloride ion, temperature, and the BZ antagonist RO 15-1788.70 An allosteric model for BZ receptor function suggests the intrinsic activity of an agonist reflects its ability to induce a conformational change in the receptor. This approach predicts that ligands with high affinity for a binding domain on both the ground state and activated conformations o f the receptor will be anxioselective agents.71 A definition of the BZ receptor as a coupling unit between the GABA receptor and chloride channel, is used to explain its interaction with the BZ tranquilizers, the f3-carbolines,RO 15-1788, and phenobarbitone. BZ agonists enhance the coupling role of the BZ receptor while f3-carbolines block the access of BZ agonists to this coupling site. This actually results in a reduced coupling mechanism in the receptor and elicits opposing pharmacological effects. The - and f$-CCM ( g ) ,are referred to as "inverse fi-carbolines, DMCM (2b)
R'
R
'
~
c
R' H
-2
o
R
R' -
2a OnCIH, H OCHI H CZH,
R' -
H H
R'
H OCHI H 2d OCzH, H H H 2e OCzHs CHzOCH' OCHzCaHm H 2f NHCH, H H H 2'C
-
R'
H OCHI H H H H
agonists" in this scheme. This distinguishes them from pure antagonists such as RO 15-1788, which blocks the access of both inverse agonists and agonists to the receptor, without inducing a functionally relevant change in it.72 Ethanol and pentobarbital may modulate the BZ binding component in the chloride ionophore-BZ-GABA receptor complex via the picrotoxinin sensitive site, and a thirsty rat conflict model indicates the anticonflict actions of pentobarbital might be mediated through BZ receptors.73' 74 A peptidoaminobenzophenone, 450088-S($), was found to effectively inhibit the labeling of BZ CHz I
CI 0
CHI
CHI
i
13
14
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Agents
Hesp, Ed.
receptors by [3H]FLU in vivo despite a lack of in vitro activit and quazepam (2) preferentially binds to BZ Type 1 Several lines of evidence indicate RO 15-1788 may not be a strict BZ antagonist but may also have some partial agonist properties; it abolishes the sedativelataxic effects of DZ, but slows the development of kindled seizures, suggesting an anticonvulsant action in its p h a r m a ~ o l o g y . ~Ro ~ 15-1788 is effective in reversing the anticonvulsant effects of DZ and CL 218,872,64’78 and is shown to block the cognitive, amnesic, psychomotor and subjective effects of DZ in man, without altering the bioavailability of this drug.79 Psychometric tests sensitive to the effects of BZs showed no intrinsic pharmacolo ical activity associated with RO 15-1788 in man up to 600 mg orally.l0 The convulsant BZ RO 5-3663 (5) and RO 15-1788, both antagonized the anticonflict effect of chlordiazepoxide, but while RO 5-3663 was distinctly anxiogenic, RO 15-1788 displayed some anticonflict properties.81 RO 5-4864 (Id), a ligand selective for the BZ low affinity (micromolar) and peripheral receptors, registered significant anxiogenic action in the social. interaction test which was reversed by phenytoin, a ligand €or the low affinity receptors.82
-
A critical review examined the Benzodiazepine Anxiolytic Agents concept that multiple BZ receptors underlie the diverse behavioral effects which are observed with this chemical class.83 A study examining the effects of rapidly increasing doses of halazepam and DZ on severely anxious hospitalized patients was reported84 and effects of DZ on short term memory” and information processingg6 were examined. DZ was found to potentiate the behavioral effect of neuroleptics in rats,87 to produce some improvement in neuroleptic resistant schizophrenics,88 and to alleviate symptoms of tardive d y s k i n e ~ i a . ~The ~ effects of long term DZ therapy and drug withdrawal effects were also Clobazam (6) and DZ were compared in their differential effects on psychomotor performance9* and anxiolytic responses .93 Lorazepam (If)was evaluated for its effects on psychomotor performance vs. clobazams4 and DZ;s5 clear performance decrements were associated with its use. 96 Several has anxiolytic efficacy comparable studies indicate alprazolam (2) to DZ but at a lower dose and with a lower incidence of sedaHalazepam and clorazepate (la) were shown to not be significantly different from each other in treating symptoms of n anxiety or tension.99
(e)
713,R = H -
7b, R = C1
-
-a
-9
Sedative -Hypnotics Midazolam (8) continues to receive substantial support as an effective hypnoticToo with neither tolerance effects nor rebound insomnia on drug withdrawal.lol A series of papers has addressed safetylo2 and psychomotor performance aspects of this drug.lo3’l o 4 The clinical literature concerning the hypnotic effi-
Chap. 2
Anti-Anxiety Agents
Yevich, New, Eison
(s),
(a).
cacy of flurazepam temazepam and triazolam (2) has been reviewed. Triazolam has been researched as a hypnotic for geriatric patients.105-107 Considerable differences were reported for the pharmacokinetics of five BZ hypnotics in healthy subjects.lo8 There is no distinguishable difference between the nocturnal effects of triazolam or flurazepam, however, the "hangover" effects of these two drugs are still under evaluation.log' 110 Triazolam appeared superior to nitrazepam (Q) in an overall evaluation of sleep.lll The h notic loprazolam ( 9 ) was found not to be potentiated by alcohol,llYPbut no direct correlation was found between its pharmacokinetic data and various sleep parameters which were scrutinized. Initial indications suggest temazepam may have less hangover potential than nitrazepam, and both nitrazepam and flunitrazepam were well tolerated in elderly patients.'15 Non-Benzodiazepines Anxiolytic Agents - Further neurochemical and/or behavioral evaluations have been reported for 10-13a which have previously been shown to exhibit anxiolytic activity in animal models. Electrophysiological and receptor binding studies suggest that etazolate
(e)
N-N-CHIPX
R'
ClH. l o p , R' N =C(CHdr, R' lob, R' = n-CeHm, R' -
E
H =H
CHi
12~1,X = CHI, Y -
CF~
11 -
-
12b, X = NH, Y
= NH CHz
1 3 ~X . 13b, X -
= CI = H
enhances BZ binding and the effects of GABA by interaction at a site distinct from the GABA and BZ recognition sites.l16 The bicucullineand picrotoxin-sensitive blockade of electrically-induced head turning in the rat by tracazolate indicates the drug's GABAmimetic a~tivity.~? Antagonism of the DZ-induced righting reflex in mice by C1 218,872 (11) has been interpreted as evidence for its partial agonist activity at the BZ receptor.ll? The quinoline derivatives PK 8165 (12a) and PK 9084 have also been hypothesized as partial B Z X c e p t o r agonists based on their antagonism of the anticonvulsant effects of DZ and their competitive inhibition of [3H]FLU binding.'18 PK 9084 showed a partial anxiolytic profile in a social interaction paradigm in rats, while PK 8165 was ineffective at the doses tested.2 9 In a pentylenetetrazol-saline discrimination model, CGS 9896 (13a) exhibited DZ-like activity which was blocked by pretreatment with the BZ antagonist CGS 8216 (=).l"
(e)
(e)
Several clinical studies have shown buspirone (=)to have anxiolytic efficacy equivalent to that of DZ with significantly less sedation. 120-122 Rats trained to discriminate oxazepam or pentobarbital from vehicle did not generalize to buspirone.123 At doses above those which :)($(CHZ)~-G?~ are anxiolytically relevant in man, the drug caused a dose-related elevation of plasma 0 prolactin in male subjects, and like the B Z ' s 14p, R, R = -(CHZ)C also increased growth hormone levels.124 Mb, R = CHa Buspirone elicits a dose-dependent rise in rat striatal dopamine (DA) metabolite and may do so by selective antagonism of presynaptic DA autoreceptors with minimal postsynaptic effects.127 Its catalepsy-reversal effects may occur
2
16 -
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Agents
Hesp, Ed.
via a DA-independent mechanism. 128 The drug increases rat locus coeruleus noradrenergic neuronal activity,129 binds to calf hippocampus serotonin type-1 receptors,130 enhances L3H]FLU ginding after oral dosing iq+rats,131 causes an increase in the K -depolarized into synaptosomes,132 and affords significant brain uptake of 45Ca levels of 1-(2-pyrimidinyl)piperazine as a metabolite in the rat. 133 The mechanistic implications of these findings are as yet unclear. The analogue MJ 13805 (=) while equipotent with & in conflict testing, has much weaker effects than the latter upon DA neurotransmission, thus suggesting that DAergic effects may not be important to the antianxiety activity of these drugs.134 The SAR of a series of aryl and heteroarylpiperazine derivatives including & and b has been reported.135 A single-blind clinical study of the GABA agonist THIP (G) found it to have weak anxiolytic effects at doses at or near those which caused sedation and other side effects.136 Members of a series of (wpiperazinyla1koxy)indanes (16) exhibited BZ-like potency Various pyrimido[5,4-d]benzazepine in the fighting mouse test. 13' derivatives (11)showed potent antimetrazol activity and were more R'
1sp, X
= NH,Y = CHI
15b, X = CHI. Y -
i .
16 -
NH
- X = Y = CI, R' = R' 3711,
= H
17b, X = Y = CI, R' = H, R' -
*
OH
potent in displacing [3H]DZ binding than DZ, with lesser effects than DZ on motor coordination and ethanol interaction.13' A member of the series (G)has been selected for anxiolytic evaluation in man and 17b, its major metabolite (in dog and man), was found to be equipotenTwith the parent drug in pharmacological tests.
-
Sedative-Hypnotics The hypnotic and anticonvulsant profile of etomidate (Is) has been reviewed.139 Based on comparative clinical effects on the photopalpebral reflex, zopiclone (19) was slightly more potent than nitrazepam with more rapid onset, shorter duration of action, and fewer side effects.140 A study in insomniac patients showed that a 7.5 mg dose of zopiclone improved the quality of sleep with a reduction in both sleep onset latency and the number of nocturnal awakenings. Following their microiontophoretic application, the "sleep" peptides, delta sleep-inducing peptide and arginine-vasotocin caused predominantly excitatory actions on neurons in the brain stems of rats and rabbits.142 Following its microinjection into the brains of rabbits, the sites of action of the sleep-promoting muramyl peptide isolated from human urine were found to be in the forebrain region.143 While parenteral administration of an adenosine deaminase inhibitor, erythro-9-(2hydroxy-3-nonyl)adenine, to I>CO,CH.CH, gNN e N n Q mice and rats caused profound ot-N N-Cnr behavior sedation, the accom@CH, 0 panying EEG changes showed 19 18 greatly enhanced time awake.144
Chap. 2
Anti-Anxiety Agents
Yevich, New, Eison 1 7
-
Anticonvulsants The effects of several marketed antiepileptic drugs including carbamazepine, phenytoin, and sodium valproate, upon cognitive function and behavior have been clinically assessed.14' Adenosine receptor binding studies of carbamazepine indicate that its anticonvulsant action may be due in part to A1 receptor agonist and A2 receptor antagonist activities.146 A computer graphics-based patternrecognition analysis of several series of 5-ethyl-5-substituted barbiturates has revealed that the region of occupied space for the low energy conformation o f the lipophilic side-chain differs between anticonvulsant and convulsant members of the series.147 N-g-prppylnorapomorphine was the most potent of a series of apomorphine-related DA agonists in protecting genetically seizure-prone mice and baboons. 148 Potential anticonvulsant activity of a-adrenergic agonists was indicated by their in vitro inhibition of interictal discharge in rat hippocampal slices; p-adrenergic agonists showed proconvulsant actions.14y Compared with placebo, the GABAergic drug progabide (20) caused a significant decrease in seizures, with mild side effects, in a double-blind clinical trial in "therapyresistant'' epileptic patients. A single-blind evaluation of THIP in epileptic patients failed to show significant differences from placebo with maximal doses of the drug.lS1 THPO a glialselective GABA uptake inhibitor had anticonvulsant activity in chicks but not in adult mice."* Denzimol (21) was shown to have a general anticonvulsant profile similar to that of carbamazepine and phenytoin in animal models.153 The triazine, LY 81067 (22) protects mice against pentylenetetrazol- and bicuculline-induced convulsions and enhances both [3H]GABA and [3H]FLU binding.154'15s Other compounds reported to have anticonvulsant activity in various animal models include the p-carboline ZK 91296 (g) ,lS6 2-phthalimidoethanelS7 eugenol analogues (g), lS8 pyrido[3,4-b] [1,4] sulfonamides benzothiazines (25)lS9 and oxoquinazolines (26).160
'''
(e),
(c),
Q
cb
C = N(CHiJs-CONHi
D
O
H
& = ( 6" 0 +Hi
CHzCHi \ /
F
zo
21
HsC \ /
1 y ) - N D OH
N=N
Lz
X = CHorN
References 1. 2. 3. 4.
5. 6. 7. 8. 9.
R.I.Altesman and J.O.Cole, J.Clin.Psychiat., 44, 12 (1983) W.Janke and P.Netter, Neuropsychobiol., 9, 33 (1983) M.Williams, J.Med.Chem., 26, 619 (1983) J.Schopf, Pharmacopsychiat., 16, 1 (1983) D.J.Greenblatt, R.I.Shader, and D.R.Abernethy, The New England J.Med., 309, 410 (1983) T.Roehrs, F.J.Zorick, J.H.Sicklestee1, R.M.Wittig, K.M.Hartse, and T.Roth, J.Clin. Psychopharmacol., 3 , 310 (1983) A.Kales, C.R.Soldatos, E.O.Bixler, and J.D.Kales, Pharmacol., 26, 121 (1983) D.Ladewig, Pharmacopsychiat., 16, 103 (1983) R.Miller, The Mt.Sinai J.Med., 50, 289 (1983)
18 -
Sect. I
- CNS Agents
10, K.G.Lloyd, Therapie 1983, 3 8 , 355 (1983) 11. M.J.Croucher, B.S.Meldrum, and P.Krogsgaar-d-Larsen,Eur.J.Pharmacol.,
Hesp, Ed.
e,
217 (1983) 12. M.M.Airaksinen, Acta Neurol.Scand., 67,31 (1983) 13. R.G.Fariello and M.K.Ticku, Life Sci., 33, 1629 (1983) 14. A.Chapman, P.E.Keane, B.S.Meldrum, J.Simiand, and J.C.Vernieres, Prog.Neurobiol., 19, 315 (1982) 15. K G .Corda . W.D.Blaker . W.B.Mendelson. A.Guidotti. and E .Costa. Proc. Natl .Acad.Sci. USA, 80, 2072 (1983) ' 16. T.J.McCown. R.A.Voge1, and G.R.Breese, Pharmacol.Biochem.Behav.,- --18, 277 (1983) 17. J.D.Leander, Drug 6ev: Research, 2, I85 (1983) 18. J.B.Pate1, C.Martin, and J.B.Malick, Eur.J.Pharmacol., 86, 295 (1983) 19. M.Gerecke, Br.J.Clin.Pharmacol., 16, 11s (1983) 20. S.Banfi, W.Fonio, E.Allievi, and S.Raimond, Pharmacol.Res.Commun.,15, 553 (1983) 21. T.Haug, Eur.J.Pharmacol., 93, 221 (1983) 22. M.W.Emmett-Oglesby, D.G.Spender, M.Lewis,Jr., F.Elmesallamy, and H.Lal, Eur.J. Pharmacol., 9 2 , 127 (1983) 23. M.W.Emmett-Oglesby, D.G.Spender,Jr., F.Elmesallamy, and H.Lal, Life Sci., 3 3 , 161 (1983) 24. L.J.Herberg and S.F.Williams, Pharmacol.Biochem.Behav., 19, 625 (1983) 25. Y.Ichimaru, M.Moriyama, and Y.Gomita, Life Sci., 2_?, 43771983) 26. Y.Gomita, Y.Ichimaru, and M.Moriyama, Jap.J.Pharmacol., 2 , 498 (1983) 27. M.Davis, Psychopharmacol.Bull., 19, 457 (1983) 28. N.M.Blampied and R.C.Kirk, Life Sci., 33, 695 (1983) 29. S.E.File and R.G.Lister, Pharmacol.Biochem.Behav., 9, 185 (1983) 30. S.E.File and R.G.Lister, Neurosci.Letters, 3 9 , 91 (1983) 31. M.Mariotti and E.Ongini, Arch.Intl.Pharmacodyn., 264, 203 (1983) 32. Y.Mizuk, M.Hashimoto, T.Tanaka, K.Inanaga, and M.Tanaka, Psychopharmacol., 80, 311 (1983) 33. J.P.Laurent, M.Mangold, U.Humbe1, and W.Haefely, Neuropharmacol., 11. 501 (1983) 34. L.Pieri, Br.J.Clin.Pharmaco1., 16, 175 (1983) 35. M.C.Cassone, L.Molinengo, and M.Orsetti, Life Sci., 33, 1215 (1983) 36. R.Scherschlicht and J. Marias, Br.J.Clin.Pharmacol., l6, 29s (1983) 37. J.M.Goldstein, L.G.Knobloch, and J.B.Malick, Life Sci., 32, 613 (1983) 38. V.E.Grimm and A.Jancourt, Intl.J.Neurosci., 8 , 127 (198q 39. S.J.Cooper, Life Sci., 32, 1043 (1983) 40. S.J.Cooper, Life Sci., 32, 2453 (1983) 41. K.Yamaguchi, K.Suzuki, T.Niho, M.Shimora, C.Ito, and H.Ohnishi, Can.J.Physio1. Pharmacol., 9, 619 (1983) 42. H.C.Rosenberg, S.Smith, and T.H.Chiu, Life Sci., 32, 279 (1983) 43. E.H.Ellinwood,Jr., M.Linnoila, M.E.Easler, and D.W.Molter, Psychopharmacol., 79, 137 (1983) 44. G.P.Ryan and N.R.Boisse, J.Pharmacol.Exp.Ther., 226, 100 (1983) 45. L.F.McNicholas, W.R.Martin, and S.Cherian, J.Pharmacol.Exp.Ther., 226, 783 (1983) 46. S.T.Mernoff, H.M.Cherwinski, J.W.Becker, and A.L.deBlas, J.Neurochem., 41, 752 (1983) (1983) 47. E.Sigel , F.A. Stephenson, C.Mamalaki, and E.A.Barnard, J.Bio1.Chem., 258,6965 48. J.H.Skerritt and G.A.R.Johnston, Eur.J.Pharmacol., 89, 193 (1983) c 315 (1983) 49. J.H.Skerritt and G.A.R.Johnson, Neurosci.Letters, L 50. B.K.Koe, Drug Dev.Res. 2, 421 (1983) 51. C.Martini, T.Rigacci, and A.Lucacchini, J.Neurochem., 41, 1183 (1983) 52. P.F.Morgan and T.W.Stone, Br.J.Pharmacol., 2,973 (1983) 53. R.Thyagarajan, R.Ramanjaneyulu, and M.K.Ticku, J.Neurochem., 41, 578 (1983) 54. M.Gavish, Life Sci., 33, 1479 (1983) 55. D.L.Niehoff, R.D.Masha1, and H.J.Kuhar, Eur.J.Pharmacol., 92, 131 (1983) 56. M.M.S.Lo, D.L.Niehoff, M.J.Kuhar, and S.H.Snyder, Neuroscixetters, 39, 37 (1983) 57. R.Mitchel1 and L.E.Wilson, Neuropharmacol., 22, 935 (1983) 58. K.W.Gee, F.J.Ehlert, and H. I .Yamamura, J.Phazacol.Exp.Ther., 225, 132 (1983) 59. K.P.Lippke, W.G.Schunack, W.Wenning, and W.E.Miiller, J.Med.Chem., 26, 449 (1983) 60. G.Neef, U.Eden, A.Huth, D.Rahtz, R.Schmiechen, and D.Seidelmann, Heterocycles, 20, 1295 (1983) 61. C.Braestrup, M.Nielsen, and T.HonorC, J.Neurochem., 41, 454 (1983) 62. A.Concas, M.Salis, and G.Biggio, Life Sci., 32, 1175 (1983) 63. A.Concas, M.Salis, M.Serra, M.Giuseppa Corda, and G.Biggio, Eur.J.Phamacol., 9 , 179 (1983) 64. L.Prado de Carvalho, G.Grecksch, G.Chapouthier, and J.Rossier, Nature, 301, 64 (1983) 65. 1.L.Martin and A.Doble, J.Neurochem., 40, 1613 (1983) 66. R.Dorow, R.Horowski, G.Paschelke, and KAmin, Lancet, July 9, 98 (1983) 67. M.M.Schweri, J.V.Martin, W.B.Mendelson, J.E.Barrett, S.M.Pau1, and P. Skolnick, Life Sci., 3,1505 (1983) 68. L.M.F.Leeb-Lundberg and R.W.Olsen, Mol.Pharmacol., 23, 315 (1983) 69. A.Doble, J.Neurochem., 40, 1605 (1983) 70. T.H.Chiu and H.C.Rasenberg, Trends in Pharmacol. Sci., 4, 348 (1983) 71. F.J.Ehlert, W.R.Roeske, K.W.Gee, and H.I.Yamamura, Biochem.Pharmacol., 32, 2375 (1983)
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Yevich, New, E i s o n
9
72.
P.Polc, E.P.Bonetti, R.Schaffner, and W.Haefely, Nauyn.Schiedebergs Arch.Pharmacol.,
13. 74. 75. 76. 77. 78. 79.
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M.M.Singh, R.E.Becker, R.K.Pitman, H.A.Nasrallah, and H.Lal, Brain Res.Bull.,
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90. 91. 92. 93. 94. 95. 96. 97. 98.
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(1983) 109. D.Bliwise, W.Seide1, I.Karacan, M.Mitler, T.Roth, F.Zorick, and W.Dement, Sleep, 5, 156 (1983) 110. C.Gorenstein and V.Genti1, Psychopharmacol., 80, 376 (1983) 111. P.A.Ellingsen, Acta Psychiat.Scand., 67, 154 n 9 8 3 ) 112. I.C.McManus, S.I.Ankier, J.Norfolk, M.Phillips, and R.G.Priest, Br.J.Clin.Pharmacol., 16, 291 (1983) 113. ZKrieger, M.Perianu, C.Bertagna, P.Mangin, and D.Kurtz, Drug Dev.Res., 3 , 143 (1983) 114. M.Matejcek, C.Neff, K.Abt, and W.Wehrli, Neuropsychobiology, 2, 52 (19837 115. M.Viukari, P.Jaatinen, and T.Kylmamaa, Curr.Ther.Res., 33, 828 (1983) 116. D.M.Barnes, W.F.White, and M.A.Dichter, J.Neurosci., 5 , 7 6 2 , (1983) 117. K.W.Gee, R.E.8rinton and H.L.Yamamura, Life Sci., 21, 1037 (1983) 118. K.W.Gee, R.E.Brinton and H.L.Yamamura, Brain Res., 264, 168 (1983) 119. D.G.Spencer and H.Lal, Drug Dev.Res., 3 , 365 (1983)120. K.Rickels, K.Weisman, N.Norstad, M.Singer, D.Stoltz, A.Brown and J.Danton, J.Clin. Psychiatry, 2 , 12 (sec. 21, 81 (1982) 12 (Sec. 2 1 , 92 (1982) 121. D.Wheatley, J.Clin.Psychiatry, 9, 12 (Sec. 2 ) , 122. J.P.Feighner, C.H.Merideth and G.A.Hendrickson, J.Clin.Psychiatry, 9, 103 (1982) 123. J.S.Hendry, R.L.Balster and J.A.Rosecrans, Pharmacol.Biochem.Behav., 1 9 , 97 (1983) 124. H.Y.Meltzer, R.Flemming and A.Robertson, Arch.Gen.Psychiat., 40, 109971983) 125. M.Cimino, F.Ponzio, G.Achilli, G.Vantini, C.Perego, S.Algeri, and S.Garattini, Biochem.Pharmacol., 2, 1069 (1983) 126. P.L.Wood, N.P.V.Nair, S.Lal, and P.Etienne, Life Sci., 2 , 269 (19831 127. B.A.McMillen, R.T.Matthews, M.K.Sanghera, P.D.Shepard, and D.C.German, J.Neurosci., 3 , 733 (1983)
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128. B.A.McMillen and C.C.McDonald, Neuropharmacol., 2 2 , 273 (1983) 129. M.K.Sanghera, B.A.McMillen and D.C.German, Eur.J.Pharmacol., 3, 107 (1983) 130. T.Glaser and J.Traber, Eur.J.Pharmacol., 88, 137 (1983) 131. N.R.Oakley and B.J.Jones, Eur.J.Pharmacol., 87, 499 (1983) 132. S.M.Pau1 and P.Skolnick, Pharmacol.Biochem.Behav., 17 (Suppl. l), 37 (1982) 133. S.Caccia, S.Garattini, A.Mancinelli and M.Muglia, JT-dhromatography, 252, 310 (1982) 134. B.A.McMillen and L.A.Mattiace, J.Neura1 Transmission, 57, 255 (1983) 135. J.P.Yevich, D.L.Temple,Jr., J.S.New, D.P.Taylor and L.A.Riblet, J.Med.Chem., 26, 194 (1983) 136. R.Hoehn-Saric, Psychopharmacol., @, 338 (1983) 137. R.Kikumoto, A.Tobe, H.Fukami and M.Egawa, J.Med.Chem., 26, 246 (1983) 138. E.J.Trybulski, L.E.Benjamin, J.V.Earley, R.I.Fryer, N.W.Gilman, E.Reeder, A.Walser, A.B.Davidson, W.D.Horst, J.Sepinwal1, R.A.O'Brien and W.Dairman, J.Med.Chem., 26, 1589 (1983) 139. A.Wauquier, Anaesthesia, 38 (Suppl) 26 (1983) 140. M.Tanaka, Y.Mizuki, H.IsoGki and K.Inanaga, Eur.J.Clin.Pharmacol., 24, 469 (1983) 141. P.J.Chaudoir, N.C. Jarvie and G. J.Wilcox, J.Int.Med.Res., 11, 333 (19B). 142. J.R.Normanton and J.P.Gent, Neuroscience, 8, 107 (1983) 143. J.E.Garcia-Arraras and J.R.Pappenheimer, J.Neurophysiol., 9, 528 (1983) 144. W.B.Mendelson, A.Kuruvilla, T.Watlington, K.Goeh1, S.M.Pau1 and P.Skolnick, Psychopharmacol., 2,126 (1983) 145. M.R.Trimble and P.J.Thompson, Epilepsia, 24 (Suppl. l), S55 (1983) 146. J.H.Skerritt, L.P.Davies, and G.A.R.Johnston, Epilepsia, 24, 634 (1983) 147. P.R.Andrews, L.C.Mark, D.A.Winkler and G.P.Jones, J.Med.Chem., 26, 1223 (1983) 148. A.M.Anlezark, D.H.R.Blackwood, B.S.Meldrum, V.J.Ram and J.L.Neumeyer, Psychopharmacol., 81, 135 (1983) 149, zL.Mueller and T.V.Dunwiddie, Epilepsia, 2, 57 (1983) 150. P.Loiseau, L.Bossi, M.Guyot, B.Orofiamma and P.L.Morselli, Epilepsia, 24, 703 (1983) 151. H.R.Petersen, 1.Jensen and M.Dam, Acta Neurol.Scand., 5, 114 (1983) 152. J.D.Wood, D.D.Johnson, P.Krogsgaard-Larsen and A.Schousboe, Neuropharmaco ., 22, 139 (1983) 153. G.Graziani, F.Tirone, E.Barbadoro and R.Testa, Arzneim-Forsch., 33 (II), 155 (1983) 154. D.T.Wong, R.C.Rathbun, F.P.Bymaster and W.B.Lacefield, Life Sci., 2,917 (1983) 155. D.T.Wong, F.P.Bymaster and W.B.Lacefield, Drug Dev.Res., 3 , 433 (1983) 156. B.S.Meldrum, H.C.Evans and C.Braestrup, Eur.J.Pharmacol., 9 l , 255 (1983) 157. I.B.Linden, G.Gothoni, P.Kontro and S.S.Oja, Neurochem.Internatioaa1, 5 , 319 (1983) 158. K.R.D.Zelger, J.L.Zelger and E.A.Carlini, Pharmacol., 27, 40 (1983) 159. R.J.Chorvat, B.N.Desai, S.E.Radak, J.Bloss, J.Hirsch and S.Tenen, J.Med.Chem., 26, 845 (1983) 160. N.A.Vaidya, C.H.Panos, A.Kite, W.B.Iturrian and C.D.Blanton, J.Med.Chem., 26, 1422 (1983)
Chapter 3.
Antipsychotic Agents
Tomas de Paulis and Sten Ramsby, Astra Lakemedal AB, Sweden Introduction - Schizophrenia continues to be one of the most fascinating and challenging research topics for medicinal chemists and neurochemists. Tools such as 3H-spiperone binding assays and an understanding of the biochemical abnormalities associated with schizophrenia may provide the path to future discoveries Haloperidol-induced dopamine (DA) receptor supersensitivity in schizophrenics appears to be attenuated by concurrent treatment with lithium.2 Noninvasive radioimaging techniques have brought new insight into the role of the regional distribution and receptor binding of antipsychotic agents .3 Studies of the blood flow in the brains of schizophrenic patients with positron-emitting Xenon-133 have revealed an over-activation in the left hemisphere during performance of a spatial line orientation test.4 PET scanning with oxygen-15 (half-life 123 s ) in some schizophrenic patients failed to support the hypothesis of reduced blood flow in frontal cortex. Enlarged ventricles and sulcal widening were correlated with type I1 schizophrenia, which is characterized by predominance of negative symptoms.6 The DA hypothesis of schizophrenia has been reviewed in the light of recent findings.’l Perioral dyskinesias can be induced acutely in rats by the DA antagonists spiroperidol and sulpiride, and by the selective D agonist SKF 38393, but not by the selective D2 agonist LY 141865.18 Coadministration of 1.Y 141865 with SKF 38393 did not induce dyskinesias, implying that the perioral movements are mediated by the D1 receptors and by an imbalance in D1_D2 responsivity.8 It has been hypothesized that the D-3 binding site is the high-affinity agonist state of the D-1 r e ~ e p t o r . ~Accumulated experience with depot neuroleptics, such as clopenthixol decanoatelO and pipotiazine palmitate, l1 has been reviewed. Phenothiazines and related riRid compounds - The antihallucinator effect of chlorpromazine correlates with serum levels of prolactin,18 consistent with the DA hypothesis of schizophrenia. No regional sitespecificity for limbic areas was found with thioridazine or with clozapine.13 An excellent correlation between daily clinical dose and the dose-dependent increase in cocaine self-administration in rats was found for seven typical and atypical neuroleptics. Clozapine, however, produced a dose-dependent decrease in cocaine intake. l4 HH2 calculations of the energies of the butaclamol structure have been utilized to suggest that a cisoid trans conformer (1) is responsible for the DA antagonist activity of this drug.15 The previously assumed active conformer was found to have 2.7 kcal/mol higher energy.
Unlike taclamine, the thiophene analog QM 7184 (2) blocks apomorphine or amphetamine induced stereotypy in the rat.36 It is equipotent with chlorpromazine but less potent than the structurally related compound butaclamol. Binding studies showed that QM 7184 is 30-50 times
ANNUAL REPORTS IN MEDICINAL CHEMISTRY-I9
Copyright 0 1984 by Academic Press. Inc. All nghts of reproduction in any form reserved. ISBN 0-12-0?10519-9
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less active than butaclamol or haloperidol in displacing 3H-spiperone from rat striatum. In contrast to the reference compounds, Ql4 1184 has a high affinity for a-noradrenergic receptors, as shown in binding studies with 3H-wB-4101.16.
I
Me 1 -
3
2
Attempts to separate the multitude of effects shown by cyproheptadine have included introduction of nuclear substituents and replacement of a benzene ring by a pyrrole ring.17 In an extension of this study to include 9-substituted pyrrolobenzapine analogs, compound 3 was found to be 2-3 times more potent than chlorpromazine versus G-spiperone and also showed high affinity for the serotonin S-2 binding The conformational flexibility was higher for these new pyrrolo-compounds than for the corresponding cyproheptadine derivatives as evidenced from the rates of racemization of the atropisomers.
:i:fta
The tetracyclic derivative GP 50302 (4)blocks DA receptors in the same dose range as chlorpromazine, and also shows strong 5-HT blocking properties. Thus in contrast with haloperidol, GP 50302 is much more potent h -viva in dis lacing 3H-spiperone from cortical, versus striatal, binding sites. lg However, in clinical trials GP 50302 had no therapeutic effects in acute schizophrenic patients, but was beneficial in withdrawn and apathetic schizophrenics. 2o The imidazole derivative CGS 10146 B (21, structurally related to clozapine, blocked Sidman avoidance at a dose of 10 mg/kg p.0. in squirrel monkeys.21 However, 1. did not produce the acute dyskinetic syndrome, characteristic of classical neuroleptic agents. In an open multicenter study in 104 schizophrenic patients, fluperlapine (NB 106-689, 6) showed significant improvement from the fifth day of treatment.n Fluperlapine has demonstrated little or no effect in a number of tests, such as catalepsy and inhibition of apomorphine- and amphetamine-induced behavior, which 3re considered specific for antipsychotic agents.23 It is a muscle relaxant and an uptake inhibitor of norepinephrine.
Me
4
5
6
Diphenvlpiperidines and butyrophenones - Haloperidol decanoate, lOOmg once per month, in chronic schizophrenic patients provoked few Parkinson-like side effects.24 DA receptor supersensitivity induced by chronic pimozide treatment was found to be dependent on the dose and not the duration, if measured as an increase in density of spiperone binding sites. When the induced supersensitivity was measured as increased inhibition of apomorphine-induced stereotypy, it was dependent on the duration and not on the dose. Thus, in the absence of pharmacologically-induced DA receptor stimulation, the functional consequences
3
Chap.
Antipsychotic Agents
de Paulis, Ramsby
3
of neuroleptic-induced supersensitivity at the receptor level remain unknown.25 X
2
X = CF3 R =CH20H
X=F
R=H
Structure-activity studies of 1-piperazino-3-phenylindanes led to the development of the potent and long-lasting neuroleptic, tefludazine (B). The %-isomer of tefludazine has 1/1000 the activity of 71 in blocking methylphenidate-induced stereotypy in mice. One member of the tefludazine series, the bisfluoro derivative (7&) was resolved: the antidopaminergic activities resided in the (+)-enantiomer. The (-)-enantiomer, however, proved to be an active uptake inhibitor of DA.Z~
(a)
is a highly potent neuroleptic agent. Positron Spiperone Emission Tomography (PET) imaging with lk-labelled N-methyl-spiperone (half-life 20 min) was used to study the distribution of DA receptors in the human brain. There was a high accumulation of activity in the basic ganglia in comparison to the rest of the brain.27 In another experiment, using 18F-spiperone (half-life 110 min) administered to a baboon 2h before the tomography imaging, it was clearly demonstrated that the ratio of binding in striaturn to cerebellum was ten times higher for 18F-spiperone than for 18F-haloperidol. 28
(m)
Cyclic oxime ethers of butyrophenones exhibit neuroleptic properties. Thus, the benzisoxazole analog of haloperidol, 9, has an ED50 of 1.6 lmol kg in blocking apomorphine-induced climbing in mice .29 The corresponding benzisoxazole analog of benperidol, HRP 913 (lo),seems to be a more selective DA antagonist than those presently in clinical use. HRP 913 is twice as potent as haloperidol in displacing spiperone from striatal binding sites and equipotent in the antiapomorphine stereotypy assay.30 It induces a biphasic atypical catalepsy at a dose 45 times higher than haloperidol, resembling thioridazine in this respect. A similar isosteric arrangement of the aromatic part of the molecule is found in FfJ 13859 (11) which in contrast to buspirone exhibits a clear neuroleptic p r 0 f i 1 T ~ ~ The molindone-butyrophenone hybrid Ro 22-6600 (12) is both a preand postsynaptic DA-antagonist.32 There are differences in the SAR of the Ro 22-6600 series and typical butyrophenones since hydrogenation of the chain carbonyl group in the former only halves activity in conditioned-avoidance response.33 Milenperone (R 34009, 12) displayed antiaggressive activity in a group of mentally retarded patients, with no greater incidence of side effects than with placebo.34 Double blind studies with timiperone (DD-3480, 14) versus ~ l o c a p r a m i n e , ~ ~
Sect. I
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s ~ l p i r i d e ~or~ h a l o p e r i d 0 1 ~ ~ showed an equal or superior efficacy against schizophrenia. The separation between catalepsy and anti-a o morphine activity for timiperone in the monkey seems to be small. 87Another new butyrophenone, HR 375 ( s ) , shows weak and short-lasting muscle relaxing properties besides being active on neuroleptic parameters such as block of climbing and conditioned avoidance behavior. However, it neither blocks stereotypy nor does it induce catalepsy in the rat.38
13 -
Substituted benzamides - The unique behavioral profile in animals of substituted benzamide neuroleptics and their selective action on a subpopulation of D-2 receptors might explain their clinical usefulness in the treatment of s c h i ~ o p h r e n i a . ~ The ~ Ki-value for sulpiride displacement of spiperone binding was approximately 20 times greater than the KD value for sulpiride binding in the presence of 120 mM Na+, suggesting that sulpiride and spiperone label different receptor sites .40 In a comparative study with classical neuroleptics, benzamides were generally 1000 times more potent in stimulating rat prolactin secretion than would have been predicted from their potencies in displacing 3H-spiperone from bovine anterior pituitary membranes .41
16 -
17 -
18 -
In structural analogy with the retro-amide i s ~ s u l p r i d e ~(16). ~ the retro-amide of clebopride, BRL-20596 (u),retains the antiapomorphine activity but totally lacks effect on gastric stimulation,43 thereby showing that separation is possible between the central and peripheral activity. Introduction of a methoxy group in the piperidine ring of clebopride abolishes the central effects. In a series of 3-piperidine substituted benzamides, cisapride (R 51619, 18) is a potent peripheral 5-HT antagonist which shows high potency in increasing gastric p r ~ m o t i l i t y . ~Another ~ compound, BRL 20627 (E),with potent peripheral stimulatory action on the gastric motility of the rat, is only a weak central DA receptor antagonist.45 Conformational studies of clebopride and YH-08050 (m)imply that it is the six-membered pseudo-ring, formed by the hydrogen bond between the amide proton and the ether oxygen atom, rather than the benzene ring, that interacts with the aromatic binding site of the DA re~ e p t o r . ~YU~ 09151-2 was the most potent and selective D-2 receptor antagonist of a series tested: the Ki value for 20b versus 3H-spiperone binding in rat striatal membranes was 0.1 nUT" X-ray studies with Yn 09151-2 have revealed that the tertiary m i n e nitrogen is i n a c c e ~ s i b l e . ~Thus ~ the active conformer must be different from that in the crystalline state if the lone pair on the tertiary nitrogen interacts with the receptor.
(m)
Chap. 3
Antipsychotic Agents
de Paulis, Ramsby
2
In a double blind study, amisulpride (DAN 2163, displayed the same antipsychotic efficacy as haloperidol with favorable effects on certain symptoms.49 At low doses in man, amisulpride behaves like a DA agonist with stimulating properties, but at doses over 600 mg the
20a 20b
19 -
R =H R=CH3
neuroleptic properties predominate.50 Similar to amisulpride, but in contrast to haloperidol, the N-cyclopropylmethyl analog LUR 2366 (m) inhibits apomorphine-induced climbing at a 10 times lower dose than that which blocks stereotypy: in fact LUR 2366 potentiates the gnawing behavior in mice.51 In this series, the n-ally1 substituted sulfone derivative alpiropride ( R I V 2093, displays the largest separation between high-affinity apomorphine (D-4) receptor sites and domperidone (D-2) receptor sites, thereby indicating a lack of effect on the pituitary and no consequent release of p r ~ l a c t i n . ~ ~
a)
clgNHO[ f-+m "*A? a.
G
R
N
OMe
3
Me0
CI
OMe
Me NI - . y $ N ; e
Me
Me0
CBS 22a -
R
=
H
R=CH3
24
23
(z),
A tetrasubstituted benzamide, A 32799 was equipotent with chlorpromazine in'blocking apomorphine-induced stereotypies in the rat, showing that introduction of sterically bulky substituents in the aromatic ring does not abolish antidopaminergic activity.53 However, the pentasubstituted compound, was only 1/50th as active as its corresponding des-4-methyl substituted analog,54 indicating the requirement of a free La= position in this series. The ability of a substito increase the basal tritium outtuted nicotinamide, CGP 11109 flow from caudate slices of the rabbit preincubated with 3H-DA, was comparable to that of s ~ l p i r i d e . ~The ~ metabolic and pharmacokinetic properties of a bis-N-methyl derivative of veralipride, sulverapride (24) were investigated in man. No radioactivity was found in the brain, indicatin very poor penetration of sulverapride across the blood-brain barrier. 5%
(a),
(a)
A new substituted salicylamide, FLB 131 exhibited potent anti-DA properties in the rat.57 Selective labeling of the striatum was demonstrated with both FLB 13158 and the N-methyl analog (25b)59 in PET imaging experiments in cynomolgus monkeys. In a series of 3-substituted 6-methoxysalicylamides, compound FLA 965 (26) showed optimal activity in displacing 3H-spiperone from rat striatal preparations ,60 A remarkable increase in the antiapomorphine activity through introduction of an alpha-hydroxyethyl group was demonstrated in the substituted benzamide MD 781124 (21). Thus, MD 781124 was 540 times more potent in antagonizing apomorphine-induced stereotypy in the rat than the corresponding 5-sulfonamide analog, sulpiride.61
26 -
Sect. I
- CNS Agents
Hesp, Ed.
The tropylamine derivative tropapride (MD 790501, 28) shows a neurophacmacological profile equivalent to that of haloperidol but with substantially less sedative, cataleptogenic or dyskinetic properties.62 In the rat, i.p., tropapride was four times more potent than
26
27 -
haloperidol on antiapomorphine activity. Among a series of azabicyclo(3.3.lJnonylbenzamide derivatives with neuroleptic properties, compound 29 was one of the most potent agents. Its EDSO value in blocking apomorphine-induced climbing in the mouse was 63 times that of clebopride. 63
30 -
28 -
The weak blocking properties of tiapride (FLO 1347, 30) at receptors labelled by various neuroleptics could not be explained solely by its poor penetration into the brain.64 Instead it was suggested to be due to a specific action of tiapride on receptor sites selectively labelled by 3H-sulpiride. Further clinical experience with tiapride in the treatment of tardive dyskinesia has shown that it is an effective agent. In a double-blind comparison with placebo, tiapride significantly improved dyskinesia in 15 patients who had received neuroleptics for 15 years.65 Miscellaneous compounds - Similar to dihydro-oxypertine (CL 77328) ,66 the structurally related isochroman derivative U-54537 E, (2)displays an atypical neuroleptic profile.67 Both compounds are weak in displacing 3H-spiperone binding and in antagonizing the behavioral syndrome induced by apomorphine. Yet, U-54537E is three times more potent than thioridazine in decreasing conditioned avoidance response and is equipotent with chlorpromazine in blocking adenylate cyclase coupled D-1 receptors.67 A thiazoledione analog MJ 13980-1 (32) of buspirone (MJ
32 -
33 -
9022-1, 33) has shown promise as a. potential antipsychotic in that it possesses antidopaminergic activity but does not induce catalepsy. In fact, MJ 13980-1 is potent in reversing phenothiazine-induced catalepsy in the rat.68 The physicochemical parameters of the aromatic orthosubstituent do not correlate with the anti-DA activity in this series.69
Chap. 3
Antipsychotic Agents
de Paulis, Ramsby
7
A double-blind phase I study of zetidoline (DL-308, 34a) revealed a strong sedative effect as judged by self-reported performance scores on logical reasoning and coordination tests.70 In an open study in 40 schizophrenic patients, doses of 10 to 90 mg daily of zetidoline exerted a good antipsychotic efficacy with akathisia as the most notable side effect.71 Substitution in the aromatic nucleus with a p-hydroxy group (E) substantially increases both duration and antiapomorphine activity in rodents.IZ m
NHCONEt,
34a R = H -
35
R=OH
2-Bromolisuride (35) is the first ergot derivative with a definite antagonist effect at central DA receptors.73 In comparison with haloperidol, 21 shows ten times larger separation between drug-induced catalepsy and apomorphine-induced stereotypy in the rat.74 S(+)-N-propylnorapomorphine shows DA receptor blocking activity75 as was shown previously for S(+)-ap~morphine.~~ A new pyrazole derivative 36 also exhibits DA antagonist properties as evident from its prolactin elevating effect on the pituitary.77 Both molindone and its rigid derivative Ro 22-1319 (21) were confirmed to be more active as presynaptic DA-antagonists than postsynaptic ,32 thereby indicating an explanation for the relatively weak anti-amphetamine activity of molindone.78 Ro 22-1319 was equipotent with haloperidol in elevating serum prolactin levels in the rat.79 More receptor-specific agonists and antagonists of DA continue to appear. A new benzazepine, SCH 23390 is claimed to be the first selective D-1 antagonist and promises to be a useful neuropharmacological tool.80 SCH 23390 is equipotent with flupentixol in displacing 3H-piflutixol (D-1) binding, but 1200 times less active in displacing 3H-spiperone (D-2) binding.81 It is virtually free from blocking effects on apomorphine-induced hypothermia and emesis and does not cause hyperprolactinemia,82 which suggests that these effects are related to the D-2 receptor.
(s)
EEDQ (2)is a potent and irreversible D-2 anta onist which apparently binds covalently to DA receptors in the CNS.8el EEDQ produces catalepsy and inhibits both amphetamine-induced stereotypy and conditioned avoidance behavior. HO
A 37 -
38
A six week study with the GABA agonist progabide (SL 76002, g ) , in schizophrenic patients on neuroleptic maintenance therapy, revealed improvement on items such as vigilance and affective i n ~ o l v e m e n t . ~ ~ There is evidence that SL 76002 blocks striatal cholinergic neurons through a gabergic mechanism. Thus repeated treatment with SL 76002 in rats prevents tolerance to the cataleptogenic action of haloperidol.85
28
Sect. I
- CNS Agents
Hesp, Ed.
Unlike TL99 (41), neither (*)-3-PPP ( 4 3 8 6 9 8 7 nor its (-)-enantiomer UH 106(-) nappear to be presynaptic agonists in vitro: thus they do not inhibit evoked release of 3H-DA from rat striatal slices. HO
"omN"" no
H"
42 -
40 -
Instead, both the r a ~ e m a t e and ~ ~ ( - ) - e n a n t i 0 m e r 8 ~ * ~behave ~ as postsynaptic D-2 antagonists in vitro. For example, the latter prevents the blocking action of the D-2 agonist LY 141865 on potassium-induced However , the (-)-enanrelease of 3H-acetylcholine from striatum.88 tiomer does behave as a presynaptic DA agonist at low doses in V ~ V O . ~ References 1. R. Rodnight, J. Neurochem., 41, 12 (1983). 2. D. E. Sternberg, M. B. Bowers, Jr., G. 8. Heninger and D. S. Charney, Psychiat. Res., lo, 79 (1983). 3. N. D. Heindel and N. Foster, Ann. Rep. ned. Chem.. H, 293 (1983). 4. R. E. Cur, B. E. Skolnick, R. C. Gur, S. Caroff, W. Rieger, W. D. Obrist, D. Younkin and M. Reivich, Arch. Gen. Psychiat., 40, 1250 (1983). 5. G. Sheppard, J. Gruzelier, R. kanchanda, S. 8. Hirsch, R. Wise, R. Frackowiak and T. Jones, Lancet 21, 1448 (1983). 6. H. A . Nasrallah, S. Kuperman, C. G . Jacoby. M. McCalley-Whitters and B. Hamra. Psychiat. Res., lo, 237 (1983). 7. Y. Sayed and J. W. Garrison, Psychopharm. Bull., 19. 283 (1983). 8. H. Rosengarten, J. Y. Schweitzer and A . J. Friedhoff, Life Sci., 33. 2479 (1983). 9. S. E. Leff and I. Creese, Trends Pharmacol. Sci., 5 , 463 (1983). 170 (1982). 10. S. Sieberns and H. Spechtmeyer, Int. Phacmacopsychiat., 11. E. A. Burch, Jr. and 9. J. Ayd, Jr., J. Clin. Psychiat., 44, 242 (1983). 12. H. Y. Meltzer and D. Busch, Psychiat. Res., 2, 285 (1983). 13. P. Seeman and C. Ulpian, Eur. J. Pharmacol., 94, 145 (1983). 14. D. C. S. Roberts and G. Vickers, Psychopharmacol., 82 135 (1983). 15. M. Froimowitz and S. Mathysse, nol. Pharamcol., 24, 243 (1983). 16. E. Arribas, S. Vega, C. Benito, M. P. Pernandez-Tome and J. del Rio, Arzneim.Forsch, 33, 1417 (1983). 17. D. C. Remy, S. F. Britcher. P. S. Anderson, P. C. Belanger. Y . Girard and B. V. clineschmidt, J. Med. Chem. 25, 231 (1982). 18. D. ,.C Remy, S. F. Britcher, S. W. King, P. S. Anderson, C. A . Hunt, W. C. Randall, P. Belanger, J. G. Atkinson, Y. Girard, C. S. Rooney, J. J. Puentes, J. A . Totaro, J. L. Robinson, E. A. Risley and M. Williams, J. Wed. Chem., 3,974 (1983). 19. R. Ortman. S. Bischoff, E. Radeke, 0. Buech and A . Oelini-Stula, Arch. Pharmacol.. 321, 265 ii982). 20. A . Delini-Stula, P. Waldmeier, P. Baumann, L. Maitre, H. Blattner and P. Schmidlin, 11th C.I.N.P. Congress (Vienna, July 9-14, 1978) Abst. p . 373. 21. J. W . Liebman, G. P. Quinn, R. A . Lovell, I. Vlattas and T. W. Glenn, Fed. Proc., 42, 1152 (1983). Abst. 5037. 22 * K. A. Fisher-Cornelssen, Arzneim-Forsch., 34, 125 (1984). 23. H. R. Burki, 186th Meet. Am. Chem. SOC. (Washington, Aug. 22-26, 1983) Abst. 765. 24. Y. G. Gelders, A . J. M. Reynjiens, C. W. Ash and T. J. L. Aerts, Int. Pharmacopsychiat., l7, 247 (1982). 25. K. J. Dewey and H. C. Fibiger, Arch. Phacmacol., 322, 261 (1983). 26. K. P. Bogeso, J. Ned. Chem., 3,935 (1983). 27. H. N. Wagner, H. 0. Burns, R. F. Dannals, D. F. Wong, B. Langst&m. T. Duelfer. J. J. Frost, H. T. Ravert, J. W. Links, S. B. Rosenbloom, S. E. Lukas, A . V. Kramer and M. J. Kuhar, Science 221, 1264 (1983). 28. M. J. Welch, M. 8. Kilbourn, C. J. Hathias, M. A . Mintun and W . E. Raichle, Life Sci., 33, 1687 (1983). 29. L. Davis, 8. C. Effland and J. T. Klein, Eur. Pat. Appl. EP 63799 (1982). Chem. Abst. 98 179353 r (1983). 30. S. Fielding, W. J. Novick, Jr., H. 1. Gayer, W. W. Petko, J. C. Wilker, L. Davis, J. T. Klein and 1. Cornfeldt. Drug Dev. Res., 3, 233 (1983).
u,
-
~
Chap. 3 31. 32. 33. 34. 35. 36. 37. 38. 39. 40. 41. 42. 43. 44. 45. 46. 47. 48. 49. 50. 51. 52. 53. 54. 55. 56. 57. 58.
59. 60. 61. 62, 63, 64. 65. 66. 67. 68. 69,
A n t i p s y c h o t i c Agents
d e P a u l i s , Ramsby
J . P. Yevich. D. L. Temple, J. S. New, W. G. Lobeck, J . D. Catt, D. P. Taylor,
3
n. s.
Eison and L. A. Riblet, 186th Meet. Am. Chem. SOC., (Washington. Aug. 22-26, 1983) Abst. 69. H. H. Keller, Arch. Pharmacol., 324, Suppl. R 19 (1983) Abst. 74. G. L. Olson, H.-C. Cheung, E. Chiang and L. Berger in "Dopamine Receptors", ACS Symposium Series 224. C. Kaiser and J. W. Kebabian, Eds., Washington D. C., 1983, p. 251. H. De Cuyper, H. M. van Praag and D. Verstraeten. VII World Congr. Psychiat., (Vienna, July 11-16, 1983) Abst. P 714. T. Nakasawa, K. Ohara, Y. Sawa, VII World Congr. Psychiat., (Vienna, July 11-'16, 1983) P 722. K. Mori, M. Miyasaka and Y. shimazono, VII World Congr. Psychiat., (VIenna, July 11-16, 1983) P 72. T. Shibuya, T. Nishimori, H. Matsuda and P. C. Chen, Int. J. Clrn. Pnarmacol. Ther. Toxicol., 20. 251 (1982). F. J . Hock, H. J. Kruse. H. Gerhards and E. Konz, Pharmacologist. 25. 130 (1983). Abst. 103. P. Jenner and C. D. Marsden, Acta Psychiat. Scand. Suppl. 69. 109 (1984). A. E. Theodorou. P. Jenner and C. D. Marsden, Life Sci., 32. 1243 (1983). H. Y. Melzer, M. Simonovic and R. So, Life Sci.. 32, 2877 (1983). G. Le Fur, F. Guilloux and A. Uzan, Biochem. Pharmacol., 29, 267 (1980). F. E. Blaney, M. S. G. Clark, D. V. Gardner, M. S. Hadley, D. Middleton and T. J . White, J. Med. Chem., 26. 1747 (1983). H. J. Cooke and H. V. Carey, Eur. J. Pharmacol., 98, 147 (1984). M. S. Hadley, F. D. King, B. McRitchie, D. H. Turner and E. A. Watts, 2nd SCI-RSC Med. Chem. Symp. (Cambridge. Sept. 12-14, 1983) Abst. p 9. H. van de Waterbeemd and B. Testa, J. lied. Chem., 26. 203 (1983). M. Terai, S. usuda, I. Kuroiwa, 0. Noshiro and H. Maeno, Jap. J. Pharmacol., 33. 749 (1983). T. Furuya, S. Iwanami. A. Takenaka and Y. Sasada. Bull. Chem. SOC. Jap. 55. 2321 (1982). H. E. Klein, D. Dieterle. E. Ruther, E. Eben, N. Nedopil and H. Hippius. VII World Congr. Psychiat. (Vienna, July 11-16, 1983). Abst. S 1090. J. Grunberger, 8. Saletu, L. Linzmeyer and H. Stohr, VII World Congr. Psychiat. (Vienna, July 11-16, 1983). Abst. S 1089. M. P. Martres, P. Sokoloff, M. Delandre, J.-C. Schwartz, P. Protais and J. Costentin, Arch. Pharmacol., 325, 102 (1984). P. Sokoloff, M. Delandre, M. P. Martres, J.-C. Schwartz, P. Laibe and C. Wermuth, 5th Catecholamine Symp. (Gothenburg, June 12-16, 1983). Abst. 459. L. Florvall, M.-L. Persson and S.-0. Ogren, Acta Pharm. Suec., 20. 365 (1983). L. Florvall and S.-0. Ogren, J. Med. Chem., 2 5 , 1280 (1982). K. Starke, L. Spath, J. D. Lang and C. Adelung, Arch. Pharmacol.. 323, 298 (1983). A . Benakis, F. 8. Sugnaux, C. Plessas, C. Rey. C. Gallay, R. Happe and P. Allais, Pharmacologist, 25. 115 (1983) Abst. 28. L. Florvall. J. Lundstram, S. Ramsby and S.-0. &ran, Eur. Pat. Appl. EP 60235 (1982). Chem. Abst. 98. 53687 j (1983). G . Sedvall, G. Blomqvist, T. de Paulis, E. Ehrin, L. Eriksson, L. Farde, T. Greiz. C. ,G. Hedstrom, D. H. Ingvar, J.-E. Litton, J. L. G . Nilsson, S. Stone-Elander, L. Widen, F. A. Wiesel and G. Wit, Ann. Meet. Am. Coll. Neuropsychopharmacol. (San Juan, Dec. 12-16, 1983). G. Sedvall, G. Blomqvist, T. de Paulis et al., in "Proceedings of VII World Congress of Psychiatry", P. Berner, Ed., Plenum Press, N.Y 1984. T. de Paulis, U. H. Lindberg, L. Florvall, S.-0. Ogren and H. Hall, 2nd SCI-RSC Med. Chem. Symp. (Cambridge. Sept. 12-14, 1983). Abst. p. 14. M. Langlois, T. Imbert and B. Bucher, Fr. Pat. Appl. 2446274 (1980). Chem. Abst. 94. 208572 m (1981). M. Jalfre, B. Bucher, N. Donne, G. Mocquet and 8. D. Porsolt, Arch. Int. Pharmacodyn.. 264. 232 (1983). M. S. Hadley and F. E. Blaney, Eur. Pat. Appl. EP 76592 (1983). Chem. Abst. 99, 70583 k (1983). J. K. Chivers, W. Gommeren, P. Jenner. J. Leysen. C. D. Marsden, C. Reavill and A. Theodorou, Brit. J. Pharmacol., 78, 398 P (1983). VII World Congr. Psychiat. (Vienna, July 11-16, 1983). Abst. S 1085. E. N. Greenblatt, J. Coupet, E. Rauh and V. A. Szucs-Myers, Arch. Int. Phannacodyn., 248. 105 (1980). P. F. von Voigtlander, R. A. Lahti, A. H. Tang and V. H. Sethy. Arch. Int. Pharmacodyn., 266, 23 (1983). D. P. Taylor, M. S. Eison, D. L. Temple and J. P. Yevich, Fed. Proc., 42, 1153 (1983). Abst. 5045. J. P. Yevich, D. L. Temple, J. S. New, D. P. Taylor and L. A. Riblet. J. Mad. Chem., 26, 194 (1983).
...
30 -
Sect.
I
- CNS Agents
Hesp, Ed.
70. L. A . Riblet, M. Herbert, P. J. Standen, A. H. Short and A. T. Birmingham, Psychopharmacol., 8l, 335 (1983). 71. M. Zapletalek, J. Libiger, I. Tuma and K. Kubicek. VII World Congr. Psychiat. (Vienna, July 11-16, 1983). Abst. P 685. 72, L. Fontanella, E. Martinelli and M. A . Assandri, Ger. Pat. DE 3307172-A (1983). 73. H. Wachtel, Arch. Pharmacol. Suppl., 324. R 90 (1983). Abst. 360. 74. H. Wachtel, W. Kehr and G. Sauer, Life Sci.. 33, 2583 (1983). 75. J. L. Neumeyer, D. Reischig, C. W. Arana, A . Campbell, R. J . Baldessarini, N. S. Kula and K. J. Watling, J. Med. Chem., 26, 516 (1983). 76. R. V. Smith, R. E. Wilcox, K. Young, U. H. Riffee and Y. C. Clement-Cornier, Fed. PCOC., 4 l , 105 (1982) Abst. 71. 77. E. 8. Smalstig, E. C. Kornfeld and J. A . Clemens, Fed. Proc., 4 l , 162 (1982). Abst. 118. 78. R. W. Fuller and H. D. Snoddy, Life Sci., 21, 2357 (1983). 79 * S. J. Kolis, T. H. Williams, T. F. Mowles, B. Burghardt, V. Toome and H. A. Schwartz, J. Pharmacol. Exp. Ther., 227, 652 (1983). 80. J. Hyttel, Eur. J. Pharmacol., 91, 153 (1983). 81. L. C. Iorio, A . Barnett, P. H. Leitz, V. P. Houser and C. A . Korduba, J. Pharmacol. Exp. Ther., 226, 462 (1983). 82. A . Hilditch, G. M. Drew and 8. J. Naylor, Eur. J . Pharmacol., 9 7 , 333 (1984). 83. H. W. Hamblin and I. Creese, Life Sci., 32, 2247 (1983). 84. P. Sevestre, N. Seguier, J. Lagrange, V. Fournier, B. Musch and P. L. Morselli, VII World Congr. Psychiat. (Vienna, July 11-16, 1983) Abst. P 730. 85. G. Bartolini, B. Scatton and B. Zivkovic in "Adv. Biochem. Psychopharmacol.", Vol. 24, B. Cattabeni et al., Eds., Raven, New York, 1980, p. 207. 86. P. Siminia and A. H. Mulder, Euc. J. Pharmacol., 89, 183 (1983). 87. R. Markstein and D. Lahaye, J. Neural Transm., 5 8 , 43 (1983). 88. J. F. Plantje, A . H. Mulder and J. C. Stoof, Pharm. Weekblad Sci. Ed.. 5 , 265 (1983). 89. S. Hjort, A . Carlsson, D. Clark, K. Svensson, M. Wikstr'd.m, D. Sanchez, P. Lindberg, U. Hacksell, L.-E. Arvidsson, A. Johansson and J. L. G. Hilsson, Psychopharmacol. 81, 89 (1983).
CHAPTER 4.
COGNITIVE
DISORDERS
F r e d M. Hershenson and John G. M a r r i o t t
Warner-LambertIParke-Davis, Ann Arbor, M I
48105
I n t r o d u c t i o n - The treatment of c o g n i t i v e d i s o r d e r s remains one of t h e g r e a t c h a l l e n g e s f a c i n g medicine and science. I n s p i t e of impressive advances i n our understanding of t h e b r a i n , s a f e and e f f e c t i v e treatments f o r t h e major d i s o r d e r s of c o g n i t i o n , e.g., mental r e t a r d a t i o n , l e a r n i n g d i s a b i l i t i e s , amnes t i c syndromes, and t h e dementias, have y e t t o be demonstrated. To t h e s e major d i s o r d e r s of c o g n i t i o n can be added many d i s o r d e r s and d i s e a s e s of t h e c e n t r a l nervous system (CNS) which have c o g n i t i v e sequelae, although they are n o t p r i m a r i l y c o g n i t i v e d i s o r d e r s , e . g . , e p i l e p s y and schizophrenia. Research i n t o human d i s o r d e r s of c o g n i t i o n i n r e c e n t y e a r s has focused t o a l a r g e degree upon Alzheimer's d i s e a s e o r Primary DegenRecent c l i n i c a l evidence,1'l6 a l o n g w i t h e r a t i v e Dementia (PDD) earlier l a b o r a t o r y s t u d i e s , 17-21 i n d i c a t e s t h a t t h e c o g n i t i v e d e f e c t s i n Alzheimer's d i s e a s e , t h e primary cause of dementia i n t h e e l d e r 1 are due t o a l o s s of f u n c t i o n i n g of c h o l i n e r g i c neurons i n t h e S i n c e many a r t i c l e s have r e c e n t l y a p p e a r e d summarizing c h o l i n e r g i c approaches t o t h e treatment of dementia,22-26 w e w i l l only b r i e f l y review t h i s a c t i v e r e s e a r c h area and then d i s c u s s o t h e r chemotherapeutic approaches which show promise i n the treatment of c o g n i t i v e dysfunction.
.
The C h o l i n e r g i c System - Following t h e precedent of 2-dopa therapy f o r P a r k i n s o n ' s d i s e a s e , many s t u d i e s have attempted t o treat t h e c h o l i n e r g i c d e f i c i t presumed t o account f o r t h e c o g n i t i v e impairments i n Alzheimer's d i s e a s e , by t h e use of p r e c u r s o r s t o a c e t y l c h o l i n e (ACh), such a s c h o l i n e c h l o r i d e o r l e c i t h i n . R e s u l t s from t h e s e s t u d i e s have been almost unif ormly negative.27-33 One r e c e n t long-term, six-month study u s i n g high doses of l e c i t h i n showed b e n e f i c i a l e f f e c t s i n PDD p a t i e n t s on a mental t e s t and a p a i r e d - a s s o c i a t e l e a r n i n g task.34 A r e p o r t demonstrating g r e a t e r enhancement of memory i n aged r a t s w i t h a combination of c h o l i n e c h l o r i d e and piracetam than w i t h e i t h e r compound a l o n e has s timula t e d s e v e r a l c l i n i c a l s t u d i e s w i t h combinations of p r e c u r s o r therapy and o t h e r drugs.35 An i n i t i a l s t u d y w i t h piracetam and c h o l i n e c h l o r i d e , i n p a t i e n t s f o r whom t h e d i a g n o s i s was mild t o moderate PDD, found g r e a t e r improvement w i t h t h e combination therapy than w i t h piracetam alone.36 Recently, t h e s u g g e s t i o n has been made t h a t p r e c u r s o r therapy be combined w i t h c h o l i n e s terase i n h i b i t o r s i n a l l f u t u r e t r i a l s of t h e l a t t e r i n Alzheimer's d i s e a s e , and i n t r i a l s of o t h e r a g e n t s which a f f e c t c h o l i n e r g i c f u n c t i o n , e.g. n a l o ~ o n e . ~ T h~i s s u g g e s t i o n i s supported by r e c e n t animal s t u d i e s showing s t r i k i n g reduct i o n s i n t h e optimal doses of c h o l i n e r g i c a g o n i s t s r e q u i r e d t o produce improved r e t e n t i o n on a T-maze t a s k in'mice, when given as two o r threedrug combina ti on^.^^ However, c l i n i c a l trials w i t h combinations of a n t i c h o l i n e s terases and ACh p r e c u r s o r s have shown l i m i t e d success. I n i t i a l p o s i t i v e e f f e c t s were r e p o r t e d i n e l d e r l y dementia p a t i e n t s , 3 9 and two r e c e n t s t u d i e s using o r a l physos tigmine supplemented by l e c i t h i n
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Copyright 0 1984 by Academic Preas. Inc All rights of reproduction in any form reserved. ISBN 0-12-040519-9
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reported o s i t i v e e f f e c t s upon c o g n i t i v e performance i n Alzheimer patients.g0,41 Others f a i l e d t o f i n d p o s i t i v e e f f e c t s upon c o g n i t i o n i n normal aged s u b j e c d 2 o r i n p a t i e n t s w i t h Alzheimer's d i s e a s e t r e a t e d i n t h i s manner.43 Many s t u d i e s have reported b e n e f i c i a l e f f e c t s i n p a t i e n t s t r e a t e d w i t h a n t i c h o l i n e s t e r a s e s alone,22,25,44-47 though o t h e r s have f a i l e d t o f i n d improvement i n c o g n i t i v e performance i n similar patients.48949 4-aminopyridine (4-AP) and 3,4-diaminopyridine (3,4-DAP) have been s t u d i e d i n animal models f o r p o t e n t i a l e f f e c t s upon cognition. These drugs enhance release of ACh, presumably by blocking potassium channels i n nerve cells, thereby promoting calcium flux.50 3,4-DAP p a r t i a l l y r e v e r s e s t h e d e f i c i t s i n ACh metabolism and motor behavior ( t i g h t - r o p e t e s t ) produced by hypoxia i n mice.51 It a l s o improved performance on a n 8-arm radial maze i n aged rats.52
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Studies of the biochemical and h i s t o p a t h o l o g i c a l changes Animal Models i n Alzheimer's p a t i e n t s , 4 - l 6 p a r t i c u l a r l y i n t h e c h o l i n e r g i c system, have opened new approaches t o developing animal models f o r t e s t i n g hypotheses and new chemical e n t i t i e s . Already s e v e r a l s t u d i e s have appeared d e f i n i n g t h e r o l e of v e n t r a l p a l l i d a l l e s i o n s i n animals. Acquisition of a c t i v e and passive avoidance was impaired i n r a t s with u n i l a t e r a l elect r o l y t i c a l l e s i o n s i n t h e ventromedial p o r t i o n of t h e pallidum, a n area which corresponds t o the nucleus b a s a l i s of Meynert i n primates.53 Responses learned before l e s i o n i n g were n o t impaired. Ventral p a l l i d a l l e s i o n s produced by i b o t e n i c a c i d d i d n o t a l t e r performance of rats on a b a t t e r y of psychomotor t a s k s o r a f f e c t s e n s i t i v i t y t o shock.54 Howe v e r , severe d e f i c i t s i n r e t e n t i o n of a passive avoidance response were found i n these lesioned animals. S i m i l a r d e f i c i t s i n passive avoidance r e t e n t i o n were found i n rats lesioned b i l a t e r a l l y i n t h e v e n t r a l pallidum using another e x c i t a t o r y .neurotoxin, k a i n i c acid.55 Ethylcholine mustard a z i r i d i n i u n i o n (AF64A, l-), a neurotoxic c h o l i n e analog, h a s been shown t o produce long-lasting hypofunction of c e n t r a l c h o l i n e r g i c systems i n mice56 and a reduction of presynaptic c h o l i n e r g i c markers i n r a t hippocampus57 without a f f e c t i n g post-synaptic muscarinic r e c e p t o r binding. It has been proposed t h a t AF64A-lesions may provide a n animal model of Alzheimer's d i s e a s e , although behavioral evidence t o support t h i s view i s q u i t e preliminary.58 A n t i c h o l i n e r g i c i n d u c e d c o g n i t i v e def i c i t s have a l s o been used as a model of age-related impairments, with a g e n t s being t e s t e d f o r t h e i r a b i l i t y t o antagonize o r r e v e r s e t h i s a c t i v i t y . Systemically administ e r e d a t r o p i n e has been shown t o produce i n c r e a s e s i n running time and working memory e r r o r s i n mice t r a i n e d on a six-arm r a d i a l maze.59 I n a s w i m maze, a t r o p i n e - t r e a t e d rats were impaired with r e s p e c t t o f i n d i n g a hidden escape platform.60 S i m i l a r d e f i c i t s were found i n rats with t o t a l hippocampalectomy.61 Atropine was found t o d i s r u p t a c q u i s i t i o n of a l i g h t l d a r k d i s c r i m i n a t i o n and a tonelno tone d i s c r i m i n a t i o n i n r a t s . b 2 Physos tigmine, conversely, f a c i l i t a t e d performance on t h i s task. Anticholinergics are a l s o e f f e c t i v e i n d i s r u p t i n g memory when i n j e c t e d d i r e c t l y i n t o the b r a i 1 1 . ~ ~ , 6 4 Conversely, c h o l i n e r g i c a g e n t s such as a r e c o l i n e , physostigmine, oxotremorine, and muscarine, a l l improved r e t e n t i o n on a n a c t i v e avoidance t a s k when administered ICV a f t e r t r a i n i n g and p r i o r t o r e t e n t i o n t e s t i n g one week l a t e r . 6 4 was 4-( o-Benzy1phenoxy)-Nme thylbutylamine hydrochloride (MCI-2016, found t o r e v e r s e scopolamine-induced impairments of spontaneous a l t e r a t i o n of responding i n r a t s similar t o t h e e f f e c t s of physostigmine, chol i n e and a m ~ h e t a r n i n e . ~ ~
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Old animals continue t o be used e x t e n s i v e l y as models of agerelated c o g n i t i v e d i s o r d e r s . Old mice, f o r example, were found t o be impaired on p a s s i v e avoidance compared t o young mice.66 I n c o n t r a s t w i t h c l i n i c a l d a t a , phosphatidylcholine i n t h e d i e t enhanced performance of o l d mice i n a shuttle-box avoidance test.67 Aged rats performed a t chance l e v e l s a f t e r 15 t r a i n i n g t r i a l s using a 12-arm r a d i a l maze, wherea s young rats had mastered t h e task.68 I n t h i s study, p o s i t i v e c o r r e l a t i o n s i n aged rats were found between maze performance and hippocampal c h o l i n e a c e t y l t r a n s f e r a s e a c t i v i t y . Aged primates, b o t h cebus and rhesus monkeys, have been employed i n s t u d i e s of age-related memory i m p a i r m e n t ~ and ~ ~ drug e f f e c t s upon memory.7a Drug t r i a l s i n monkeys have demonstrated e f f e c t s w i t h c h o l i n e r g i c a g e n t s and neuropeptides similar t o those r e p o r t e d i n human t r i a l s , adding t o t h e v a l i d i t y and u t i l i t y of t h e s e models. 71
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While c h o l i n e r g i c approaches t o t h e treatment of cogniBiogenic Amines t i v e d i s o r d e r s have been t h e focus of much c l i n i c a l work, cumulative labo r a t o r y d a t a i m p l i e s a n i m o r t a n t r o l e i n l e a r n i n g and memory f o r t h e a d r e n e r g i c nervous ~ y s t e m . ~ I ~ n ,a d~d ~ i t i o n , t h e r e i s a dramatic l o s s of c e l l s i n t h e l o c u s c o e r u l e u s i n b r a i n s of Alzheimer p a t i e n t s , b u t n o t i n m u l t i i n f a r c t dementia, s u g g e s t i n that t h i s d i s e a s e i s n o t simply a d i s o r d e r of c h o l i n e r g i c There i s a l s o a s u g g e s t i o n t h a t c h o l i n e r g i c i n f l u e n c e s upon memory may involve dopaminergic mechanisms .76
rigi in.^^,^!
C l i n i c a l t r i a l s of amphetamine, methylphenidate, p e n t y l e n e t e t r a z o l , and p i p r a d o l have g e n e r a l l y f a i l e d t o f i n d s i g n i f i c a n t e f f e c t s upon cogn i t i o n i n impaired e l d e r l y s u b j e c t s , although mood improvement and reduct i o n i n f a t i g u e have been noted.74 These results c o n t r a s t w i t h t h e p o s i t i v e e f f e c t s upon performance induced by psychostimulant drugs i n h y p e r a c t i v e c h i l d r e n w i t h a t t e n t i o n a l d e f i c i t d i ~ o r d e r . ~ Only ~ , ~ ~ Hyderginem, which has both alpha-adrenergic and dopaminergic a c t i v i t y , h a s been shown t o have c o n s i s t e n t e f f e c t s i n improving mental performance i n dementia p a t i e n t s , although t h e o v e r a l l degree of improvement i s usually Medical and psychological improvements have a l s o been r e p o r t e d i n normal e l d e r l y v o l u n t e e r s t r e a t e d w i t h Hyderginem.80 Two r e c e n t c l i n i c a l s t u d i e s have found impairment of c o i t i v e f u n c t i o n i n s u b j e c t s t r e a t e d w i t h a n t i h y p e r tens i v e medica t i o n .
r8
I n animals, p r o p r a n o l o l a t t e n u a t e d b o t h t h e memory f a c i l i t a t i o n and amnesia produced by post- t r a i n i n g s t i m u l a t i o n of f r o n t a l c o r t e x i n r a t s t r a i n e d using two l e v e l s of footshock.83 Amphetamine attenuated C02induced amnesia f o r o n e - t r i a l i n h i b i t o r y avoidance i n young r a t s , b u t had no e f f e c t i n o l d animals suggesting a change i n t h e neuromodulatory i n f l u e n c e of catecholamines on memory processes i n s e n e ~ c e n c e . ~A s~ has been r e p o r t e d f o r c h o l i n e r g i c drugs, combinations of a d r e n e r g i c and dopaminergic drugs have produced more improvement i n performance than i n d i v i d u a l drugs alone. The a c t i o n s of t h e c e n t r a l stimulants, c a f f e i n e , s t r y c h n i n e , o r methylphenidate, on r e t e n t i o n of shuttle-box avoidance i n r a t s were much more e v i d e n t when combined w i t h o r o t i c acid.85 p-Chlorophenylalanine f a c i l i t a t e d l e a r n i n g and/or memory when admins t e r e d i n con-
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j u n c t i o n w i t h c a f f e i n e , s t r y c h n i n e , o r amphetamine.86 Apomorphine prot e c t e d a g a i n s t t h e d i s t u r b a n c e s i n conditioned avoidance responding produced by hypobaric h y p ~ x i a . T ~ h~i s a c t i v i t y of apomorphine was blocked by adrenalectomy b u t n o t by domperidone. Apomorphine i n j e c t e d i n t r a hippocampally immediately a f t e r a c q u i s i t i o n of a b r i g h t n e s s discrimi n a t i o n t a s k improved re t e n t i o n and increased incorpora t i o n of L-f ucose i n t h e hippocampus of rats.88
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A number of s t u d i e s have been conducted aimed a t c o r r e Neuropeptides l a t i n g abnormal l e v e l s of a p a r t i c u l a r neuropeptide w i t h Alzheimer's d i s e a s e . The following neurope t i d e s have been s t u d i e d r e c e n t l y : s o m a t o s t a t i n 89990 substance-P,gl n e u r o t e n s i n g2 CCK,g3,g4 VIP,93 TRH,92 g l ~ c a g o n and , ~ ~b e t a - e n d ~ r p h i n . ~Only ~ somatostatin l e v e l s i n CSF and b r a i n appear t o be reduced i n r e l a t i o n t o t h e s e v e r i t y o f t h e d i s e a ~ e . ~ Angiotensin ~ , ~ ~ , ~ c~o n v e r t i n g enzyme a c t i v i t y has been r e p o r t e d t o be e l e v a t e d i n d i s c r e t e b r a i n r e g i o n s from Alzheimer p a t i e n t s when compared t o age-ma tched c o n t r o l s .97
The neurophysiological e f f e c t s g 8 of v a s o p r e s s i n and oxytocin and t h e e f f e c t s of v a s o p r e s s i n on memory99 have been r e c e n t l y reviewed. Behavioral s t u d i e s w i t h t h e s e p e p t i d e s i n d i c a t e t h a t v a s o p r e s s i n enhances, w h i l e oxytocin i m a i r s memory,100,101 and that t h e s e effects a r e c e n t r a l l y mediated. 105 Arginine v a s o p r e s s i n (3) improved p a s s i v e avoidance i n c h i c k s l o 3 and l y s i n e v a s o p r e s s i n (2) was found t o r e v e r s e anisomycin-induced amnesia i n mice. l o 4 Recently, i t h a s been suggested that p e r i p h e r a l l y administered v a s o p r e s s i n e l i c i t s i t s e f f e c t on memory through a n a v e r s i v e property, r e s u l t i n g i n a n a r o u s a l response. l o 5 C l i n i c a l s t u d i e s w i t h v a s o p r e s s i n i n head-injured106 and Alzheimer p a t i e n t s 1 0 7 have f a i l e d t o demonstrate d i f f e r e n c e s i n performance on l e a r n i n g and memory t a s k s between drug and placebo-control groups. A r e p o r t that oxytocin produces amnestic e f f e c t s i n normal human s u b j e c t s h a s been r e p l i c a t e d i n a r e c e n t study.108 A number of c l i n i c a l i n v e s t i g a t i o n s have been conducted w i t h t h e v a s o p r e s s i n analog, 1-desamino-8-D-arginine v a s o p r e s s i n (d esmopres s i n , DDAVP, 51, and have y i e l d e d c o n f l i c t i n g results. P o s i t i v e e f f e c t s on learnin- and memory were observed i n c o g n i t i v e l y unimpaired subj e c t s , l E g * l l o depressed i n d i v i d u a l s , l l O and p a t i e n t s w i t h d i a b e t e s i n s i p i d u s . l l l A 25 pg i n t r a n a s a l dose of DDAVP, however, f a i l e d t o r e v e r s e t h e amnesia i n p a t i e n t s r e c e i v i n g elec troconvulsive shock therapy.l12 T h i s may be d u e t o t h e s i n g l e , r e l a t i v e l y low dose t h a t was used. Other s t u d i e s of DDAVP i n i n d i v i d u a l s w i t h memory d i s o r d e r s f a i l e d t o show any s i g n i f i c a n t i m p r ~ v e r n e n t . l l ~ One , ~ ~ p~ a t i e n t developed a paranoid-like s c h o s i s a f t e r r e c e i v i n g 30 pg of DDAVP by i n t r a The v a s o p r e s s i n analog, desglycinarnide n a s a l administration. a r g i n i n e v a s o p r e s s i n (DGAVP, 5 ) improved l e a r n i n g i n rats on a foodmotivated task.116 However, DGAVP f a i l e d t o produce any b e n e f i c i a l e f f e c t s on memory i n p a t i e n t s s u f f e r i n g from Korsakoff's Syndrome.l17
I
H-Cis-Tyr-Phe-Gln-Asn-Cys-X X = Pro-Arg-Gly-NH2
Pro-Lys-Gly-NH2 Pro-Arg-OH
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(Am) (LVP) 6 (DGAVP)
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22
Vasopressin may s e r v e a s t h e p r e c u r s o r of s m a l l e r molecules with e f f e c t s on l e a r n i n g and memory. The c y c l i z e d d e r i v a t i v e of t h e C-termi n a l d i p e p t i d e of a r g i n i n e v a s o p r e s s i n ( c clo-(Arg-Gly)) was found t o block puromycin-induced amnesia i n mice. lf8 The C- terminal t r i p e p t i d e of oxy t o c i n (L-prolyl-L-leucyl-glycineamide o r PLG) has been r e p o r t e d t o c a u s e a n enhancement of memory i n c h i c k s . l l g I t has been suggested t h a t while v a s o p r e s s i n i s involved i n memory p r o c e s s e s , t h e neuropeptides ACTHIMSH a f f e c t m o t i v a t i o n a l and a t t e n t i o n a l processes.120 However, s t u d i e s comparing t h e e f f e c t s of a number of neuropep t i d e s on s h o r t - term memory i n young a d u l t primates i n d i c a t e d t h a t a-MSH produced some f a c i l i t a t i o n of memory.lZ1 Use of aged primates t o a s s e s s t h e e f f e c t s of neuropeptides on memory suggested t h a t a r g i n i n e and l y s i n e v a s o p r e s s i n produced t h e b e s t o v e r a l l e f f e c t s followed by ACTH4-10. l Z 2 Somatostatin was v i r t u a l l y i n a c t i v e and oxytocin caused a n impairment i n memory i n h a l f t h e aged animals tested. ACTH4-10 h a s a l s o been r e p o r t e d t o be involved i n r e t r i e v a l processes of memory. In r a t s t r e a t e d w i t h a n t i v a s o p r e s s i n serum, which d i s r u p t s s t o r a g e l r e t r i e v a l p r o c e s s e s , ACTH4-10 r e s t o r e d t h e r e t r i e v a l of memory, p o s s i b l y by i n c r e a s i n g t h e s t a t e of arousal.123
The ACTHq-g analog (ORG 2766) h a s been compared to methylpheni d a t e i n h y p e r k i n e t i c c h i l d r e n w i t h a t t e n t i o n a l d e f i c i t d i s o r d e r . 1*4 Although a n e c d o t a l r e p o r t s i n d i c a t e d a n improvement i n t h e s o c i a b i l i t y of t h e s e c h i l d r e n , t h e r e d i d n o t appear t o be a n improvement i n a t t e n t i o n . ACTHq-g h a s a l s o been administered t o p a t i e n t s w i t h severe senile dementia. No improvement was observed a f t e r f o u r weeks of drug t h e r apy.125 There appears t o be some r e l a t i o n s h i p between t h e b e h a v i o r a l e f f e c t s of both ACTH126 and t h e ACTHq-g analog127 and t h e o p i o i d s ys tem. The r o l e of enkephalins and endorphins i n l e a r n i n g and memory pres e n t s a somewhat confusing p i c t u r e . It has r e c e n t l y bee? suggested t h a t t h e a p p a r e n t c o n f l i c t s i n the l i t e r a t u r e may be due t o t h e d i f f e r e n t doses of o p i a t e s used i n p a r t i c u l a r s t u d i e s ; high doses appear t o f a c i l i t a t e l e a r n i n g w h i l e l o w doses cause a d e ~ r e m e n t . ~The ~ ~e,f f~e c~t s~ o f t h e endorphins on animal l e a r n i n g have been r e c e n t l y reviewed, and t h e p r o p o s a l made that d i s t u r b a n c e s i n the endor h i n system may l e a d t o a t t e n t i o n a l d e f i c i t d i s o r d e r s i n c h i l d r e n . 138 Actions of o p i a t e a n t a g o n i s t s , such as naloxone, on l e a r n i n g and memory have been s t u d i e d i n both l a b o r a t o r y animals and humans. Again, c o n f l i c t i n g results have been reported. Naloxone and diprenorphine have been r e p o r t e d t o enhance t h e s p a t i a l memory of rats t r a i n e d on a n 8-arm r a d i a l maze.131 Another i n v e s t i g a t i o n , however, found t h a t naloxone had a d i s r u p t i v e e f f e c t on a l e a r n e d behavior i n r a t ~ . 1 Cognitive ~ ~ impairments were e v i d e n t a f t e r i n f u s i o n s of 2 mg/kg naloxone i n normal i n d i v i d u a l s ; 33 no changes i n c o g n i t i v e performance were observed a t lower doses. R e s u l t s from a double-blind, placebo-controlled study of naloxone i n seven p a t i e n t s w i t h Alzheimer's Disease i n d i c a t e d a t l e a s t temporary p o s i t i v e e f f e c t s on cognition,134 however o p e n - t r i a l s conducted a t f o u r separate c e n t e r s d i d n o t show any e f f e c t 1 3 . l ~ ~ nootropic (noos = thought o r reason; t r e p o = t u r n drug which s e l e c t i v e l y a f f e c t s higher c e r e b r a l chemistry and pharmacology of n o o t r o p i c s have been r e c e n t l y reviewed.139 Most examples of t h i s c l a s s a r e comprised o f gamma lactams which a r e chemically related t o t h e folded conforma t i o n
36 -
Sect. I
- CNS Agents
Hesp, Ed.
of GABA. These include piracetam etiracetam (8), pramiracetam (CI-879, 9 ) , oxiracetam (ISF-2522, and aniracetam (Ro 13-5057, 11). ~ ~ ,neurochemicall42 1~~ e f f e c t s of praThe n e u r o ~ h a r m a ~ o l o g i c a l ~and miracetam s u g g e s t t h a t t h e compound i s a c o g n i t i o n a c t i v a t o r . A c l i n i c a l study involving computerized q u a n t i t a t i v e EEG a n a l y s i s i n d i c a t e d t h a t t h i s drug a c t s a s a v i g i l a n c e enhancer, 143 Preliminary f i n d i n g s from a n open-label study of pramiracetam i n Alzheimer p a t i e n t s w i t h mild t o moderate co n i t i v e d e c l i n e i n d i c a tea a n improvement i n goal-direc ted behavi o r 14& Oxiracetam has a l s o been s t u d i e d i n e l d e r l y p a t i e n t s w i t h o r g a n i c b r a i n syndrome, and p o s i t i v e e f f e c t s have been n 0 t e d . 1 ~ ~Aniracetam improved impaired l e a r n i n g and memory i n rodents,146 and produced some b e n e f i c i a l e f f e c t s i n aged p a t i e n t s i n a g e r i a t r i c n u r s i n g home s e t t i n g . 147 F i n a l l y , c o g n i t i o n a c t i v a t i n g p r o p e r t i e s have been 3-Phenoxypyridine (CI-844, d e s c r i b e d f o r a series of 3 - a r y l o q p y r i d i n e s . 1 2 ) was found t o be the most a c t i v e member of t h i s series i n enhancing l e a r n i n g and/or memory i n a s i n g l e t r i a l passive avoidance t a s k i n mice r e t e s t e d one week a f t e r training.148
(l), 10)
.
R
0
I
Q
I
0
R' -CHCNHR"
-7,
a
R = R' = R" = H
8, R = R" = H; R' = E t 9, -
Q3
no N
R = R' = H; R" = CH CH N(i-Pr)* 11 12 10, R OH;2R'2= R" = H Summary and Conclusions - The l a s t few y e a r s have seen s i g n i f i c a n t p r o g r e s s made i n e f f o r t s t o understand and t r e a t c o g n i t i v e d i s o r d e r s . With t h i s progress has come a n even g r e a t e r a p p r e c i a t i o n of t h e complexity and r i c h n e s s of t h e p h y s i o l o g i c a l e v e n t s underlying c o g n i t i v e f u n c t i o n and t h e d i f f i c u l t i e s f a c i n g t h e physician and s c i e n t i s t i n f i n d i n g e f f e c t i v e treatments f o r c o g n i t i v e d i s o r d e r s . 3
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-
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Cognitive Disorders
P. L. Wood, P. Etienne, S. Lal, S . Gauthier, S . Cajal and N. P . V. Nair, L i f e S c i . , 2, 2073 (1982). P. Davies and R . D. T e r r y , Neurobiol. Aging, 2, 9 (1981). H. A . C r y s t a l and P. Davies, J. Neurochem., 1 7 8 1 (1982). J. A . Biggins, E . K. P e r r y , J. R . McDermott, A . I . Smith, R . H. Perry and J. A . Edwardson, J . Neurol. S c i . , = , 117 (1983). R . H . P e r r y , G . J . Dockray, R . Dimaline, E. K. P e r r y , G. Blessed and B . E . Tomlinson, J . Neurol. S c i . , 465 (1981). D. J. Sanders, S . Zahedi-As1 and A . P. Marr, Prog. Brain Res., 55, 465 (1982). H . Kaiya, T . Tanaka, K. Takeuchi, K. Morita, S . Adachi, H. Shirakawa, H . Ueki and M . Namba, L i f e S c i . , 33, 1039 (1983). E. K. P e r r y , G . Blessed, B . E . Tomlinson, R . H. P e r r y , T . J. C r o w , A . J. Cross, G . J . Dockray, R . Dimaline and A. Arrequi, Neurobiol. Aging, 2, 2 5 1 ( 1 9 8 1 ) . A. Arrequi, E. K. P e r r y , M. Rossor and B. E. Tomlinson, J . Neurochem., 38, 1490 (1982). R. E. J. Dyball and A. T. Paterson, P h a m c . Ther., 20, 419 (1983). D. M . Gash and G. J. Thorns, Trends Neurosci., 197 (1983). T . B . van Wimersma Greidanus, J. M. van Ree and D. de Wied, Pharmac. T h e r . , 20, 437 (1983). G. L. Kovacs and G. Telegdy, Pharmac. T h e r . , 375 (1982). G. Meisenberg and W. H . Simmons, Neurosci. Biobehav. Rev., 7, 263 (1983). J. L. Davis, R. M . Pic0 and A. Cherkin, Behav. Neur. B i o l . , 242 (1982). M . E. Judge and D. Quartermain, Pharmacol. Biochem. Behav., 463 (1982). '' A . E t t e n b e r g , D. van der'Kooy, M. Le Moal, G. F . Koob and F . E. Bloom, Behav. Brain Res., 3 3 1 (1983). W. D. Fewtrell, A . 0. House, P. F. Jamie, M. R. Oates and J. E. Cooper, Psychol. Med., 2, 423 (1982). R. Durso, P. Fedio, P. Brouwers, C. Cox, A. J. Martin, S. A . Ruggieri, C . A. Tamminga and T. N. Chase, Neurology, 32, 6 7 4 (1982). D. J. Kennett, M . C. Devlin and B. M. F e r r i e r , L i f e S c i . , z , 273 (1982). B. E. Beckwith, T . P e t r o s , S. Kanaan-Beckwith, D. I. a u k , R. J. b u g , and C . Ryan, P e p t i d e s , 2, 627 (1982). H. Weingartner, P. Gold, J. C. Ballenger, S. A . Smallberg, R. Summers, D. R. Rubinow, R . M . P o s t and F. K. Goodwin, Science, 211, 6 0 1 (1981). F. L a c z i , J. M . van Ree, A. Wagner, 2s. Valkusz, T . Jardanhazy, G. L. Kovacs, G. Telegdy, J. S z i l a r d , F. A. Laszlo and D. d e Wied, Acta. Endo205 (1983). crinol., B. L e r e r , T. Zabow, N . Egnal and R. H . Belmaker, Biol. P s y c h i a t . , Is, 8 2 1 (1983). J . S . J e n k i n s , H . M . Mather and A . K. Coughlan, J . Neurol. Neurosurg. P s y c h . , '5, 8 3 0 (1982). J . R . Tinklenberg, A . Pfefferbaum and P. A. Berger, Psychopharmacol. Bull., 206 (1981). G . B . C o l l i n s , D. J. Marzewski and M . B . R o l l i n s , Lancet, 11, (8250) 808 (1981). R. B . Messing and S . B . Sparber, E u r . J. Pharmacol., E, 43 (1983). F. L a c z i , J. M . van Ree, L. Balogh, A . Szasz, T . Jardanhazy, A . Wagner, L . Gaspar, Z . Valkusz, I . Dobranovics, J . S z i l a r d , F. A , Laszlo and D . de Wied, Acta. Endocrinol., 177 (1983). R . Walter, P. L. Hoffman, A . C . Church, J. B . Flexner and L. B . Flexner, Horm. Behav., 3, 234 (1982). J . L. Davis, R . M . Pic0 and A. Cherkin, Pharmacol. Biochem. Behav., 893 (1982). D. de Wied and J. M . van Ree, L i f e S c i . , s , G O 9 (1982). G. A . Olson, R . Roip-Smith, M. D. Mauk, G . J . La Hoste, D. H . Coy, C . W . Hill and R . D. Olson, P e p t i d e s , 2, 1 3 1 (1981). R. T. Bartus, R : L. Dean and B . Beer, Neurobiol. Aging, 6 1 (1982). T . B . van Wimersma Greidanus, P e p t i d e s , 2, 7 (1982). H . J . B u t t e r , Y. L a p i e r r e , P . F i r e s t o n e and A. Blank, J . Clin. Psychopharmacol., 3, 226 (1983).
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J. C. Martin, B. R . B a l l i n g e r , L. L. Cockram, F. M. McPherson, R. M. Pigache and D. Tregaskis, Acta. P s y c h i a t r . Scand., 7 ,205 (1983). 126. I . Izquierdo and R . D. Dias, Psychoneuroendocrinol., 8, 81 (1983). 127. M . Fekete, F. Drago, J. M . van Ree, B. Bohus, V. M . Wiegant and D. d e Wied, L i f e S c i . , 32, 2193 (1983). 128. J. D. B e l l u z z i and L. S t e i n , Ann. N.Y. Acad. S c i . , 398, 221 (1982). 129. C. C a s t e l l a n o , Behav. Neur. B i o l . , 36, 189 (1982). 130. S. R . Rose and J. Orlowski, J. Dev. Behav. P e d i a t r . , 6 , 131 (1983). 131. M. Gallagher, R . A . King and N. B. Young, Science, 221, 975 (1983). 132. W. C. Lynch and S . Clark, L i f e S c i . , 32, 2801 ( 1 9 8 3 r 133. M . R. Cohen, R. M . Cohen, D. P i c k a r , H . Weingartner and D. L. Murphy, Arch. Gen. P s y c h i a t . , 0, 613 (1983). 134. B. Reisberg, S. H. F e r r i s , R. Anand, P. Mir, V. Geibel and M . J. De Leon, N. Engl. J. Med., 308, 721 (1983). 135. J. P. Blass, M. J. Reding, D. Drachman, A. M i t c h e l l , G. Glosaer, R. Katzman, L. J. Thal, S . G r e n e l l , J. E. Spar, A . Larue and E. L i s t o n , N. Eng. J. Med., 309, 556 (1983). 136. T. M. I t i l , Biol. P s y c h i a t r . , 521 (1983). 137. S. Kancwski, Drug Dev. Res., 2, 433 (1982). 138. C. E. Giurgea, Drug Dev. Res., 2, 441 (1982). 139. B. J. R . Nicolaus, Drug Dev. Res., 2, 463 (1982). 140. T. A . Pugsley, B. P. H. Poschel, D. A. Downs, Y. H. S h i h and M. I. Gluckman, Psychopharmacol. B u l l . , 2, 721 (1983). 141. B. P. H. Poschel, J. G . M a r r i o t t , and M. I. Gluckman, Psychopharmacol. Bull., 720 (1983). 142. T. A . Pugsley, Y. H . Shih, L. Coughenour and S . F. S t e w a r t , Drug Dev. Res., 2, 407 (1983). 143. T. M. I t i l , S. Mukherjee, G. Dayican, D. M. Shapiro, A. M. Freedman and L. A. Borgen, Psychopharmacol. B u l l . , 19, 708 (1983). 144. R. J. Branconnier, J. 0. Cole, E. C. Dessain, K. F. Spera, S. Ghazvinian and D. D e V i t t , Psychopharmacol. B u l l . , 726 (1983). 145. T. M. I t i l , G. N. Menon, M. Bozak and A. Songar, Drug. Dev. Res., 2, 447
125.
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Chapter 5 . Adaptive Changes in Central Nervous System Receptor Systems B. Kenneth Koe and Fredric J. Vinick Pfizer Central Research, Groton, CT 06340
Introduction -The phenomenon that repeated (subacute, chronic) administration of drugs can induce pharmacological effects that become progressively a t t e n u a t e d or accentuated compared t o the original effects of a single (acute) dose is well known. In the past decade, the molecular events involved in the rapid loss o f the ability o f the norepinephrine (NE) receptorcoupled adenylate cyclase system of intact cells to generate cyclic AMP (CAMP) f o l l o w i n g stimulation o f P-adrenoceptors by P-agonists have been w e l l studied.l.2 In such cells, isoproterenol stimulates the formation of CAMP, but continual exposure t o this agonist induces a refractoriness, characterized by a rapid desensitization of agonist-stimulated adenylate cyclase and a slower loss o f P-adrenoceptors. The reduction in sensitivity is measured as a change in maximal.response of the adenylate cyclase, while t h e loss in padrenoceptors (down-regulation) is ascertained as a decrease in receptor density (determined by Scatchard analysis of saturation binding o f antagonists). The K0.5 values for adenylate cyclase activity and the KD values (affinity-1) for antagonist binding are unchanged i n desensitization and down-regulation, respectively. Prior t o the irreversible disappearance of 0-adrenoceptors, an uncoupling process occurs; although the number of receptors remains essentially the same, the sensitivity of these receptors t o isoproterenol is less than t h a t o f untreated cells. A rapid and reversible desensitization and down-regulation of the p-receptors of rat cerebral cortex is observed on incubation of tissue slices with isoproterenol3 or desipramine (DMI), an NE uptake blocker.4 Similar adaptive changes in the receptors o f the NE system also occur in wiwo in the brain when NE levels are altered. Prolonged NE depletion can be effected by drugs (reserpine) or selective lesions (6-hydroxydopamine [6-HDA] or electrolytic), whereas prolonged elevation can be achieved by preventing metabolism ( M A 0 inhibitors) or increasing synaptic concentrations through inhibition o f reuptake.fi.6 This chapter examines the recent literature on adaptive changes in central NE, serotonin (5-HT) and dopamine (DA) receptor systems and concentrates on drug-induced effects which could relate to therapeutic modes o f action in human diseases. Reviews o f related topics have appeared .7-10
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Norepinephrine Receptors Depression is a CNS disease believed to be associated with a perturbation of central NE transmission. The first effective drugs in depression were M A 0 inhibitors and tricyclic uptake blockers which increase the concentration o f neuronal or synaptic NE, respectively. With the recognition that the onset of action of antidepressants was several weeks, attention turned t o studying the effects o f subacute and chronic administration of antidepressant drugs. In rats, multiple treatments w i t h antidepressant drugs1 1-14 and electroconvulsive shock (ECS)11.15-17 desensitized the NE receptor-coupled adenylate cyclase system in limbic forebrain slices and decreased the number of ji-adrenoceptors measured by antagonist binding. In contrast t o the subsensitivity observed after these chronic treatments, acute administration showed n o effects. In general, antidepressants,6.l1.12.18-34 M A 0 inhibitors,5.9,21,23.37.38 lithium3536 and REM sleep deprivation39 have all been shown t o induce desensitization andlor down-regulation of NE receptors. Administration of multiple daily doses of drug produces down-regulation faster than single daily dosing and allows a lower dose per injection.6.29 In a time course study in rats, D M I (10 mglkg b.i.d.) elicited maximal desensitization of CAMP accumulation by isoproterenol in cortical slices in 5 t o 7 days and down-regulation o f P-adrenoceptors in cortical membranes after 7 days. After D M I withdrawal, the subsensitive NE receptors returned to normal within a week.6 After multiple ECS treatments, 0-adrenoceptors remain down-regulated for at least a
ANNUAL REPORTS IN MEDICINAL CHEMISTRY-19
Copyright 0 1984 by Academic Press, Ino. All rights of reproduction in any form reserved. ISBN 0-12-040519-9
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week.15 These studies indicate that faster onset of therapeutic effects might be achieved by rapidly increasing the dose of antidepressants. Since side effects may preclude such a strategy, other ways t o facilitate the desensitization/down-regulationhave been investigated. The coadministration o f a2-adrenergic antagonists (yohimbine, d i h y d r o e r g o t a m i n e or phenoxybenzamine) w i t h D M I and other antidepressants increased t h e rate o f downregulation.a-42 In contrast, the al-antagonist, prazosin, was ineffective in accelerating downregulation.42 Rapid desensitizationldown-regulation of Padrenoceptors by co-administration of iprindole and amphetamine occurred at doses of each which were ineffective alone.43.44 On the other hand, co-administration o f chlorpromazine with imipramine failed t o enhance the down-regulation of 6-receptors observed w i t h imipramine alone, even though this combination is reportedly more effective in treating delusional depression.45 Unlike most studies, some recent reports claim down-regulation of 6-adrenoceptors following a single ECS treatment or single doses of DMI on days 1 and 17.46 One explanation for the facilitation of down-regulation o f 6-adrenoceptors by a2adrenergic antagonists i s as follows: the expected synaptic build-up of NE after DMI is attenuated by concurrent stimulation of a2-autoreceptors, which inhibits NE release. Only when these a2-autoreceptors become down-regulated will synaptic levels of NE increase, thus accounting in part for the slow onset of therapeutic defects.47 The notion that repeated administration of antidepressants or ECS can induce subsensitivity of a2-autoreceptors is based on the tolerance t o various actions of low doses of clonidine in mice or rats induced by such regimens.48.49 Chronic administration of clorgyline desensitized a2-autoreceptors and downregulated &adrenoceptors before a decrease in the CAMPresponse t o NE could be observed.38 The a2-adrenoceptor adaptation elicited by clorgyline reverted t o normal very gradually.50 One potential disadvantage of using a2-antagonists i s that autoreceptors may quickly become supersensitive. Reduced antagonism of responses t o clonidine after long-term administration of mianserin, an a2-antagonist, may be the result of the development o f supersensitive autoreceptors.s1,52 Some authors propose that depression may be caused in part by supersensitivity of a2-adrenoceptors and report evidence t h a t chronic antidepressant treatments elicit down-regulation of these receptors.53.54 In other studies, however, an upregulation of a2-adrenoceptors has been observed.55.56 In contrast, al-adrenoceptor binding is not affected by chronic administration of antidepressants.21 The subsensitive P-adrenergic system resulting from chronic administration o f antidepressants implies that postsynaptic responsiveness t o NE is diminished, e.g., long-term administration of DMI, clomipramine, maprotiline or tranylcypromine reduced the sensitivity of cingulate cortical neurons t o iontophoretically applied NE.57 Similarly, chronic DMI markedly decreased the responsiveness of Purkinje cells in cerebellum t o applied NE.58 In contrast, other studies have shown that chronic antidepressant treatments enhanced postsynaptic NE and 5-HT responses in the facial motor neurons.59 Possibly related t o the enhanced NE responses is the finding that chronic antidepressant treatment can apparently facilitate the interaction o f the guanine nucleotide binding protein w i t h the catalytic protein.60 The subgroup of antidepressants comprising the selective 5-HT uptake blockers, zimelidine,24.31 fluvoxamine33 and sertraline34 also down-regulate j3-adrenoreceptors. Such agents may induce this subsensitivity by the removal of an inhibitory 5-HT constraint on NE neurons through inhibition of firing of raphe 5-HT neurons34 or enhancement o f 5-HT reuptake.6’ The notable exception is fluoxetine.21.25,26,30 In rats deprived of a 5-HT input, via lesions with 5,7-dihydroxytryptamine (5.7-DHT) or by p-chlorophenylalanine (PCPA), chronic administration of DMI failed t o down-regulate 6adrenoceptors, although the usual desensitization of NE sensitive adenylate cyclase was still observed.62.63 When NE receptors were desensitized and reduced in number by the DMI treatment, PCPA reversed the decrease o f preceptors without affecting the cyclase.14 These persistent &receptors may be “uncoupled”, since their affinity for isoproterenol is reduced.64 Possibly, the step from uncoupled receptors t o actual disappearance o f NE receptors is interrupted by the loss o f 5-HT input. An interesting corollary is the observation that depressed patients benefiting from either imipramine or tranylcypromine showed
Chap. 5
A d a p t i v e Changes i n CNS Receptor Systems Koe, V i n i c k
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reemergence of their depressive symptoms when PCPA was administered along w i t h the antidepressant.65.66 Since drugs for the treatment of depression appear t o increase NE activity, P-agonists may be therapeutically effective in this disorder. Antidepressant activity has been reported following administration o f the P-agonist, salbutamol, w i t h a suggestion of more rapid onset of acti0n.67~68 The time course of clinical improvement w i t h oral salbutamol paralleled the induction of subsensitivity of a plasma NE-sensitive adenylate cyclase.68 Subacute infusion of the P-agonist, clenbuterol, was found t o desensitize isoproterenol-stimulated adenylate cyclase and down-regulate P-adrenoceptors i n the rat cerebellum.69 In contrast, the pantagonist, propranolol, not only blocked the effects o f chronic DMI but also elicited upregulation of P-receptors and enhanced sensitivity o f isoproterenol-sensitive adenylate cyclase.6 Subacute administration of phenoxybenzamine or propranolol also increased the cAMP response t o NE in mouse limbic forebrain.70 Interestingly, major depressive episodes have been reported for several patients on propranolol therapy.71 Unlike the rat experiment described above,6 propranolol did not attenuate the therapeutic effects of an antidepressant in a human study.72 Pharmacological treatments which deplete neurons of NE,70.73 and 6-HDA74 or DSP-4 induced lesions,75 increased the cAMP response t o NE or isoproterenol, and j3-adrenoceptor density. Unilateral electrolytic lesions o f the locus ceruleus prevented the desensitizing effects of D M I or iprindole on the lesioned side only.76 The P-adrenoceptors involved in down-regulation or up-regulation in rat cortex belong mainly t o t h e p1 subtype.77 A subpopulation of receptors w i t h neither a nor P characteristics is also coupled t o the adenylate cyclase of rat limbic forebrain78 Desensitization o f the P-receptor system by repeated administration of antidepressants also occurs in the pineal gland, which i s innervated by postganglionic sympathetic fibers. The stimulation of the P-receptor/adenylate cyclase system increases the synthesis of serotonin-Nacetyl transferase, a critical enzyme for melatonin production. Thus, the subsensitivity o f pineal P-adrenoceptors found after chronic D M I or nialamide was accompanied by a fall in melatonin formation stimulated by isoproterenol or darkness.79 Chronic administration o f amitriptyline, maprotiline and clenbuterol also attenuated the increase of pineal melatonin stimulated by isoproterenol, but iprindole, mianserin, fluoxetine and repeated ECS had n o effect80 Attempts t o detect subsensitivity of the P-receptors in patients treated w i t h D M I by monitoring night-time plasma melatonin levels failed.81 The exclusive P-adrenergic regulation of melatonin synthesis seen in rats may not extend t o man.82 Stress applied t o rats (electric foot shock) induced desensitization o f NE receptorcoupled adenylate cyclase in cortical slices.83 This f i n d i n g suggests t h a t a similar desensitization could be a natural response t o prolonged emotional stress. Thus, the NE receptors i n depressed patients may be unable t o down-regulate i n response t o stress, necessitating antidepressant therapy.84 In another study, however, t h e number o f padrenoceptors in rats was not altered by chronic foot shock stress.15 Most studies on adaptive changes in the P-adrenergic system focus on cerebral cortex, frontal cortex, limbic forebrain and hippocampus. Although ECS did n o t down-regulate P-adrenoceptors i n striatum, hypothalamus or cerebellum,15 chronic drug treatments d o cause a decrease in the density of P-receptors in these brain regions.85.86.69 Adaptation of NE receptors is also observed after long-term administration of other types of drugs. For example, chronic chlorpromazine desensitized cortical NE-sensitive adenylate cyclase in rats18 but caused supersensitivity in mouse limbic forebrain.70 Chronic haloperidol did not desensitize cortical NE-stimulated adenylate cyclase73 b u t apparently induced up-regulation of a2-adrenoceptors (enhanced locomotor response t o clonidine) in mice.87 The methylxanthines (caffeine,88 pentoxyfylline89), which increase turnover of brain NE, caused rapid down-regulation of p-adrenoceptors. Serotonin Receptors - The relationships between 5-HT receptors and the actions of drugs are much less clear than those for NE or DA receptors. Numerous conflicting findings result from the inherent difficulties of relating behavioral and electrophysiological effects. Also, the aforementioned effects d o not correlate well with drug-induced changes in binding sites that
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are poorly defined functionally. Serotonin receptors are termed 5-HT1 (based on 3[H]5-HT bindingso) or 5-HT2 (based on 3[H]spiperone binding in frontal cortical membranes).21.91 The latter sites are also labeled by 3[Hlketanserin92 or 3[Hlmianserin.93,94 Serotonin agonists exhibit nanomolar affinities for 5-HT1 receptors but only micromolar affinities for 5-HTz binding sites. A 5-HT sensitive adenylate cyclase has been characterized, but i t s connection with 5-HT1 or 5-HT2 receptors is not clear.91.95.96 Increases in hippocampalso and cortical21 5-HT1 receptors are found i n rats lesioned w i t h 5,7-DHT. Similar lesions increased 3[H]mianserin binding sites61 but not 3[Hlspiperone97 or 3IHlketanserin binding.98 Chronic PCPA treatment effected increases in 5-HT199 and 5-HT2 receptors (3[H]spiperone binding)loo and 3[H]mianserin binding sites.61 The binding of 3[H]imipramine t o brain membranes i s supposedly associated with 5-HT uptake sites, but there is a question whether 3[H]imipramine labels a physiologically relevant site.101 3[Hllmipramine binding sites are decreased in rats with 5,7-DHT lesions but remain unchanged in rats following chronic treatment with chronic PCPA.61 The role of serotonergic changes in depression and antidepressant therapy has been a subject of intense study. For example, brains of suicide victims exhibited an increase in cortical 5-HT2 binding sites102.103 and either an increase103 or a decrease104 in ,[H]imipramine binding. Furthermore, cortisol secretion stimulated by 5-hydroxytryptophan was found t o be greater for depressed and manic patients than normal controls, suggesting the involvement of supersensitive 5-HT receptors.105 Thus, an increased risk t o suicide and depression may be characterized by a decrease in serotonergic function.106 If up-regulation of 5-HT receptors is associated with suicide and depression, then the question arises as t o whether antidepressant therapy normalizes 5-HT receptor density. Several investigators have, reported that chronic administration of M A 0 inhibitors results in a decrease in the number of 5-HT1 receptors (KO unchanged) in rat cortex or brain stem,99.107 inhibits the "5-HT syndrome" induced by 5-HT agonistslos and attenuates the response of cortical neurons t o microiontophoretically applied 5-HT.109 However, variable results have been obtained with tricyclic antidepressants. Many studies have indicated that chronic administration of these drugs does not down-regulate 5-HT1 receptors,21-23.lo7.111.112 but there are a few e x c e p t i o n s . ~ ~ ~ ~ One ~ ~ group . ~ 1 3 reported a decrease in t h e number o f 3[H]5-HT binding sites after long-ter DMI, imipramine, amitriptyline or dimetacrine treatments.113 Another group found that chronic treatment with imipramine or DMI produced high and low affinity 5-HT1 receptors in rat dorsal cortex.112 Even 5-HT specific uptake blockers have led t o conflicting findings: chronic clomipramine had no effect on 3[H]5-HT binding,llo whereas D-fenfluramine led t o a decrease in 3[H]5-HT binding (lowered KD and Bmax).114.115 Fluoxetine was found by several groups not t o alter 5-HT1 receptors;l9,2l.lll.l16 however, others have reported either a reduction in the number of high affinity 5-HT1 receptors in frontal cortex117 or a decrease in the affinity of 5-HT1 receptors in dorsal cortex.112 Similarly, one chronic study with zimelidine failed t o show downregulation of 5-HT1 receptors in rat cortex,llE while a different group observed an induction of high and low affinity 3[H]5-HT binding sites, amounting t o a "selective down-regulation" of 5-HT1 receptors.112 Chronic lithium treatment can also alter S-HTI receptors as evidenced by the fact that receptor density decreased in the rat hippocampus; no change was observed in the cortex.119 The 5-HT autoreceptor blocker, methiothepin, facilitated the down-regulation of 5-HT1 receptors induced by imipramine or mianserin, presumably by increasing 5-HT release.120 Chronic administration, of imipramine, DMI, amitriptyline, iprindole or pargyline down-regulated 3[H]spiperone-labeled5-HT2 receptors in rat brain.21.97.121 Pretreatment with the serotonergic neurotoxin, p-chloroamphetamine (PCA), did not lessen the effectiveness of amitriptyline, which suggests that the drug itself may be interacting with postsynaptic receptors. Interestingly, the PCA treatment, while reducing 5-HT uptake drastically, had no effect on 5-HT2 receptors.97 Chronic administration of mianserin decreased the number of 5-HT2 sites (3[H]spiperone binding) in rat cortex.122 Even a single dose of this drug lowered 5-HT2 binding.100.122 Similarly, amoxapine and loxapine were able t o reduce the density of 5-HT2 receptors after both acute and chronic treatment.100 Clozapine blocked the binding of 3[H]spiperone t o frontal cortex in intact mice as effectively as amitriptyline;123 long-term administration of clozapine decreased the number of 5-HT2 sites labeled by 3[H]ketanserin.124 Rats with lesioned 5-HT neurons did not show alterations in 5-HT2 receptor density as
Chap. 5
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measured by 3[H]spiperone,97 3[H]LSDl25 or 3[H]ketanserin binding.98 In contrast, chronic PCPA treatment increased 5-HT2 receptor binding.100 Chronic administration of imipramine decreased 3[H]ketanserin binding and 3[H]spiperone binding21 without affecting the binding of 3[H]mianserin,98 which also labels 5-HT2 receptors.94 Repeated administration of mianserin itself did n o t down-regulate 3[Hlmianserin binding61 nor did lesions of presynaptic 5-HT neurons.93 In fact, up-regulation of these binding sites has been reported after either lesions or PCPA treatment61 Electroconvulsive shock, unlike antidepressant drugs, caused an increase in the number of 5-HT2 binding sites labeled by 3[H]spiperone in rat cortex.126.127 Receptor affinity was unchanged as was 5-HT1 binding.128 Amitriptyline administered together w i t h the a2-antagonist, yohimbine, decreased 5-HT2 receptor binding after 4 days compared to 10 days for amitriptyline alone.129 Phenoxybenzamine and dihydroergotamine were also effective, whereas the specific al-antagonist, prazosin, was not. Very recently, it has been shown that subacute co-administration of imipramine and chlorpromazine produced a greater reduction of 5-HT2 binding in rat cortex than either drug alone;130 in contrast, this same drug combination had no effect on a2- and 0-receptor densities.45 Repeated administration of imipramine diminished 3[H]imipramine binding in rat hippocampus but not in cereb.ellum or cortex; in the same animals, down-regulation o f preceptors was induced in the latter regions but n o t in the hippocampus.131 The downregulation of 3[H]imipramine binding sites by chronic imipramine was accompanied by an increase in 5-HT reuptake in hippocampal slices.61 It was proposed that chronic administration of imipramine and DMI may effect the desensitization/down-regulation of 0-adrenoceptors by eventually facilitating 5-HT reuptake, thereby attenuating the inhibitory effects of 5-HT on NE neurons.61 This diminished 5-HT function could also lead t o the enhanced responses t o 5-HT observed after chronic tricyclics.132 The down-regulation of 3[H]imipramine binding sites I S supposedly a reduction in the effector sites for an "endogenous modulator'' (inhibitory) of 5-HT reuptake.61 It is notable that either deprivation of REM sleep or chronic ECS treatment can down-regulate 3[H]imipramine sites as well as P-receptors.39 However, iprindole and mianserin, w h i c h produce subsensitivity o f NE receptors, d o n o t d o w n - r e g u l a t e 3[H]imipramine sites.61.131 Chronic tricyclic antidepressants, but not fluoxetine, femoxetine, or chlorpromazine, increased sensitivity of postsynaptic forebrain neurons t o microionotophoretically-applied 5-HT.132 Similar treatments did not affect the sensitivity of raphe 5-HT autoreceptors, as assessed by the effect o f LSD on neuronal firing rates.133 Electrophysiological studies following chronic zimelidine treatment revealed enhanced 5-HT neurotransmission o n electrical stimulation o f rat hippocampus but not an enhanced responsivity of hippocampal neurons t o applied 5-HT.134 These results are at variance w i t h the notion t h a t long-term zimelidine may attenuate 5-HT transmission112 and w i t h the observation t h a t chronic antidepressants increase postsynaptic functional sensitivity.59.132 Acute zimelidine inhibited the firing of raphe 5-HT neurons, but the effect became progressively less w i t h continued drug administration. After 14 days of zimelidine, the firing rate returned t o normal, but the response o f raphe 5-HT autoreceptors t o intravenous LSD was much less.134 These findings suggest that the autoreceptors mediating the firing rate of 5-HT neurons become desensitized by continuat exposure t o this 5-HT uptake blocker. The postsynaptic 5-HT f u n c t i o n a l supersensitivity elicited by chronic clomipramine was n o t blocked by chronic l i t h i u m administration.135 Acute administration of amitriptyline, imipramine, trazodone, mianserin or viloxazine (but not D M I or iprindole) antagonized the "head-twitch" response t o 5-methoxy-N,Ndimethyltryptarnine (5-MeODMT) in mice. Chronic administration o f the same agents, including D M I and iprindole but excluding mianserin or viloxazine, e l i c i t e d enhanced responsiveness t o this 5-HT agonist.136 Recently, the head twitch response has been ascribed t o 5-HT2 receptor stimulation, whereas the 5-HT syndrome may involve activation of 5-HT1 receptors.137 Antagonism of the head twitch response by acute tricyclic drugs but enhanced responses after chronic treatments suggest that "receptors" blocked after one dose should be up-regulated by multiple doses. Instead, the effects of chronic tricyclic antidepressants o n 5-HT2 receptors (down-regulation) and the behavior presumably mediated by these receptors are opposite. As noted earlier, mianserin can down-regulate 5-HT2 receptors after acute or
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chronic administration; both treatments also antagonized the head twitch response in mice.122.136 The up-regulation o f 5-HT2 receptors by multiple ECS treatments i s accompanied by enhanced behavioral responses t o 5-HT agonists.126 Thus, the effects on 5-HT2 receptors seem t o parallel a behavioral response for mianserin and ECS but not other antidepressant drugs. However, a recent investigation showed parallel increases in the 5-HT head twitch response and 5-HT2 receptor density following chronic DMI treatment.138 Finally, another study reported that 5-HT1 receptor binding was unaltered, whereas 5-HT2 binding was decreased after chronic administration of clomipramine or fluoxetine. The 5-HT syndrome in the clomipramine and fluoxetine-treated rats from 5-MeODMT was also diminished.111 This result supports other findings that postsynaptic activity appears t o be attenuated following long-term administration of 5-HT uptake blockers,ll2 but the behavioral subsensitivity t o 5-HT agonists is in contrast t o the supersensitivity t o microiontophoretically-applied 5-HT in chronic clomipramine treated rats.135 Amitriptyline, trazodone and other agents, in fact, may function as antagonists of 5-HT at the postsynaptic receptor.139-141 Chronic treatment with zimelidine induces a lower affinity 5-HT1 site in both rat cortex and hypothalamus, in concert with the persistent reduction o f 5-HT synthesis. These effects might result in reduced presynaptic and postsynaptic activity.112.142 A similar decrease in 5-HT transmission after chronic 5-HT uptake blockers could be responsible for their ability t o desensitize and downregulate P-adrenoceptors.34 Other investigators have proposed that antidepressant drugs alter the structure of the postsynaptic 5-HT receptor t o give a higher affinity 5-HT binding form which is inactive, i.e., no longer coupled t o a 5-HT sensitive adenylate cyclase.143 The relevance of these findings to the action o f antidepressant drugs is obscure, since M A 0 inhibitors were ineffective. Dopamine Receptors - All antipsychotic drugs (neuroleptics) are known to block DA receptors. The number of receptor subtypes claimed for DA, based on binding studies, include "D-1, D-2, D-3 and D-4".144.145 However, only t h e postsynaptic D-2 receptors (labeled by 3[H]butyrophenones) are most consistently relatable t o the effects of drugs. The D-1 subtype, a DA-stimulated adenylate cyclase site, has no clearcut function in brain.146 Recent studies on agonist binding disclosed that 3[H]DA binds t o t w o sites, both postsynaptic; one is related t o D-2 sites and the other t o D-1 sites.147 Changes in DA receptors are of particular relevance t o three major types o f CNS disorders, schizophrenia, tardive dyskinesia and Parkinson's disease. Strong evidence points t o the involvement of the DA system in many of the clinical manifestations of schizophrenia. Several studies have been conducted t o determine whether this disease ultimately causes alterations in DA receptors. In one investigation, 3[H]spiperone binding in 19 postmortem brains from schizophrenic patients showed an increase in the density of postsynaptic DA receptors (D-2) versus a control group.148 A concurrent increase in KD of 3(H]spiperone binding was attributed t o residual neuroleptics. Two patients had never received antipsychotic drugs, and five others had been drug-free for at least a year prior t o death (this latter group had normal K,I values). Thus, up-regulation of postsynaptic receptors in the caudate nucleus, putamen and nucleus accumbens may be an aspect of the disease process. Another study gave similar values, but it was difficult t o rule out supersensitivity induced by prior neuroleptic treatment.149 In Parkinson's disease, DA receptor supersensitivity i s postulated as a compensatory response t o the degeneration of nigrostriatal DA neurons. Examination of postmortem brains of Parkinson patients revealed an increase in 3[H]haloperidol binding in the putamen and a similar trend in the caudate.150 In the brains o f such patients on long term L-dopa therapy, 3[H]haloperidol binding was normal. L-Dopa thus normalizes the DA system. It was proposed that deliberate up-regulation by decreasing or withdrawing L-dopa treatment may be beneficial t o patients who become refractory t o this drug due t o the development of subsensitivity.lso.l51 The more disabled Parkinson patients showed a decreased number o f striatal D-2 receptors and loss o f therapeutic response t o L-dopa.152 In Huntington's disease, postmortem brain tissue from patients showed a significant decrease in the number o f 3[H]spiperone 0-2 sites (unchanged KD) in the putamen, caudate nucleus, and frontal cortex.153 This diminished dopaminergic function in the basal ganglia and frontal cortex may be a cause of the motor and mental disorders, respectively, in Huntington's disease.
Chap. 5
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2
Many animal studies have been carried out t o determine the effects of antipsychotic drugs on DA receptors.7.144.145 Rats treated chronically with haloperidol exhibited increases in 3[H]haloperidol binding in the striatum and mesolimbic region.154 Also, 3[H]apomorphine binding was increased in the aforementioned brain areas. Enhanced 3[H]haloperidol binding after chronic haloperidol, fluphenazine and reserpine was reflected by an increase in receptor density, not in affinity.155 In accord with the greater number of D-2 receptors, an augmented behavioral response t o apomorphine,l56.157 amphetamine157 or tranylcypromine + L-dopalss was observed. In general, chronic administration of neuroleptics alters the number of D-2 receptors; except for thioxanthenes, the number of D-1 sites, i.e., DA sensitive adenylate cyclase activity, is unchanged.156-158 The thioxanthene, cis-flupenthixol, which exhibits similar a f f i n i t i e s f o r D-1 a n d 0 - 2 receptors, u p - r e g u l a t e d D-1 receptors a f t e r c h r o n i c administration.l56,159 Longer treatment (18 months) increased the number o f D-2 sites without affecting that of D-1 sites (labeled by 3[H]piflutixol) despite increased DA-sensitive adenylate cyclase activity.160.161 Receptor plasticity was demonstrated by the fact that withdrawal after 18 months of cis-flupenthixol treatment resulted in the return of enhanced apomorphine-induced stereotypy t o normal and elevated Bmax for 3[H]spiperone binding t o control values after 1 month and 3 months, respectively. However, DA-stimulated adenylate cyclase activity remained elevated for 6 months, returning t o normal activity 1 year following drug withdrawal.161 Similar e f f e c t s were f o u n d upon continual administration o f trifluoperazine (1 year). This drug initially acted as a DA antagonist, but behavioral supersensitivity t o high dose apomorphine, increased density of D-2 receptors and enhanced DA-stimulated adenylate cyclase activity was observed at 6 and 12 months and persisted for 3 months after drug withdrawal.160 These receptor sensitivity changes appeared t o parallel the development o f drug-induced human tardive dyskinesia. Administration of haloperidol, trifluoperazine, fluphenazine or pifluthixol for 6-9 months resulted in an enduring functional blockade of DA receptors, as measured by low dose apomorphine elicited responses; D-2 binding was elevated.162 Other investigators have reported that neuroleptic-induced DA receptor supersensitivity can be reversed by chronic L-dopa administration, presumably by increasing the concentration o f DA, which in turn down-regulates the receptors.151.163 Concurrent chronic treatment of rats w i t h haloperidol and lithium did not lead t o the development of DA receptor supersensitivity.164 This prophylactic effect .of lithium i n preventing up-regulation o f t h e DA receptors may be involved in i t s antimanic properties. Studies of receptor regulation have also been useful in attempts t o understand the mechanism of action of antipsychotic drugs. In monkeys, it was found that after chronic haloperidol treatment, DA receptor sensitivity t o a challenge dose of haloperidol is maintained in the frontal and cingulate cortex, whereas tolerance is seen i n t h e putamen and caudate.165.166 Thus, these cortical regions may represent the site of therapeutic action of antipsychotic drugs. Furthermore, the unique effects of neuroleptics on the mesocortical DA neurons may be caused by the lack o f autoreceptors i n this brain region.167.168 In electrophysiological studies, DA autoreceptors (substantia nigra) were found t o be 6 t o 10 times more sensitive t o DA agonists than postsynaptic receptors (caudate nucleus); therefore, low doses of agonists may act preferentially at autoreceptors t o inhibit DA release.169 Thus, selective autoreceptor agonists could be effective antipsychotic agents; the development of tolerance may be a limiting factor.170.171 Conflicting results have been reported regarding the effects of chronic antidepressant administration on DA receptors: imipramine and iprindole induced subsensitivity o f autoreceptors,l72 b u t efforts t o repeat these findings have failed.173-175 Also, some investigators have observed a decrease in D-2 binding following tricyclic antidepressant treatment,lO whereas others have claimed no change.21 Conclusion - Recent years have witnessed an increased interest in the study of receptor plasticity i n the CNS following drug administration or i n relation t o disease processes. Generally, stimulation of receptors with agonists causes desensitization/down-regulation; antagonists produce the opposite effects. The NE system seems t o exhibit the best correlation between therapeutic drug response and receptor alteration. Many contradictions in the literature remain t o be explained. A better understanding of the functional significance of receptor subtypes would help answer many questions regarding adaptive mechanisms.
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E. C. Hwang. J. Magnussen and M. H. Van Woert. Res. Comm. Chem. Pathol. Pharmacol.. 29,79 (1980). 0. T. Wong and F P. Bymaster. Rer. Commun. Chem. Pathol. Pharmacol., 32.41 (1981). 5.8. ROSS. H. Hall, A. L. Renyi and D.Werterlund. Psychopharmacology,72,219 (1981). 5. Treiser and K . 1. Kellar. Eur. 1. Pharmacol.,64,183 (1980). T. Segawa and M. Uehara. Life kr..30,809 (1982) S. J. Peroutka and S. H Snyder, 1. Pharmacol. Exp. Ther.. 215,582 (1980). M. A. Blackshear and E. Sanders-Bush.1. Pharmacol. Exp. Ther.. 221.303 (1982). S Clernents-Jewery and P A. Robron. Neuropharmacology.19.657 (1980). G. P Reynolds. N. J. Garrett. N. Rupnlak. P. Jenner and C. D Marsden. Eur. 1 Pharmacol.. 89.325 (1983). M. A. Blackshear. L. R. Steranka and E. Sanders-Bush. Eur. 1.Pharmacol.. 76,325 (1981). K. J Kellar. C. S. Cascio, J. A. Butler and R. N. Kurtzke. Eur. f Pharmacol..69,515 (1981). 1. Vetulani. U. Lebrecht and A. Pilc, Eur. 1.Pharmacol., 76,81 (1981). K. J Kellar and D A. Bergstrom, Neuropharmacology,22.401 (1983) F. T. Crews, J. A. Scott and N. H. Shorrtein, Neuropharmacology,22, 1203 (1983). M. Mikuni and H. V Meltzer, Life SCI. 34,87 (1984). W. 1. Kinnier, 0.-M. Chuang and E. Costa, Eur. 1. Pharmarol,67,289(1980). C. De Montigny and G. K. Aghajanian, Science, 202, 1303 (1978). P. Blier and C. De Montigny. Naunyn-Schmiedeberg’sArch. Pharmacol ,314.123 (1980) P Blier and C. De Montigny. 1. Neurosci.,3,1270 (1983). D W Gallager and W. E. Bunney. Jr . Naunyn-Schmied~~erg’s Arch Phdrmacol , 307,129 (1979). E Friedman. T B Cooper and A. Dallob. Eur. 1. Pharmacol., 89.69 (1983) I Lucki.M S Nobler and A. Frazer, 1. Pharmacol Exp. Ther..228,133(1984). A. R Greene, D 1. Heal, P Johnson.8. E. Lawrence and V L. Nirngaonkar, 8r 1 Pharmacol.. 80.377 (1983) K. Fuxe. S -0 Ogren. L. Agnati. 1. A. Gustafsson and G. Jonsson. Neurosci. Len.. 6,339 (1977). H . Nagayama. 1. N. Hingtgen and M H. Apriron. Pharmacol. Biochem. Behav.. 13,575 (1980). 1 N. Hingtgen. H C Hendric and M. H. Aprison. ibid., in press (1984). K. Fuxe, S.-0 Ogren and L. F . Agnati. Neurosci. Lett., 13,307 (1979) G. Fillion and M. P. Fillion. Nature. 292,349 (1981). P. Seeman. Pharrnacol. Rev., 32,229 (1980). I. Creese. M. W. Harnblin. S. E Leff and D. R. Sibley, in “Handbook of Psychopharmacology”,VoI 17, L. L. Iversen, 5 . D. lverren and S. H . Snyder, Ed..Plenum Press, New York. NV. 1983, p. 81. B Costall and R. 1. Naylor. Life Sci..28.215 (1981) N. G Bacopoulos. Life ScJ ,34,307 (1984). F Owen, T. J. Crow, M. Poulter, A. J. Cross, A Longden and G. J. Riley, The Lancet. 223 (1978). T Lee, P. Seeman, W. W Tourtello. I 1. Farley and 0 Hornykeiwicz. Nature. 274.897 (1978) T. Lee, P. Seeman. A. Rajput, 1.1. Farley and 0.Hornykeiwicz. Nature, 273.59 (1978). A. 1. Friedhof and J. C. Miller. Ann. Rev. Neurosci.. 6. 121 (1983) u. K. Rinne, 1.0.Rinne. 1. K Rinne, K. Laakso, A. Laihinen and P Lonnberg. 1. Neural Jransm. (Suppl.), 18,279 (1983). T. 0.Reisme. J. 2 . Fields. L 2 . Stern, P. C. Johnson, E. D. Bird and H. I. Yamamura, Life Sct., 21.1123 (1977). P. Muller and P. Seeman, Life Sci., 21, 1751 (1977). D. R. Burt, I.Creere and 5 . H. Snyder. krence. 196.326 (1977). S. Fleminger, N. M.l. Rupniak, M. D.Hall, P. lenner and C. D.Marsden. Biochem. Pharmacol.. 32.2921 (1983). P. von Voigtlander, E . C. Lorey and H. Triezenberg, J. Pharrnacol. Exp. Jher., 193,88 (1975). D J. Heal, A. R. Green, D. 1. Boullin and 0. G. Grahame-Smith. Psychopharmacology,49,287 (1976). J. Hyttel and A.V. Christenren. 1. Neural Transm. (Suppl.). 18. 157 (1983). N M. J. Rupniak, P. Jenner and C. D.Marrden, Life Sci.. 32,2289 (1983). K Murugaiah. S. Fleminger. A. Theodorou, P. Jenner and C. D. Marsden, Biochem. Pharmacol., 32.2495 (1983) 1. L. Waddington. A. 1. Cross, S. 1. Gamble and R. C. Bourne. Adv. Btochem. Psychopharmacol.,37,299 (1983) A. 1. Friedhoff. K . Bonnet and H. Rorengarten. Res. Commun. Chem. Pathol. Pharmacol.. 16,411 (1977). A. Pert, J. E Rosenblatt.C. Sivit, C. B Pert and W. E. Bunney. Jr.. Science, 201, 171 (1978). N. G. Bacopoulos. G. Burtos. D. E. Redmond. J. Baulu and A. H . Roth. Brain Res.. 157,396 (1978). N. G. Bacopoulos. 0. E. Redmond. 1. 8aulu and R. H. Roth, 1. Pharmacol. Exp. Tber.,212, 1 (1980) M.J. Bannon, 1. F. Reinhard. Jr., E. 8. Bunney and R. H. Roth. Nature. 296,44 (1982). M. 1. Bannon. R. L. Michaud and R. H . Roth, Mol. Pharmacol.. 19,270 (1980). L. R. Skirboll. A. A. Grace and 8.5. Bunney. Science. 206.80 (1979). H. V . Meltzer. Pharmacol. Biochem. Behaw., 17, Suppl. 1. 1 (1982). H. V . Meltzer. T. Kolakowrka, A. Robertson and 8.1. Tricou. Rychopharmacolopy, 81.37 (1983). L. A. Chiodo and S. M. Antelman, Nature, 287.451 (1980). 1. welch. H. Kim, 5. Fallon. 1. Liebman. D.A. McNeill and M. Cower, Nature. 298.301 (1982). H. H. Holcomb, M. 1. Bannon and R . H. Roth, Eur. 1. Pharmacol.. 82. 173 (1982). I. Creese, R. Kuczenrki and D. Segal, Pharmacol. Biochcm. Behav.. 17.375 (1982).
Chapter 6. CNS Autoreceptors as Targets for Drug Design Pieter B. U. W. U. T i m e r m a n s , Department of Pharmacy, Division of Pharmacotherapy, University of Amsterdam, Plantage Huidergracht 24, 1018 TV Amsterdam, The Netherlands Introduction - Several lines of evidence strongly suggest that presynaptic receptors are of physiological importance. I n contrast to somadendritic receptors, which primarily modulate impulse generation, presynaptic receptors primarily modulate synthesis and release of transmitter. Presynaptic receptors are located on, in or near the axon terminals of many neurons. Activation results in facilitation or inhibition of transmitter release/synthesis, whereas blockade generally induces the opposite of a ~ t i v a t i o n . l - ~ In view of the ubiquity of presynaptic receptors at central and peripheral sites,3 this Chapter focusses on the subset of presynaptic receptors termed autoreceptors, i.e. presynaptic receptors on a particular neuron which are activated by its own neurotransmitter. CNS autoreceptors have been postulated for, inter alia, norepinephrine (NE)/ epinephrine, dopamine (DA), serotonin (5HT), acetylcholine (ACh), histamine (H) and y-aminobutyric acid (GABA). Autoreceptors for Norepinephrine (NE) - Though the concept of autoinhibition of N E release is not universally a ~ c e p t e d , ~the , ~ consensus is that release of N E from sympathetic nerves is modulated through inhibitory a2-autoreceptors. By contrast, somadendritic a2-adrenoceptors of central noradrenergic neurons inhibit f i ~ i n g . ~ Release , ~ ~ ~ of 3H-amezinium from rat cortical noradrenergic axons has been advanced as a model for the study of the a2-autoreceptor h y p o t h e ~ i s . ~ There are a number of mechanisms possible for the link between presynaptic a2-adrenoceptor activation and transmitter release, in which a pivotal role for Ca2+ has been established.3~8 Uore recent data implicates inhibition of adenylate cyclase with receptor activation and subsequent attenuation of transmitter release. 9*10
CNS
(z),ll*lz
The a-adrenoceptor agonists 8-HT 9 2 0 (1) and 933 UK-14,304 (3),11,13 U-7 (9). l4 TL-99 DP-6.7-ADTN of Z-amin0-6,7-dihydroxybenzonorASL-7022 (9)17 and some =-isomers bornene preferentially stimulate this type of a-adrenoceptor. The antagonists yohimbine and rauwolscine,8 RS 21361 (11),19 idazoxan (RX 781094 g ) 2 0 and derivatives,21 BDF 6143 (u),22 Wy 26392 (14)
(z),l)
H
2
R = CHa-CH2-i X=O
3 -
and Wy 26703 (11)23 as well as S K W 86466 (13)24 block a2-adrenoceptors selectively. The stereochemical requirements of a2-adrenoceptors have been described in detail.25.
ANNUAL REPORTS IN MEDICINAL CHEMISTRY-19
Copynght 0 1984 by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0- 12-0405 19-9
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Presynaptic a2-adreqoceptors possibly represent only a minor fraction of the total a2-adrenoceptor population in the CNS 26 Although the general characteristics of presynaptic (auto) and postsynaptic a2-adrenoceptors with respect to drug selectivity are very similar,8 some findings suggest that in rat cerebral cortex the a2adrenoceptors for NE differ from the a2-adrenoceptors located on serotonergic terminals .27 Surprisingly, in rat frontal cortex a1adrenoceptors seem to modulate 5HT release.28 The suggestion that different recognition sites on the presynaptic a2-autoreceptor exist in
.
\
OH
9
-
4 X = 5,Mi-OH; R = CH3 X = 6,7-di-OH; R CH3 & X = 6,7-di-OH; R = n-CsH7 L X = 7-OH; R = n-C3H7 X = 8-OH; R = n-C3H7
7'
HozwN-R2 HO
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4 ? + -02 ? N j
11
R1R2-H orCH3
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14 R = n-propyl R = i-butyl N-S02-R CH3
the CNS for imidazoli(di)nes and cat echo la mine^,^^ has not found support. 30 Direct labeling of central a2-autoreceptors has been uns u c c e ~ s f u l . ~ ~The * ~ ~autoreceptors regulating NE release undergo sensitivity changes after long-term drug-induced activation or blockade: activation typically leads to a decrease in receptor number, whereas blockade produces the converse. 33 CNS Autoreceptors for Dopamine (DA)
-
DA autoreceptors, probably located on dopaminergic nerve terminals, modulate calcium-dependent stimulation evoked release of DA a negative feed-back 1 n e c h a n i s m , ~ ~ 3 as ~ ~ ~has 5 been reported for the field-stimulated release of 3H-DA from slices of the striatum/caudate nucleus of rats ,36 rabbits37 and cats.36,38 Presynaptic DA autoreceptors have also been implicated in the regulation of the synthesis of DA,39 whereas somadendritic DA receptors inhibit the firing of the dopaminergic neurons.40 In the rat, autoreceptor regulation of DA synthesis appears more significant in the mesolimbic as compared with the nigrostriatal DA pathway, while autoreceptors may be absent in the median eminence.41
Chap.
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The controversies in the classification of DA receptors have been reviewed.4 2 The majority of current receptor binding data supports two major DA receptor classes for the CNS, D-1 and D-2, with the DA autoreceptor probably belonging to the latter.42 However, the data is not sufficiently quantitative to assess conclusively whether more than one distinct DA autoreceptor is present on DA neurons.42 Furthermore, in studies with behavioral models of postsynaptic and neurochemical models of presynaptic dopaminergic activity have demonstrated that DA autoreceptors and postsynaptic DA receptors are diff ierent . 3 8 * 4 3 9 4 4 The suggestion that the so-called D-3 binding site corresponds to presynaptic DA auto receptor^^^*^^ has been refuted both on the basis of pharmacological and neurochemical evidence .47 ,48 Some agents have been proposed as selective presynaptic DA agonists, ,51 N.N-dipropyl-2-amino-7-hydroxye.g. 3-PPP (17)49,5O, RSD-127 (18) tetralin (1)and the selective a2-adrenoceptor stimulants TL-9953 and B-HT 920.54 TL-99 and 1 inhibit the release of 3H-DA from brain slices, but 3-PPP is inactive in this in vitro model for presynaptic DA a ~ t i v i t y . ~ ~The , ~ ~apparent DA autoreceptor selectivity of TL-99 as assessed in viva may be due at least partially to its a2agonist a ~ t i v i t y . 5 ~ ~ has I t been hypothesized that the presynaptic DA receptors mediating biochemical effects and those mediating effects on release are different.48*52 Both isomers of 3-PPP are selective agonists for the autoreceptor at low doses. At higher doses postsynaptic activity is also observed, with the ( + ) - and (-)-enantiomem behaving as an agonist and antagonist, r e s p e ~ t i v e l y . ~ ~ -For ~ ~ the more rigid the prelpost selectivity analogs of 3-PPP, c7-OBQ (19)and t7-OBQ (B), is lost for the trans compound whereas the compound (19)is inactive.61 Inhibition by d-amphetamine of the electrically-evoked release of 3H-DA does not involve the activation of presynaptic inhibitory DA autoreceptors .62*63
(a),
OH
I OCHq
?H
Effects of chronic neuroleptic administration on cerebral DA receptor function have been reviewed.64 Following chronic haloperidol treatment, the DA autoreceptors modulating 3H-DA release in the rabbit caudate develop supersensitivity to a p ~ m o r p h i n eor ~ ~ to DA itself .66 for Serotonin (5-HT) - Various experimental findings imply that presynaptic autoreceptors modulate the release of 5HT from the corresponding neurons in rat brain.67 In rat brain slices, or synaptosomes preincubated with 3H-5HT, the CaZ+-dependent stimulation-evoked release of the amine is inhibited by 5HT,68*69 5HT analog^,^^^^^ and numerous 5HT receptor agonists. 72 Hekitepine, metergoline and quipazine are competitive antagonists at the 5HT a u t o ~ e c e p t o r . ~ ~The - ~ ~ 5HT autoreceptors in rat cortical tissue may be different from the presynaptic excitator 5HT U - r e c e p t o r ~ ~on ~ sympathetic nerve endings of the rabbit heart,!O and from the somadendritic 5HT receptors in rat raphe nuclei75176 which mediate an inhibition of neuronal-activity. 70 Rank orders of 5HT agonists indicate that 5HT autoreceptors, inhibitory presynaptic 5HT receptors on sympathetic nerve endings of canine saphenous vein, and the binding sites of 3H-5HT in rat brain, designated as CNS Autoreceptors
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5HT1 sites, are similar,70,71,77 but relative blocking potencies of antagonists show that they are not i d e n t i ~ a 1 . 6 9 , 7 ~ , 7 2 , ~In ~ fact, 5HT1 binding sites in rat brain cortex are not homogenous, since various compounds produce biphasic or shallow displacement curves.71,79*80 This may be one of the reasons why correlations between binding affinity for 5HT1 sites and functional activit frequently difficult to interpret .81 Two subtypes of the JH-TE; recognition site, designated ~ H T ~ (high A affinity site) and 5HT18 (low affinity site), have been recognized from displacement studies with spiperone, which exhibits a 3000-fold difference in affinity between the two sites.80 Both sites are differentially distributed in rat brain.82 A subset of the 3H-5HT binding sites (low affinity, rather than high affinity sites, for antagonists) may be localized on the serotonergic neurons and may correspond to the 5HT autoreceptors. 71 The 5HT agonist PAT ( 8 ) potently and selectively displaces 3H-5HT from the rrported ~ H T ~ binding A site in rat frontal cortex.83 However. H-PAT has been reported to bind to pre and postsynaptic 5HT sites in rat b ~ a i n . ~ 4 In the hippocampus, 3H-PAT binding sites have been associated with (postsynaptic) 5HT1 sites, whereas in the striatum they exhibit some properties of 5HT auto receptor^.^^ The agonist activity of PAT on the 5HT autoreceptor h vitro appears rather limited,85*86 and so the potent central effects of PAT on 5HT turnover are unH likely to be mediated by 5HT auto receptor^.^^ Additional selective 5HT1 (A or B) agonists/ antagonists may provide further clarification of several discrepancies. The selective 5HT1 agonist RU 24969 (21) may be a possible candidate.S8
*’
Citalopram, a selective inhibitor of 5HT uptake, does not affect per the stimulation-evoked overflow of 3H-5HT from rat hypothalamic slices , but antagonizes the inhibitory effect of LSDg9 and 5-methoxyt ~ y p t a m i n e on ~ ~ 3H-5HT release , indicating an interaction between the neuronal uptake mechanism and 5HT autoreceptors. On the other hand, stimulation or blockade of 5HT autoreceptors does not modulate the 5HT uptake process .91 The autoreceptor regulating 5HT release undergoes sensitivity changes after chronic pharmacological activation or blockade.33 However, long-term treatment with metitepine does not induce changes in the sensitivity or function of 5HT autoreceptors in rat hypothalamus. 92
se
CNS Autoreceptors for Acetylcholine (ACh) - Proposals have been made for the existence of an inhibitory muscarinic feedback regulation of ACh in the CNS, and at certain locations in the p e r i p h e ~ y . l * ~ ~Evidence *~~ for a negative feedback mechanism involving ACh autoreceptors has been obtained in both brain slices and s y n a p t o s o m e ~ . ~ ~Cyclic -~~ GWP has been found to act as a second messenger for the muscarinic autoreceptor regulating ACh release. 97*98 Rat hippocampal muscarinic autoreceptors become super or subsensitive following chronic in treatment with the muscarinic antagonist, scopolamine, or the cholinesterase inhibitor, paraoxon, r e s p e c t i ~ e l y . ~The ~ inhibitory muscarinic autoreceptors in synaptosomes of rat striatal nerve terminals have a markedly lower release modulating capacity than autoreceptors in cortical or h i p pocampal nerve endings. loo Since autoregulat ion of ACh release through muscarinic receptors occurs as effectively in the striatum as in the cottex or hippocampus, it has been suggested that in the former area the autoregulation of ACh release may not necessarily require the activation
-
Chap. 6
CNS Autoreceptors
Timmermans
55
of autoreceptors on cholinergic nerve terminals .loo Unlike the periphery,lol the autoreceptors for ACh in the CNS seem to be different from postsynaptic muscarinic receptors .94 CNS Autoreceptors for Histamine (H) - The first experimental evidence for histamine autoreceptors in rat brain has been presented tecently.lo2 3H-Histamine can be released in a calcium-dependent manner with K+ or veratridine from pre-labeled pools in slices or synaptosomes from rat cerebral cortex. Histamine, as well as its N-methyl and N,N'-dimethyl congeners, produces up to 60% inhibition. Other HI- or H2-agonists, like 2-methyl and 4-methylhistamine and dimaprit, were ineffective. The partial H2-receptor agonist imptomidine also lacked effect, but competitively antagonized the action of histamine. The HI-receptor antagonists mepyramine, cyclizine and chlorpheniramine were found inactive. The H2-receptor blocker burimamide was active, but tiotidine was without effect. The relative potencies of agonists and antagonists led the authors to suggest that autoinhibition of histamine release in rat brain is mediated by an as yet unidentified class of histamine receptor, for which they propose the designation H3.1°2 CNS Autoreceptors for GABA - Studies in vitro have demonstrated that the release of GABA is subject to negative feedback control through GABA autoreceptors on GABAergic terminals. In rat cortical synaptosomes, GABA and muscimol each inhibit evoked release of 3H-GABA, and are blocked by b i c u ~ u l l i n e . ~ ~The ~ K+-evoked release of GABA from rat frontal cortex is reduced by GABA, muscimol and 3-amino-propane sulphonic acid; the effect of muscimol being antagonized by bicuculline and picrotoxin.lo4 The GABA agonist 6-aminolaevulinic acid inhibits K+induced release of GABA from preloaded synaptosomes from rat cerebral cortex, and again this effect may be blocked by bicuculline and picrotoxin.lo5 Inhibition of the K+-evoked, Ca*+-dependent release of 3H-GABA from rat substantia nigra has been demonstrated with exogenous GABA and muscimol, and antagonism quantified for picrotoxin.lo6 Bicuculline-sensitive postsynaptic GABA receptors are called GABAA receptors, whereas baclofen-sensitive GABA receptors present on eripheral sympathetic nerve terminals are termed GABAB receptors,10~-108 The GABAB site has also been detected in the CNS, but its relationship to the presynaptic receptor which modulates transmitter release remains uncertain. 109 Physiological Role of Autorecevtors in the CIS; Targets for Drugs Though autoreceptors play a key role in modulating transmitter release, it should be emphasized that many other types of prejunctional receptors can influence transmitter synthesis, release, or neuron f i r i n ~ . l - ~ For example, NE release can be inhibited by stimulation of muscarinic, dopamine, serotonin, histamine, GABA, opiate, prostaglandin and adenosine receptors located at noradrenergic nerve endings. Furthermore, in many cases the net effect of an agonist or antagonist will be the sum of presynaptic and postsynaptic actions, since the majority of drugs cannot discriminate between both receptor populations. Thus it is frequently extremely difficult to interpret the (patho)physiological role of CNS autoreceptors or their interactions with drugs. Functional and biochemical studies suggest that endogenous depression may be related to impaired central noradrenergic transmission.llo*lll Drug-induced desensitization of central a2-autore-
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ceptors, stemming from chronic inhibition of neuronal NE uptake, and hence elevated synaptic NE levels, has been postulated to play a key role in the mechanism of action of many antidepressants.111 This would lead to yet further enhanced neurotransmission, with subsequent down regulation of central B-adrenoceptors111 - possibly a ivotal event in the induction of the desired therapeutic effect.llf However, the situation is far from clear with, on the one hand, conflictin reports as to whether chronic desipramine and imipramine do, lf3 or do not ,114 9 down regulate presynaptic a2-adrenoceptors in rat brain; and on the other, the finding that mianserin treatment results in supersensitive a2-adrenoceptors. It has been suggested recently that the induction of subsensitive central presynaptic a2-adrenoceptors cannot represent the fundamental mode of action of antidepressants .'-I1 Irrespective of the mechanism of action of tricyclic antidepressants, antagonists of a2-autoreceptors per gg might have a beneficial effect in depressive illness, since they facilitate noradrenergic neurotransmission. Phenoxybenzamine, dihydroergotamine and yohimbine, but not the selective al-adrenoceptor antagonist prazosin, have been shown to be synergistic with several antidepressants in decreasing 0-adrenoceptor density in rat cerebral cortex. Thus combined administration of an a2-adrenoceptor blocker with other antidepressants may provide a rapid onset t h e r a ~ y . 1 1 ~ RS 21361 produces a more rapid onset of 0-adrenoceptor desensitization by itself than desipramine.120 Chronically administered antidepressants also down-regulate the number of 5HT2 recognition sites in rat b~ain.1~1-123 Inasmuch as 5HT2 receptors are also implicated in the therapeutic action of antidepressants, blockade of central presynaptic a2-(auto)receptors accelerates the down regulation of 5HT2 receptors by antidepressants. 224 Desensitization of presynaptic DA receptors followin chronic administration of antidepressants has been observed in s0me,185*126 but not in other s t ~ d i e s . 1 ~ ~ -However, ~~8 central DA turnover is unaltered by such regimenslll and it is too soon to assess the relevance of these findings to the mechanisms underlying the clinical efficacy of these drugs. Clonidine and some related dru s suppress narcotic withdrawal signs in the rat, monkey and in man1299630 possibly via stimulation of presynaptic a2-adrenoceptors, thereby inhibitin the firing of the locus ceruleus through reduction of NE ~ e 1 e a s e . l ~ Clonidine ~ has also been found effective in the treatment of alcohol withdrawal states.lS2 Elevated central NE activity (e.g. in the locus ceruleus) is an important correlate of human anxiety, though not the sole one.133~134 The beneficial effects of clonidine as an antianxiety agent in humans could result from its inhibitory action on NE activity by stimulation of a2-autoreceptors . , The observation that ohimbine is anxiogenic in humans is in accord with this hypothesis. 1 3 7 The ideal agent €or facilitating central cholinergic transmisson would act simultaneously as an antagonist at ACh autoreceptors and as an agonist at postsynapttc ACh-receptors .138 It has been suggested13* that compounds, with this profile would be of therapeutic value in the
Chap. 6
CNS Autoreceptors
Timmermans
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treatment of e.g. Huntington's chorea139 and Alzheimer-type demenan oxotremorine analog, is a presynaptic antagonist tia.140 BU-5 and a postsynaptic agonist at peripheral and central muscarinic synapses -in vitro.138
(a),
CH3-C
11
y 3 -N-CH-C=C-CHz-N
0
CHQ
3
22 -
The question of a physiological role for GABA autoreceptors remains open.3 GABA is involved in many CNS functions. Impaired GABA-mediated inhibition may contribute to the pathophysiology and phenomenology of epileptic seizures .Irr1 Consonant with this hypothesis, the purported GABA autoreceptor agonist 6-aminolaevulinic acid is a convulsant .Io5 "GABA replenishment" treatment has been advocated for seizures, psychosis, movement disorders, pain, hypertension and sleep disturbances Presynaptic autoreceptors for GABA have yet to be investigated as targets for potential drugs (e.g. GABA autoreceptor antagonists). The clinical significance of DA autoreceptor stimulation has been discussed. 14* Selective DA autoreceptor agonists, which reduce DA synthesis and release, represent a novel pharmacological approach to the treatment of various neurological disorders, including schizophrenia and Huntington's chorea, where an overactivity of central dopaminergic neurons is believed to be etiologic. Such drugs might lack some of the side-effects, e.g. tardive dyskinesia, associated with the currently prescribed neuroleptic agents, which are postsynaptic DA antagonists Apomorphine, N-pro ylnorapomorphine, and bromocryptine have been studied in the clinic.141-148 There is evidence for an antipsychotic effect following acute administration, particularly €or the two former agents. However, preliminary evidence suggests that the effect tolerates rapidly on repeated administration.149 References
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z,
z,
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CNS Autoreceptors
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Pharmacodynamic Agents
W i l l i a m T. Comer, Bristol-Myers Research and Development 345 Park Avenue, N e w York, New York 10154
Chapter 7.
A n t i h y p e r t e n s i v e Agents
P e t e r Sprague and James R. Powell, The Squibb I n s t i t u t e f o r Medical Research, P r i n c e t o n , New J e r s e y 08540 I n t r o d u c t i o n - A r t e r i a l h y p e r t e n s i o n c o n t i n u e s as a major r i s k f a c t o r i n t h e development of c a r d i o v a s c u l a r d i s e a s e . C u r r e n t l y i t i s n o t p o s s i b l e t o prevent e s s e n t i a l h y p e r t e n s i o n and t h i s p r e s e n t s a c h a l l e n g e t o t h e c l i n i c i a n , pharmacologist, and m e d i c i n a l chemist t o d i s c o v e r adequate t h e r a p i e s . The b e n e f i c i a l e f f e c t s of drug t h e r a p y i n t h e management of h y p e r t e n s i o n have been r e c e n t l y reviewed. A number of r e c e n t reviews d e s c r i b e t o p i c s r e l a t e d t o h y p e r t e n s i o n . The s i g n i f i c a n c e of catecholamine l e v e l s i n h y p e r t e n s i o n h a s been discussed and a c a u t i o n a r y n o t e o f f e r e d t h a t l e v e l s i n mixed venous blood a r e unreThe l i a b l e i n d i c a t o r s of sympathetic n e u r a l a c t i v i t y i n h y p e r t e n s i o n . , r o l e of t h e sympathoadrenal a x i s i n h y p e r t e n s i o n h a s been r e ~ i e w e d . ~The need f o r i n t a c t v a s c u l a r endothelium f o r v a s c u l a r responses t o a number of a g e n t s h a s been d e s c r i b e d . 5 A r e c e n t review examines t h e r o l e of r e n i n subs t r a t e i n h y p e r t e n s i o n . L a s t l y , based on t h e unimodal d i s t r i b u t i o n of blood p r e s s u r e s , t h e e x i s t e n c e of h y p e r t e n s i o n as an e n t i t y h a s been questioned. Renin I n h i b i t o r s - A review of p r o g r e s s made i n d e f i n i n g t h e s t r u c t u r e of mouse submaxillary gland r e n i n h a s appeared. The presence of two a s p a r t y l , two t y r o s y l and one a r g i n y l r e s i d u e s a t t h e a c t i v e s i t e h a s been demonstrated. Current approaches t o r e n i n i n h i b i t i o n have been reviewed. Monoclona l a n t i b o d i e s w i t h h i g h a f f i n i t y and s p e c i f i c i t y f o r human r e n i n have been developed and s e v e r a l new p e p t i d e s modeled a f t e r t h e minimal s u b s t r a t e ochave been prepared. tapeptide 1 His-Pro-Phe-His-Leu-Leu-Val-Tyr 2 His-Pro-Phe-His-Leuseu-Val-Tyr
(1)
3 Iva-His-Pro-Phe-His-Sta-Ile-Phe-NH2
4 BOC-Phe-His-Sta-Leu-(4-amido-l-benzylpiperidine) H-77 ( 2 ) , i; which t h e Leu-Leu amide i s reduced t o an amine, suppresses t h e p r e s s o r e f f e c t i n dogs of i n j e c t e d dog r e n i n (ID50=0.06 mglkgjhr) . l o * 1 1 Heptapeptide 2, i n which Leulo-Leu11 i n 1 i s r e p l a c e d by s t a t i n e ( S t a ) , i s one of t h e most p o t e n t human r e n i n i n h i b i t o r s i n v i t r o y e t d i s c o v e r e d ( I ~ o = 2 nM).12 F u r t h e r refinement of t h i s c l a s s gave 4, a p r o t e c t e d t e t r a p e p t i d e amide w i t h a 60 picomolar K i a g a i n s t human r e n i n . 1 3 Angiotensin Converting Enzyme I n h i b i t o r s - An e x c e l l e n t review on t h e mechanisms by which i n h i b i t o r s of a n g i o t e n s i n c o n v e r t i n g enzyme (CEI) reduce blood p r e s s u r e h a s appeared. l 4 Current evidence sup o r t s t h e h y p o t h e s i s t h a t CEI e x e r t t h e i r hypotensive e f f e c t s p r i m a r i l y gy i n h i b i t i o n of circu-'
ANNUAL REPORTS IN MEDICINAL CHEMISTRY-I9
Copyright 0 1984 by Academic Press, Inc. All rights of reproduction in any forni reherqd ISBN 0-12-'J40519-9
62
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- Pharmacodynamic Agents
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l a t i n g and tissue-bound c o n v e r t i n g enzyme. S e v e r a l new C E I modeled a f t e r e i t h e r c a p t o p r i l o r e n a l a p r i l have been r e p o r t e d . P i v a l o p r i l (RHC 3659, 2) h a s been shown e q u i v a l e n t t o c a p t o p r i l -i n v i t r o b u t o n l y 114 as a c t i v e i n man.I5-l7 Z o f e n o p r i l (k) r e p r e s e n t s a second g e n e r a t i o n c a p t o p r i l showing 10-fold g r e a t e r potency and a l o n g e r (WY 44,221) h a s a c t i v i t y d u r a t i o n of a c t i o n . The d i h y d r o i n d o l e analog similar to 6. 9-2 T e t r a h y d r o i s o q u i n o l i n e v a r i a t i o n 2 is e q u i v a l e n t i n act i v i t y t o c a p t o p r i l . 2 2 SA 446 given o r a l l y , i n h i b i t s angiotensin I (i.c.v.)-induced h y p e r t e n s i o n more e f f e c t i v e l y t h a n c a p t o p r i l , implying del i v e r y a c r o s s t h e blood-brain b a r r i e r . The s i d e - c h a i n benzoyl analog 10 i s more p o t e n t t h a n c a p t o p r i l w i t h a l o n g e r d u r a t i o n of a c t i v i t y . 2 4 Lactam 11, 2-fold less a c t i v e t h a n c a p t o p r i l , w a s developed u s i n g a computera s s i s t e d molecular modeling approach.25
1
(z),
FHs
R- S-CHiCH-CO-R‘
-5
b A 12
(c$cc-
-9
Ph-(C%
2al NH A
I?M*qNH
l3 p
Ph-C-
U
* q
A
CHI 8
B=8wH
0 R Ah-Pro
l!i
H-
d
H-
8
-N
COOH
A summary of t h e pharmacology of e n a l a p r i l (MK-421) h a s appeared a l o n g w i t h an account of c l i n i c a l s t u d i e s done w i t h l i s i n o p r i l (MK-521) which s u g g e s t t h i s drug w i l l be s u i t a b l e f o r once-a-day a n t i h y p e r t e n s i v e t h e r a p y . 26-28 Analogs of e n a l a p r i l which have appeared i n c l u d e t h e perhy(SCH 31,846). This compound h a s a q u i c k e r o n s e t of droindole d e r i v a t i v e a c t i o n , b e t t e r a b s o r p t i o n , and g r e a t e r b i l i a r y e x c r e t i o n t h a n e n a l a p r i l b u t i s o t h e r w i s e i n d i s t i n g u i s h a b l e i n vivo. 29-31 S y n t h e s i s of t h e c l o s e l y rel a t e d 2 h a s been r e p o r t e d . 3 2 G l u t a r i c a c i d analog g, m i s s i n g a side-chain n i t r o g e n , i s i n t e r m e d i a t e i n a c t i v i t y i n v i t r o between c a p t o p r i l and enalap r i 1 . 3 3 A r e l a t e d s i m p l i f i e d d e r i v a t i v e 15 (CGS 13,945), i s much l e s s pot e n t t h a n c a p t o p r i l i n man.34 The i n t e r n a l lactam s t r a t a g y h a s a l s o been (CGS 14,831) i s p o t e n t & employed i n t h e e n a l a p r i l series. Benzazepine 7 (CGS 14,8248) h a s a more r a p i d o n s e t of v i t r o (150’3 nM), and i t s ester 1 a c t i o n p l u s greater potency t h a n e n a l a p r i l i n dogs. 35 9 36 The phenethylphosphinamidate 1 9 refinement o f t h e phosphoramidate CEI 3 is 3-fold more po. ~ ~ 20, a d e r i v a t i v e of benzoyl-Phe-Gly-Pro i n which t e n t i n ~ i t r o Compound t h e s c i s s i l e amide bond i s reduced t o a n amine, is a p o o r l y a c t i v e C E I . 3 8
12
16
V a s o d i l a t o r s - The c e l l u l a r mechanisms 3y which v a s o d i l a t o r s lower blood p r e s s u r e have been reviewed. 39 One of t h e s e i n v o l v e s t h e a r a c h i d o n i c a c i d cascade i n which b o t h t h e cyclooxygenase and lipoxygenase pathways p l a y a r o l e . Evidence f o r i n c r e a s e d s y n t h e s i s of p r o s t a g l a n d i n s (PG) by t h e r e n a l glomerulus of SHR r e l a t i v e t o normotensive r a t s h a s been p r e s e n t e d . 4 0 The use o f s y n t h e t i c a n a l o g s of n a t u r a l PG as a n t i h y p e r t e n s i v e a g e n t s remains an active area of r e s e a r c h . The main o b s t a c l e s t o overcome are manifold s i d e e f f e c t s , s h o r t d u r a t i o n of a c t i o n and poor o r a l e f f e c t i v e n e s s .
Chap. 7
A n t i h y p e r t e n s i v e Agents
Sprague, Powell
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CL 115,347, a 16-vinyl-16-hydroxy-PGE2 analog, i s a more p o t e n t v a s o d i l a t o r ~ , s~h o~r t d u r a t i o n t h a n PGE2 w i t h l e s s emesis and d i a r r h e a p ~ t e n t i a l . ~The of a c t i o n a f t e r o r a l a d m i n i s t r a t i o n i s overcome by g i v i n g t h e drug t o p i c a l l y . Thiazolidinone i n c r e a s e s r e n a l blood flow maximally 5 h r a f t e r o r a l d o s i n g w i t h potency comparable t o PGE2.43 The ll,l2-seco-PG analog 22, i n c r e a s ed r e n a l blood flow a f t e r o r a l a d m i n i s t r a t i o n without concomitant i n c r e a s e s i n h e a r t rate o r blood p r e s s u r e . 4 4 Carbacyclin analog ZK 36,374 (23) i s 5f o l d more p o t e n t t h a n PGI2 i n r i t r o , and s t a b i l i z e d e n o l e t h e r 24 (CG 4203) a l s o mimics PGI2 i n animal t e ~ t s . ” ~ , Both ~ ~ compounds have a l o n g e r durat i o n o f a c t i o n t h a n PGI2. BW 245C (25) mimics PGD2 i n i t s e f f e c t s on platel e t s and i n v i v o h a s both PGD2 and PGIZ-like a ~ t i v i t i e s .When ~ ~ given t o man by c o n s t a n t i n f u s i o n , 25 shows a c t i v i t y and s i d e e f f e c t s similar t o PGI2 w i t h a g r e a t e r d u r a t i o n of a c t i o n . 4 8 However, t h i s compound i s s t a b l e o n l y a t h i g h pH and i s n o t a c t i v e o r a l l y .
21
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MDL 899 i s a d i r e c t a c t i n g a r t e r i o l a r v a s o d i l a t o r i n man, but c o a d m i n i s t r a t i o n of a beta-blocker i s n e c e s s a r y t o prevent r e f l e x - r e l a t e d s i d e e f f e c t s . l t g A c l i n i c a l s t u d y w i t h c a d r a l a z i n e (ISF 2469, 22) shows t h i s compound s i m i l a r t o h y d r a l a z i n e w i t h fewer s i d e e f f e c t s . 50 Two o t h e r may owe some of t h e i r acv a s o d i l a t o r s , b u d r a l a z i n e (28) and p i n a c i d i l (2) t i v i t y t o calcium channel blockade. 5 1 52 Anthranilamide 30 (WIN 48,049) lowers blood p r e s s u r e i n monkeys w i t h o u t accompanying t a c h y c a r d i a . This compound a c t s p r i m a r i l y as a d i r e c t v a s o d i l a t o r b u t a l s o has sympatholytic and dopaminergic a c t i v i t i e s . 54 Compounds 2 and 2 a l s o lower blood p r e s s u r e by a p p a r e n t l y d i r e c t v a s o d i l a t o r mechanisms. 55 3 56
n
33 The 0 - s u l f a t e 22 of m i n o x i d i l h a s been proposed as an i m p o r t a n t act i v e m e t a b o l i t e . 5 7 Unlike m i n o x i d i l , 33 i s a c t i v e i n v i t r o and i s more pot e n t w i t h a f a s t e r o n s e t of a c t i o n i n v i v o . Evidence s u g g e s t s t h e dihydroexert t h e i r e f f e c t pyrano and dihydrofuranocoumarin v a s o d i l a t o r s (e.g. by CAMP p h o s p h o d i e s t e r a s e i n h i b i t i o n . 58 F i n a l l y , t h e p i v a l i c a c i d e s t e r of i s o x s u p r i n e 35 h a s a slower o n s e t and l o n g e r d u r a t i o n of a c t i o n t h a n t h e p a r e n t phenoT5’
34)
Calcium Entry Blockers - I n t h e U.S. n i f e d i p i n e , verapamil, and d i l t i a z e m are marketed f o r a n t i a n g i n a l o r a n t i a r r h y t h m i c u s e , though none has FDA approval y e t f o r use i n h y p e r t e n s i o n . The p o t e n t i a l t h e r a p e u t i c use of calcium e n t r y b l o c k e r s (CEB) i n t h e
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treatment of a t h e r o s c l e r o s i s has r e c e n t l y been reviewed. 6 o Verapamil and n i f e d i p i n e have been shown t o s u p p r e s s a t h e r o g e n e s i s i n c h o l e s t e r o l - f e d r a b b i t s without a l t e r i n g serum c h o l e s t e r o l l e v e l s . 6 1 9 6 2
New evidence s u g g e s t s c e r t a i n CEB a f f e c t n e u r a l r e g u l a t i o n of blood v e s s e l s a t several sites. S t u d i e s i n a n e s t h e t i z e d dogs i n d i c a t e t h a t n i f e d i p i n e i n c r e a s e s and verapamil d e c r e a s e s t h e s e n s i t i v i t y of c a r o t i d s i n u s b a r o r e c e p t o r s a p p a r e n t l y by i n t e r f e r e n c e w i t h a calcium-dependent and a sodium-dependent mechanism, r e s p e c t i v e l y . A similar i n c r e a s e i n baroref l e x s e n s i t i v i t y w a s found i n man t r e a t e d c h r o n i c a l l y w i t h n i f e d i p i n e , which may e x p l a i n why t h e r e i s evidence of sympathetic a c t i v a t i o n d u r i n g i n i t i a l therapy. 6 4 Tachycardia and i n c r e a s e d l e v e l s of plasma norepinephrine a r e found d u r i n g a c u t e b u t not c h r o n i c phases of n i f e d i p i n e t r e a t m e n t . 6 5 During a c t i v a t i o n of p o s t g a n g l i o n i c sympathetic nerves t h e r e i s an e x o c y t o t i c release of norepinephrine which i s dependent upon t h e movement o f calcium a c r o s s t h e n e u r a l c e l l membrane. The e f f e c t s of CEB on stimuluss e c r e t i o n coupling i n sympathetic n e r v e s has been s t u d i e d i n s e v e r a l d i f f e r e n t p r e p a r a t i o n s . Verapamil does n o t a f f e c t t h e n e u r a l release of norepinephrine i n e i t h e r i s o l a t e d c a t h e a r t o r i n the pithed rat.66,67 I n t h e l a t t e r model n i f e d i p i n e b l u n t e d i n c r e a s e s i n blood p r e s s u r e due t o act i v a t i o n of sympathetic n e r v e s b u t d i d n o t a f f e c t c a r d i o v a s c u l a r responses t o exogenous norepinephrine s u g g e s t i n g a p r e j u n c t i o n a l s i t e of i n h i b i t i o n . A similar p r e j u n c t i o n a l CEB e f f e c t w a s found f o r d i l t i a z e m i n guinea p i g m e s e n t e r i c and dog b a s i l a r a r t e r i a l p r e p a r a t i o n s . 6 8 C i n n a r i z i n e and f l u n a r i z i n e were shown more p o t e n t than verapamil, d i l t i a z e m o r n i f e d i p i n e i n reducing t h e potassium-induced uptake of calcium i n a synaptosomal preparation.69
New s t u d i e s have examined t h e i n t e r a c t i o n of v a r i o u s CEB w i t h a-adren o c e p t o r s and t h e i r e f f e c t on p r o c e s s e s mediated by a-adrenoceptors. 70 The (+) and (-) a n t i p o d e s of verapamil, n i c a r d i p i n e and (+) D-600 showed s i m i l a r i n h i b i t i o n of r a d i o l i g a n d binding t o al-adrenoceptors, whereas d i l t i a zem and n i f e d i p i n e showed a v e r y low level of a c t i v i t y . 7 1 A d i f f e r e n t p a t t e r n was s e e n i n r e g a r d t o a2-adrenoceptors. (-) Verapamil w a s t h e most pot e n t i n h i b i t o r of binding w i t h (+) verapamil, ( 2 ) D-600, d i l t i a z e m , and n i c a r d i p i n e showing i n t e r m e d i a t e a c t i v i t y . N i f e d i p i n e was i n a c t i v e . Verapamil, b u t n o t d i l t i a z e m , impairs t h e a2-adrenoceptors p r e s e n t i n r a b b i t hypothalmus which modulate t h e release of norepinephrine. 72 The dihydropyridines nimodipine o r PY-108-068 ( 3 8 ) , a s w e l l a s verapamil nifedipine and d i l t iazem s e l e c t i v e l y i n h i b i t vasoconst r i c t i o n m e d i a t e d by a2 -adreno c e p t o r s and n o t t h a t mediated by a]-adrenoceptors. 73-79 Thus, v a s o d i l a t a t i o n (and presumably a n t i h y p e r t e n s i v e e f f e c t s ) caused by CEB may be due t o impairment of a2-adrenoceptor mediated v a s c u l a r tone. There may be some impairment of v a s c u l a r al-adrenoceptor p r o c e s s e s as w e l l . 78 N i f e d i p i n e dec r e a s e s v a s o p r e s s o r responses t o norepinephrine i n man. *O
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The dihydropyridine CEB f l o r d i p i n e
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Chap.
7
A n t i h y p e r t e n s i v e Agents
Sprague, Powell
t i e s u n l i k e o t h e r d i h y d r o p y r i d i n e s . 8 1 F l o r d i p i n e and n i f e d i p i n e showed sim i l a r p o t e n c y i n v i t r o i n r e l a x i n g potassium-depolarized c a n i n e v e i n s b u t f l o r d i p i n e was >lOO-fold less p o t e n t i n c a u s i n g r e l a x a t i o n of a r t e r i a l s t r i p s . F l o r d i p i n e w a s 1000-fold less p o t e n t t h a n n i f e d i p i n e i n c a u s i n g d e p r e s s i o n o f f e l i n e c a r d i a c t i s s u e . Thus, f l o r d i p i n e is a " v a s c u l a r sel e c t i v e ' ' CEB. I n SHR, a 30 mg/kg dose o f f l o r d i p i n e produced blood p r e s s u r e l o w e r i n g which p e r s i s t e d f o r 24 h r s and, i n dogs, o r a l d o s e s from 0.03 t o 3.0 mg/kg lowered blood p r e s s u r e due t o reduced p e r i p h e r a l v a s c u l a r res i s t a n c e . 8 2 The lowered blood p r e s s u r e was a s s o c i a t e d w i t h t a c h y c a r d i a , presumably of r e f l e x o r i g i n . O r a l d o s e s of 200 o r 300 mg lowered blood p r e s s u r e i n normotensive human s u b j e c t s and caused r e f l e x t a c h y c a r d i a . 8 3 I n h y p e r t e n s i v e s u b j e c t s , 60 mg of f l o r d i p i n e lowered blood p r e s s u r e w i t h o u t any accompanying changes i n c a r d i a c r a t e . I n a d d i t i o n t o calcium e n t r y b l o c k a d e , f l o r d i p i n e h a s been found t o i n h i b i t c y c l i c n u c l e o t i d e phosphod i e s t e r a s e . 84 Y 8 5
Another v a s c u l a r s e l e c t i v e d i h y d r o p y r i d i n e , FR34235, (40) i s a b o u t f o u r t i m e s more p o t e n t i n v i t r o t h a n n i f e d i p i n e i n c a u s i n g r e l a x a t i o n i n p o t a s s i u m - d e p o l a r i z e d r a b b i t a o r t i c s t r i p s b u t 50 times less p o t e n t i n causi n g d e p r e s s i o n of r a b b i t c a r d i a c t i s s u e . 86 FR34235 a l s o d i s p l a y e d s e l e c t i v i t y f o r r e l a x a t i o n o f a r t e r i a l s t r i p s from v a r i o u s v a s c u l a r b e d s . 8 7 Given i . v . t o a n e s t h e t i z e d dogs, BAY K 8644 (41) caused i n c r e a s e s i n m y o c a r d i a l c o n t r a c t i l i t y , blood p r e s s u r e , and p e r i p h e r a l v a s c u l a r resist a n c e . 8 8 , 8 9 These changes were r e v e r s e d by n i f e d i p i n e b u t n o t by a- o r 6a d r e n o c e p t o r blockade. BAY K 8644 i n c r e a s e d c a r d i a c r a t e and c o n t r a c t i l i t y and a l s o caused coronary v a s o c o n s t r i c t i o n i n an i s o l a t e d c a r d i a c p r e p a r a t i o n . T h i s compound may b e u s e f u l a s a t o o l t o a s c e r t a i n t h e f u n c t i o n of CEB i n t i s s u e s . SC-30552 (42) g i v e n o r a l l y t o SHR ( s p o n t a n e o u s l y h y p e r t e n s i v e r a t s ) lowered blood p r e s s u r e and caused b r a d y c a r d i a . Tachycardia caused by i s o p r o t e r e n o l w a s n o t a f f e c t e d by SC-30552, whereas i s o p r o t e r e n o l - i n d u c e d decreases i n blood p r e s s u r e were a t t e n u a t e d . G a n g l i o n i c b l o c k a d e caused by hexamethonium d i d n o t a f f e c t v a s o d i l a t a t i o n caused by t h i s CEB. A pA2 v a l u e a g a i n s t calcium-induced c o n t r a c t i o n s of r a b b i t . a o r t i c s t r i p s of 7.42 w a s c a l c u l a t e d . These e f f e c t s s u g g e s t 42 p o s s e s s e s some $2- b u t n o t $1- o r aa d r e n o c e p t o r a c t i v i t y and may d e p r e s s t h e SA node.
(g),
a new t y p e of CEB, g i v e n t o normotensive rats o r a l l y a t KB-944 10-50 mg/kg produced graded r e d u c t i o n s i n blood p r e s s u r e and i n c r e a s e d c a r d i a c rate.'l The i n c r e a s e d h e a r t r a t e was a p p a r e n t l y due t o t h e b a r o r e c e p t o r r e f l e x as i t was b l u n t e d by concomitant p r o p r a n o l o l t r e a t m e n t . KB-944 w a s i n t e r m e d i a t e i n potency (nifedipine>KB-944>diltiazem) as an a n t i h y p e r t e n s i v e when g i v e n t o SHR, DOCA-NaC1 h y p e r t e n s i v e r a t s , and r e n a l hypert e n s i v e r a t s , and had a l o n g e r d u r a t i o n of e f f e c t . KB-944 g i v e n i n t r a v e n o u s l y t o a n e s t h e t i z e d dogs produced n e g a t i v e d r o m o t r o p i c e f f e c t s r e f l e c t e d by i n c r e a s e d AV n o d a l conduction t i m e and 3' h e a r t b l o c k i n some a n i m a l s and t h u s KB-944 may be u s e f u l as a n a n t i a r r h y t h m i c e g 2
(44)
reduced blood p r e s s u r e i n S e r o t o n i n Receptor A n t a g o n i s t s - K e t a n s e r i n e x p e r i m e n t a l and c l i n i c a l h y p e r t e n s i o n a c t i n g by s p e c i f i c blockade of serot o n i n (5-HT2) r e c e p t o r s . 9 3 However, k e t a n s e r i n h a s been found t o p o s s e s s p o t e n t a-adrenoceptor b l o c k i n g p r o p e r t i e s i n v i t r o and t h e a n t i h y p e r t e n s i v e e f f e c t s of t h i s compound more c l o s e l y c o r r e l a t e w i t h a-adrenoceptor r a t h e r t h a n s e r o t o n i n antagonism. 9 4 LY53857 (45) i s a h i g h l y s p e c i f i c s e r o t o n i n r e c e p t o r a n t a g o n i s t which does n o t lower blood p r e s s u r e i n S H R . 9 5 Thus, i t a p p e a r s t h a t a n t i h y p e r t e n s i v e e f f e c t s of s e r o t o n i n r e c e p t o r a n t a g o n i s t s may be due, a t l e a s t i n p a r t , t o a-adrenoceptor blockade.
66
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Dopamine Receptor Agonists - Compounds which mimicdopamine can lower blood p r e s s u r e by pre- and p o s t j u n c t i o n a l as w e l l as c e n t r a l n e u r a l mechanisms. The R- and S-enantiomers of SKF 82526 (46) are b o t h r e n a l v a s o d i l a t o r s , b u t t h e R-enantiomer o p e r a t e s i n h y p e r t e n s i v e rats by a p o s t j u n c t i o n a l mechis a pergolide anism whereas t h e S-enantiomer does n o t . 9 6 LY141865 (48) d e r i v a t i v e which lowers blood p r e s s u r e a f t e r o r a l a d m i n i s t r a t i o n t o SHR.97 LY141865 i n low doses reduced t h e h y p e r m o t i l i t y observed i n SHR b u t not i n t h e i r corresponding normotensive c o n t r o l s . 9 8 P e r g o l i d e g i v e n i . v . lowers blood p r e s s u r e and reduces p e r i p h e r a l sympathetic n e r v e a c t i v i t y s u g g e s t i n g a c e n t r a l nervous system s i t e of a c t i o n . 9 9
(47)
-
MPV295 (49) lowers blood p r e s s u r e a-Adrenoceptor Agonists and A n t a g o n i s t s bv s t i m u l a t i o n of a-adrenoceptors i n t h e b r a i n s t e m F o o MPV295 s t i m u l a t e s o n l y c a r d i o v a s c u l a r a2-adrenoceptors whereas c l o n i d i n e s t i m u l a t e s b o t h aland a2-adrenoceptors. I n t e r e s t c o n t i n u e s i n t h e area of s e l e c t i v e a l - a d r e n o c e p t o r blockade as a mechanism t o lower blood p r e s s u r e . Compound 50 w a s less t h a n 1 / 1 0 as p o t e n t as p r a z o s i n i n v i t r o as an a-adrenoceptor b l o c k e r b u t w a s n e a r l y e q u i v a l e n t i n lowering blood p r e s s u r e i n r e n a l h y p e r t e n s i v e rats. l o l Unl i k e p r a z o s i n , t r i m a z o s i n lowered blood p r e s s u r e by some unknown mechanism i n a d d i t i o n t o blockade of a1-adrenoceptors. l o 2 Compound 51 showed pot e n c y as an al-adrenoceptor b l o c k e r i n v i t r o less t h a n t h a t found w i t h p r a z o s i n . l o 3 The compound lowered blood p r e s s u r e i n SHR and may have c e n t r a l a n t i h y p e r t e n s i v e e f f e c t s due t o blockade of b r a i n al-adrenoceptors. Like p r a z o s i n , KF-4942 (52) lowers blood p r e s s u r e due t o blockade of vasT i b a l o s i n e (53) i s a s e l e c t i v e al-adrenoceptor c u l a r a1- adrenoceptors.m a n t a g o n i s t w i t h b o t h c e n t r a l and p e r i p h e r a l a c t i o n s . O 5 T i b a l o s i n e i s about 1000 t i m e s less p o t e n t t h a n p r a z o s i n i n c a u s i n g blockade of v a s c u l a r a l - a d r e n o c e p t o r s and d i s p l a y s a n x i o l y t i c a c t i v i t y i n animals and a t r a n q u i l i z i n g e f f e c t i n man.lo6 The S-enantiomer of WB-4101 (54) w a s more pot e n t t h a n t h e R-enantiomer i n b l o c k i n g a l - and a2-adrenoceptors and w a s a l s o more s e l e c t i v e toward a l - r e c e p t o r s i n p i t h e d rats. l o 7
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67
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P-Adrenoceptor Blocking Agents Compound 55 d i s p l a ed n e a r l y a 9000-fold g r e a t e r a f f i n i t y f o r 61- than f o r $2-adrenoceptors. I C I 141,292 (56) w a s a l s o s e l e c t i v e € o r B 1-adrenoceptor mediated c a r d i o v a s c u l a r and metaboli c responses. I n t r i n s i c sympathomimetic a c t i v i t y w a s found toward c a r d i o v a s c u l a r , b u t n o t m e t a b o l i c 61-adrenoceptors. l o g K-351 (57), an a n t i h y p e r t e n s i v e a g e n t i n SHR, showed n o n - s e l e c t i v e B-adrenoceptor blockade aa d r e n o c e p t o r blockade, and d i r e c t v a s c u l a r r e l a x a t i o n i n v i t r o . l l o y
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Miscellaneous Agents A t r i o p e p t i n s I and I1 (At1 and A t I I ) , two p e p t i d e s of a t r i a l t i s s u e o r i g i n , have been i s o l a t e d and c h a r a c t e r i z e d . l 1 These s u b s t a n c e s are d i u r e t i c , n a t r i u r e t i c and r e l a x i n t e s t i n a l (At1 and A t I I ) and v a s c u l a r ( A t I I ) t i s s u e . '12 The d i s c o v e r y of t h e s e compounds s u g g e s t s a new endogenous c o n t r o l mechanism i n v o l v i n g t h e h e a r t and kidneys t h a t may b e important f o r maintenance of normal blood p r e s s u r e . The r a t i o of CNS t o a n t i h y p e r t e n s i v e a c t i v i t i e s , t y p i c a l l y from 1 t o
1 0 f o r hypotensive t e t r a h y d r o c a n n a b i n o l a n a l o g s , h a s been i n c r e a s e d t o 100 i n (58). - 113 However, t a c h y p h y l a x i s w a s observed f o r t h e a n t i h y p e r t e n s i v e activity
.
U-54,669F (2)lowered blood p r e s s u r e i n h y p e r t e n s i v e and normotens i v e r a t s and monkeys. '14 The compound i n h i b i t e d sympathetic neuronal funct i o n and d e p l e t e d catecholamines i n c a r d i a c and o t h e r t i s s u e s . However, t h e r e were no e f f e c t s on p o s t j u n c t i o n a l organ responsiveness t o c a t e c h o l a mines n o r e f f e c t s on blood p r e s s u r e d u r i n g p o s t u r a l changes. References 1. M. Moser, Hypertension, 5, 808 (1983). 2. D.S. Goldstein, Hypertension, 5, 86 (1983). 3. B. Folkow, G.F. DiBona, P. Hjemdahl, P.H. Thoren and B.C. Walin, Hypertension, 5 , 339 (1983). 4. B.C. Zimmerman, Circ. Res., 53, 121 (1983). 5. R.F. Furchott, Circ. Res., 2,557 (1983). 6. D.B. Gordon, Hypertension, 5, 353 (1983). 7. M.F. O'Roarke, Aust. NZ J. Med., 3, 84 (1983). 8. T. Inagami, J.J. Chang, C.W. Dykes, Y. T a k i i , M. Kisaragi and K.S. Misono, Fed. Proc. 47, 2729-2734 (1983). Haber, Clin. and Exper. Hyper.-Theory and P r a c t i c e , A5, 1193-1205 (1983). 9. 10. M. Szelke, B. Leckie, A. H a l l e t t , D.M. Jones, J. S u e i r a s , B. Atrash and A.F. Lever, Nature, 555-557 (1982). 11. M.J.A. A m s t e i n , H.B. B e l l , D.P. Clough, J.S. Major, A.A. Oldham and J. Saunders, B r . J. Pharmacol., 2, 253P (1983). 1 2 . J. Boger, N.S. Lohr, E.H. U l m , M. Poe, E.H. Blaine, C.M. F a n e l l i , T.Y. Linn, L.S. Paine, T.W. Schorn, B.I. LaMont, T.C. Vassil, 1.1. S t a b i l i t o , D.F. Veber, D.H. Rich and A.S. Bopari, Nature, 303, 81-84 (1983). 13. European Patent Application 82111359.6. 1 4 . T. Unger, D. Ganten and R.E. Lang, Clin. and Exper. Hyper.-Theory and P r a c t i c e , A5, 1333-1354 (1983). 15. A. Schwab, I. Weinryb, R . Marcerata, W. Rogers, J. Suh and A. Khandwala, Biochem. Pharm., 2,1957-1960 (1983). 16. M. Burnier, G.A. Turini. H.R. Brunner, M. Porchet, D. Kruithof, R.A. Vukovich and H. Gavras. B r . J. Clin. Pharmacol., 12, 893-899 (1981). 1 7 . T.A. Solomon, F.S. Caruso and R.A. Vukovich, Clin. Pharmacol. Ther., 22, 231 a b s t r . c28 (1983). 18. J . R . Powell, B. Rubin, C.W. Cushman. J. Krapcho and M . J . Antonaccio. Abstr. Eur. ~ e ~ t . Hypertens. 1 s t Milan 355 (1983).
a,
68 -
Sect. I1
- Pharmacodynamic Agents
Corner, Ed.
19. D.H. K i m , C.J. Guinosso, G.C. Buzby. Jr.. D.R. Herbst, R.J. McCaully, T.C. Wicks and R.L. Wendt, J . Med. Chem., 26, 394-403 (1983). 20. J.L. Dinish, N.K. Metz, R.W. Lappe, D.H. K i m and R.L. Wendt, Pharmacologist, 25, 102 a b s t r . 8 (1983). 21. J.L. Stanton, N . Gruenfeld, J . E . Babiarz, M.H. Ackerman and R.C. Friedmann, J . Med. C h a . , 26, 1267-1277 (1983). 22. K. Hayashi, Y. Ozaki, K. Nunami, T. Uchida, J. Kato, K. Kinashi and N. Yoneda, Chem. Pharm. Bull., 31, 570-576 (1983). 23. T. Unger, T. Yukimura, M. MarinGrez, R.E. Lang, W. Rascher and D. Ganten, Europ. J . Pharmacol., 2, 411-420 (1982). 24. F.J. McEvoy, F.M. L a i and J.D. Albright, J . Med. Chem.. 26, 381-394 (1983). 25. E.D. Thorsett, E.E. Harris, S. Aster, E.R. Peterson, D. Taub, A.A. P a t c h e t t , E.H. Ulm and T.C. Vassil, Biochem. Biophys. Res. Corn., 111,166-171 (1983). 26. C.S. Sweet, Fed. Proc. 42, 167-170 (1983). 27. H.H. Rotmensch, P.H. Vlasses, B.N. Swanson, R.O. Davies, D.D. Merill and R.K. Ferguson. Clin. Pharmacol. Ther., 3, 210 a b s t r . A24 (1983). 28. H.H. Rotmensch, P.H. Vlasses, B.N. Swanson. J . D . I r v l n . K.E. Harris, D.G. Merrill and R.K. Ferguson, Am. J. Card., 53, 116-119 (1984). 29. E.J. Sybertz. T. Baum. H.S. Ahn, S. Nelson, E. Eynon, D.M. Desiderio, K. Pula, F. Becker, c. Sabin, R. Moran, G.V. V l i e t , B. Kastner and E. Smith, J. Cardiovasc. Pharmacol., 2. 643-654 (1983). 30. M.L. Powell, K. Stabins and J.E. P a t r i c k , Fed. Proc., 42, 378 a b s t r . 499 (1983). 31. T. Baum, E . J . Sybertz, R.W. Watkins, H.S. Ahn, S. Nelson, E. Eynon, G.V. Vliet, K.K. Pula, C. Sabin, D.M. Desiderio, F.T. Becker and S. Vermulapalli, J. Cardiovasc. Pharmacol., 2, 655-667 (1983). 32. M. Vincent, G. Remond, B. Portevin, B. Serkiz and M. Laubie, T e t . L e t t . , 23, 1677-1680 (1982). 33. N . Gruenfeld. J.L. Stanton, A.M. Yuan, F.H. Ebetino, L . J . Browne, C. Gude and C.F. Huebner, J. Med. Chem., 26, 1277-1282 (1983). 34. B. J a c o t des Combes, G.A. T u r i n i , H.R. Brunner, M. Porchet, D.S. Chen and Supradip (Ben) Sen, J . Cardiovasc. Phannacol., 5, 511-516 (1983). 35. W. H. Parsons, J . L . Davidson, D. Taub, S.D. Aster, E.D. T h o r s e t t , A.A. P a t c h e t t . E.H. Ulm and B . I . Lamont, Biochem. Biophys. Res. Corn., 117, 108-113 (1983). 36. D.S. Chen, R.A. Dotsen, R.Q. B u r r e l l and B.E. Watkins, Pharmacologist, 25, 240 a b s t r . 715 (1983). 37. R.E. Galardy, V. Kontoyiannfdou-Ostrem and Z.P. Kortylewicz, Biochem., 22, 1990-1995 (1983). 38. R.G. Almquist, P.H. C h r i s t e , W. Chao and H.L. Johnson, J. Pharm. Sci., 72, 63-67 (1983). 39. R. Zelis, Amer. J. Med., 76(6B), 3-12 (1983). 40. M. Konieczkowski, M.J. Dunn, J.E. Stork and A. Hassid, Hypertension, 2, 446-452 (1983). 41. P. Cervoni, P.S. Chan, F.M. Lai and J . E . Birnbaum, Fed. Proc., 2, 157-161 (1983). 42. P.S. Chan, P. Cervoni, M.A. Ronsberg, R.C. Accomando, G . J . Quirk, P.A. Scully and L.M. Lipchuck, J. Phannacol. Exp. Ther., 727-732 (1983). 43. J.B. Bicking, M.G. Bock, E.J. Cragoe, Jr., R.M. DiPardo, N.P. Could, W.J. Holtz, T . J . Lee, C.M. Robb, R.L. Smith, J.P. Springer and E.H. Blaine, J. Med. Chem., 26, 342-348 (1983). 44. J . B . Bfcking. C.M. Robb, E . J . Cragoe, Jr., E.H. Blaine, L.S. Watson and M.C. Dunlay, J. Med. Chem., 26, 335-341 (1983). o ,197-212 45. J . Casals-Stenzel, M. Buse and W. Losert, Prostaglandins Leukotrienes Med., l (1983). 46. B. Muller, J. Schneider, K. Wilsmann, W. Lintz and L. Flohe, Prostaglandins Leukotrienes l , 361-372 (1983). Med., l 47. B.J. Whittle, S. Moncada, K. Mullane and J . R . Vane, Prostaglandins, 2,205-223 (1983). 48. M.A. Orchard, J.M. Ritter, G.L. Shepherd and P . J . L e w i s , B r . J . Clin. Pharmacol., 15. 509-511 (1983). 49. C.W. Howden, H.L. E l l i o t . C.B. Lawrie and J . L . Reid. J. Cardiovasc. Phannacol.. 2. 552556 (1983). 50. M. Catalano, J . P a r i n i and A. L i b r e t t i . Eur. J. Clin. Pharmacol., 2 5 , 157-161 (1983). 51. T. Chiba, M. Hirohashi, I. S u d C f , S. Tanaka, K. Watanabe and A. Akashi, Arzneim.Forsch., 33(1), 112-116 (1983). 52. M.J.M.C. Thoulen, J.C.A. van Meel, B. W i l f f e r t , P.B.M.W.M. Timermans and P.A. vanZweiten, Pharmacology, 27, 245-254 (1983). 53. H. Lape, A. DeFelice and D. Bailey, Pharmacologist, 25, 101 a b s t r . 1 (1983). 54. A. DeFelice and H. Lape, Pharmacologist, 25, 101 a b s t r . 2 (1983). 55. J.M. Evans, C.S. Fake, T.C. Hamilton, R.H. Poyser and E.A. Watts, J. Med. Chem., 26, 1582-1589 (1983). 56. L. Davio, M.N. Agnew, R.C. Effland, J.T. Klein, J.M. Kitzen and M.A. Schwenkler, J . Me& Chem., 26. 1505-1510 (1983). 57. J.M. McCall, J.W. Aiken, C.G. Chidester, D.W. DuCharme and M.G. Wendling, J . Med. Chem.. 26, 1791-1793 (1983). 58. Thastrup, B. Fjalland and J. Lernich, Acta Pharmacol. e t Toxicol., 2,246-253 (1983).
u,
Chap. 7
A n t i h y p e r t e n s i v e Agents
Sprague, Powell
Q
59. A. S a l imb e n i . E. Manghisi. G. Fern1 and G.B. Fregnan. I1 Farmaco.-Ed. S c i . , 38, 904910 ( 1 9 8 3 ) . 60. F.V. DeFeudis, L i f e S c i . , 557 (1983). 61. J.L . Rouleau, W.A. Parmley, J . S t evens. I. Wikrnan-Coffelt, R. S i e v e r s , R.W. Mahley and R.J. Havel, J. Am. C o l l . C a r d i o l . , 1.1453 (1983). 62. P.D. Henry and K . I . B e n t l e y , J. C l i n . I n v e s t . , 68, 1366 (1981). 63. C.M. Heesch. B.M. Miller, M.D. Thames, and F.M. Abboud, Am. J. P h y s i o l . , 245, H653 (1983). 64. W. Kiowski, 0. Bertel, P. E rne, P. B o l l i , U.L. H ulthe n, R. R i t z and F.R. B u h l e r , Hy p e r te n si o n , 5 (S uppl I), 1-70 (1983). 65. R.A.B. McLeay, T . J . S t o l l a r d , R.D.S. Watson, and W.A. L i t t l e r , C i r c u l a t i o n , 67,1084 (1983). 66. G. H a e u s l e r , J. Pharmacol. Exp. T herap., 180,672 (1972). 67. J . R . Powell and L . J . K e w i n , Fed. Proc. 62. 842 (1983). 68. H. Kuriyama, Y. I t o . H. S uzuki , K. Kitamura, T. I t o h , M. Kajwara and S. F u j i w a r a , C i r c . Res., 2 (Suppl I ) , 92 (1983). 69. M. Wibo, I. D e l f o s s e and T . Godfraind. Arch. i n t . Pharmacodyn., 263, 333 (1983). 70. P.A. van Zweiten and P.B.M.W.M. T i mermans. J. Mol. C e l l . C a r d i o l . , 15, 717 (1983). 71. W.G. N a y l e r , J . E . Thompson and B. J a r r o t t , J . Mol. Cell. C a r d i o l . , i 15
ANNUAL REPORTS IN MEDICINAL CHEMISTRY- 19
Copyright 0 1984 by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0-12-040.519-9
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Effects of kukotrienes on Respiratory Function 1) In Vivo Actions - The in vivo pulmonary response to leukotrienes varies among species. Rats are often resistant to the effects of leukotrienes, but aerosolized LTD causes a bronchospasn in an inbred Basenji-Greyhound dogs , an strain with bronchial hyperresponbiveness. inbred strain with hyperreactive airways, develop an atropine-inhibitable bronchospasm to inhaled LTD while there is no response in mongrel dogs. 2 7 Furthermore, aerosolized LTh4 is 300-900 times more potent than histamine in causing a bronchospasm in Ascaris-sensitive rhesus monkeys, whereas non-allergic monkeys are relatively non-responsive. * The bronchial response in monkeys to aerosol LTD4 is inhibited by inhaled FPL-55712 Administered into the right while intravenous FPL-55712 is inactive. atria of monkeys, LTC4 causes a bronchospasm and a transient increase in atrial pressure followd by a prolonged hypotension.
’
In healthy humans, inhaled LTC4 and LTD4 are more potent than histamine at causing a decrease in maximum expiratory flow rate.31’32 The effect of LTD4 is independent of the production of cyclooxygenase products. In contrast to airway hypersensitivity to histamine among human asthnatics, there is no increased sensitivity to inhaled LTD4. 3 3 ?he importance of cyclooxygenase products, particularly thranboxane A2, in the bronchospastic response to leukotrienes depends on the species and route of administration. The bronchospasn resulting from intravenous LTCq in guinea pigs3’ and LTD4 in cats3 is blocked by cyclooxygenase inhibitors. The response to inhaled LTC in guinea pigs is either not inhibited or is enhanced by cyclooxygenade blockade.
,’
2) In Vitro Actions - Studies using a superfusion technique suggest that a major portion of the contractile response of guinea pig lung parenchymal strips to LTC and LTD is due to the generation of thrcmboxane A2.37 In contrast, rabhit and r& parenchymal strips do not develop a marked contractile response or synthesize thromboxane A2 in response to LTC4 or LTD4. More recent work has shown that under non-flow conditions, the contractile responses of guinea pig parenchyma to LTC and LTD4 are LTB ib 5-10 times more indepedent of thrclmboxane A generati~n.~*’~~ potent than histamine in con&acting guinea pig ludg strip^.^ Intravenous or aerosol administration of LTB4 to guinea pigs produces a These in vitro and in vivo effects of LTB4 are secondary bron~hospasm.~~ to the generation of cyclooxygenase products.
’
Conversion of LTC to LTD and subsequent blockade of the contractile response by FPL-55712 &ring ikxbation with guinea pig trachea has been reported. 4 2 This LTD4-induced contraction may utilize predaninantly intracellular calcium stores because it is inhibited by 1MB-8, an inhibitor of intracellular calcium mobilization, but not by nifedi~ine.~ In contrast, on the guinea pig ileum, the slowness of the contractile response to LTD4, compared to histamine, and the sensitivity to blockade by a slow calcim channel blocker, D.600, suggests that LTD4 utilizes extracellular calcium.4 The Merck-Frosst group prepared the sulfones of LTC4, LTD4 and LTE4 and found that they are almost equipotent to the sulfides on contracting guinea pig trachea and intravenous administration to guinea pigs produces an indanethacin and FPL-55712 inhibitable bronchospasm. 14 The contractile activity of LK4 and LTD analogs has been examined. The rank order of potency is similar on guine4 pig trachea, lung parenchyma and ileum where the 5R, 65 isaners have 0.01-times the potency of the natural
Chap. 24
Biology of Leukotrienes
Kreutner, Siege1
243
5 S , 6R i~aners.~~~48 Eight biosynthetically formed sulfidopeptide leukotrienes (LTC3, 8,9-LTC3, LTC4, 11-trans LIT4, LTC5, LTD4, LTE4 and 11-trans LTE ) were studied for contractile activity on guinea pig lung parenchyma.sif All canpounds are full agonists and 5000-times more potent than histamine. There is no potency difference between 11-cis and 11-trans i s m r s of LTC4 or LTE4. Incubation of LTE4 with glutathione and gm-glutamyltranspeptidase results in the formation of LTF .50 LTF4 is equipotent to LTD on guinea pig trachea, but only 0.01-timed as potent on ileum and in v~vo!’~
Purified human lung mast cells release SRS-A upon stimulation with anti-IgE. 5 2 Eosinophils (horse) release leukotrienes upon stimulation with ionophore A-23187 5 3 and platelets (rabbit) release leukotrienes on stimulation with thranbin or PAF.54 Rat alveolar macrophages 5 5 and human peritoneal macrophages 5 6 stimulated with A-23187 release LTC4 and LTB4, but not LTD4. The production of LTB4 by human alveolar macrophages has also been demon~trated.~~ IgE mediated release of SRS-A occurs with alveolar but not peritoneal rat macrophages.55 Sensitized h w n lung challenged with antigen, releases sufficient amounts of LTC4, D4 and E4 to cause contraction of human bronchi in v i t r ~ . ~ ~ A major finding has been the importance of lipoxygenase products in the secretion of airway mucus. Mucus glycoprotein secretion fran human bronchi in culture is stimulated by l o w concentrations (1-10 nM) of various H!iTEs, with 12- and 15-HETE m s t active.59’60 Antigen-provoked mucus release is inhibited by the lipoxygenase inhibitors ETYA, nordihydrcguiaretic acid and alpha naphthol.59 A recent report claims a significant decrease in mucociliary clearance after oral ingestion of aspirin by healthy volunteers.61 This effect presumably is due to an augmented production of lipoxygenase products after cyclooxygenase inhibition. Other reports have shown increased secretion of mucus after intraarterial injection of LTC4 or LTD4 into dog^,^^-^' intratracheal administration of LTC4 into cats 6 5 or in vitro addition of LTC4 or LTD4 to human bronchial mcosa .6 Leukotriene Actions in the Gastrointestinal System - Large species differences have been found in the response of gastrointestinal tissue to sulfidopeptide leukotrienes. Human gastrointestinal muscle (ileum, stanach, jejunum, colon) does not contract to SRS-A fran guinea pig lung.67 In contrast, both LTCq and LTD contract the rat stomach and colon but not the duodenum or ileum6’ 4LTC4, D4 and E4 produce cyclooxygenase independent-Sontractionsof the guinea pig gall bladder.6 At high concentrations (10 MI, LTC4 and LTD also provoke acid secretion from isolated rabbit gastric parietal c e l l ~ . ~A7 ~role for leukotrienes in inflamnatory bowel disease is suggested by the release of SRS-A fran antigen challenged sensitized colonic m u c o ~ a . ~ ~ Lipoxygenase products may also be essential for insulin release by pancreatic islets. Glucose-induced insulin release is inhibited by lipoxygenase inhibitors, including BW-755CI nordihydrcquairetic acid and 15-HETE.72,73 Also, 5-HETE augments insulin release triggered by low concentrations of glucose.73 Rat pancreatic islets produce 5-, 12- and 15-HETE fran arachidonic acid.73 Actions of Leukotrienes on Cardiovascular Tissue - Purified SRS-A generated fran guinea pig lung has been shown to increase vascular permeability in guinea pig skin, especially if injected together with a vasodilator like EGE2.74 Intradermal LTC4 and LTD4 cause a wheal and
244
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flare75C76and increase microvascular blood flow 77 in human skih. In a hamster cheek pouch preparation, law concentrations of LTC and LTD4 cause intense arteriolar constriction followed by a marked incretse in vascular permeability. 7 8 Airway edema associated with an anaphylactic reaction may be a leukotriene dependent response, since local application of LTC4, D4 or E to guim7$ pig trachea in vivo produces an increase in microvascular perm8ability
.
Low concentrations of LTD (1-lOnM) cause a reversible cessation of beating of rat heart cells in hlture. Evidence was presented by Burke -et al. for a role of leukotrienes in cardiac anaphylaxis. The cardiac dysfunction associated with anaphylaxis is partially duplicated by addition of LTC , D4 or E4. A l l leukotrienes produce a negative inotropic effect, decreasi coronary flow of isolated guinea pig heart, and decrease the contractile force of pectinate muscle f m n human heart. LTD4 also potentiates the tachycardia and arrhythnias caused by histamine. agents on Sulfidopeptide leukotrienes also appear to be potent contra:\ile canine renal arteries8* and rat and cat coronary arteries. These effects are independent of thronboxane A2 generation.
The in vivo effects of leukotrienes on blood pressure and heart rate are quite canplex. Generally, there is a biphasic response consisting of an initial transient increase in systemic blood pressure followed by a sustained systemic hypotension after a bolus of 1-10 pg/kg of LTCq or LTD4. 84-86 Intravenous LTC4 decreases cardiac output and rate while Pretreatment with indmthacin increasing total peripheral resistance. potentiates the initial increase in blood pressure and attenuates the Low sustained hypotension and changes in cardiac function due to LTC doses of LTD4 injected into the left circmflex coronary artery 8f sheep result in coronary vasoconstriction and impaired ventricular function.a7
’
.
Leukotrienes may also play a pathogenic role in hypxic pulmonary hypertension. Hypoxic pulmonary vasoconstriction in sheep is prevented or LTC and LTD are present ‘inlung lavage fluid reversed by FPL-55712. obtained from human newborns &th a clfnical diagnosis of persistent pulmonary hypertension and hypoxemia. Leukotrienes in Inflamnatory Disorders - Leukotrienes are produced by cells ( W s and macrophages) that are present in large numbers at inflmtory sites. If stimulated with the calcium ionophore A-23187, PMNs produce LTB4, which has potent chemokinetic activity and is also an aggregating agent for neutrophilsPl The potency of LTB as a chemotactic agent is similar to that of the canplement ccmponent C5a40r the synthetic peptide F-met-leu-phe. 2In a hamster cheek pouch preparation, LTB4 causes adhesion of leukocytes to vascular endothelim. 76 ’78 Evidence has also been presented that LTB4 is eosinophil chemotactic factor (ECF).95 However, ECF stimulates the migration of eosinophils, but not neutrophils while LTB4 is equally chemotactic for both cell types. 9 6 The neutrophil chanotactic activity of LTB is blocked canpetitively by acetyl LTB 9 7 Intradermal injection of Ld4 (0.2-1.5 m l e ) into human skin caused an imnediate erythema and wheal followed by a delayed reaction of erythema and induration at 1-4 hr with perivascular neutrophil infiltrates. 76
.
LTB has been shown to cause the release of enzymes f m n human 98.4 9 9 Canpared to C and F-met-leu-phe, LTB is a weak secretogcgue and is active’8nly at concentrations 14-fold greater t h a n pMB.
Biology of Leukotrienes
Chap. 24
Kreutner, Si ege1
2115
needed to stimulate chemotaxis or adherence of neutrophils. Neutrophil degranulation caused by LTB4 is inhibited by LTB4-dimethylamide (KD = 0.2 In addition, it has recently been reported that LTB4 may have pM). h u n d u l a t o r y activity For example, LTJ34 appears to cause specific suppression of human lymphocyte function, possibly by inducing suppressor cells. Moreover, LTB4 augments natural cytotoxic cell activity. These latter activities may play important roles in chronic inflamnatory disease. Injection of LTJ3 into rabbit skin,lo6 rabbit eye,l07 guinea pig peritoneal cavityg4 4and human skin results in leukocyte accumulation at the injection site. In a hamster cheek pouch preparation, the movement of leukocytes through the vascular endothelium as a result of the local application of LTB has been directly observed.lo6 The increase in vascular permeability due t8 LTB4 is augmented by the co-administration of a vasdilator prostaglandin and is dependent upon the presence of neutrophils
.
.
The role of LTB4 as an inflammatory mediator is supported by studies showing high amounts of LTB4 in the skin c h e fluid of involved skin from psoriatics and in gouty effusions. In contrast, LTC4 and LTD4, but not LTB4, are detected by bioassay and HPLC in carrageenaninduced pleurisy in rats. 1 1 2 Leukotrienes may also be involved in the . modulation of pain in inflamnatory lesions. In the rat paw, LTB4 causes However, the mechanism of action of this response slight hyperalgesia. is not understood. Leukotrienes C4, D4 and E4 may also be important mediators of inflamnatory processes. LTD4 and LTE4 influence vasopermeability in guinea pig skin and their activities are potentiated by the presence of prostaglandin vasodilators. In addition, LTC4 and LTD increase neutrophil adherance and may also affect release reaction4 of macrophages
.
Elevated levels of both LTBq and LTC4 are found in psoriatic lesions in man.114’11 Ulcerative colitis has among its characteristic features an accumulation of neutrophils in mucosal sites. Samples of tissue f m In inflamed sites in such patients contain elevated levels of LTB4. addition, these samples have an increased ability to synthesize 5-lipoxygenase products ccmpared to normal tissue. Synovial fluids from rheumatoid arthritic or gouty patients also contain large numbers of neutrophils and elevated levels of LTB4. Receptor Binding of Leukotrienes - Initial reports suggested the presence of tissue specific heterogeneity of LTD4 receptors.ll* This is supported by more recent experiments on guinea pig trachea utilizing FPL-55712.119 The availability of high specific activity radiolabeled LTC4 and LTD4 has permitted t p detection of specific binding sites 1~lung for these ligands. [ Hl-LTDq exhibits high affinity (K,,= 10 OM), saturable binding to membrane3+of gy)nea @g lung.lz0 Binding i$ enhanced by divalent cations (Ca , Mg , Mn 1 and inhibited by Na LTE , but not LTC , has high affinity for the binding site suggesting q a t L d 4 and LTD4 ineeract at different sites. High affinity binding of [ HI-LTC was also and guinea pig 122,923 and on an intact demonstrated on lungs from rat snooth muscle cell line, DITl MF-2, derived fran Syrian hamster vas deferens.lZ4 The LTC4 binding is biochemically distinct from LTD binding sites.120’122 The KD for FPL-55712 is greater against LTC than &D4. Binding of LTD4, but not L K 4 is altered in the presence o# guanine nucleotides.
.
24 6
Sect. V
- Topics
i n Biology
Egan, Ed.
Evidence has also been presented that LTB interacts with a specific and secretogogue cell receptor. 9 3 - 1 2 5 The difference in chemot&ic activity of LTB4 has been explained in tern of the former being associated with high affinity receptors and the latter with lcw affinity receptors. Pharmacology of Leukotriene Biosynthesis Inhibitors and Antagonists 1) Biosynthesis Inhibitors - BW-75% (3-1 and phenidone ( 4 ) are inhibitors in of the lipoxygenase and cyclooxygenase pathways 127,128 aEd inhibit vitro anaphylactic contractions of airway smooth muscle fran guinea 7127c129 P1gs’ W 7 5 % inhibits SRS-A, but not histamine, release fran antigen challenged sensitized human lung 1 3 0 and when given by inhalation, attenuates antigen-induced bronchospasm in Ascaris-sensitive monkeys. 1 3 1 Bw-755C inhibits prostaglandin synthesis in inflamnatory exudates but does not inhibit PGI synthesis by gastric mucosa.132 BW-75% also reduces the concentr&ion of Lm4, thrclmboxane B2 and PGE2 in exudates derived fran the subcutaneous implantation of carrageenan impregnated PMN migration into the inflamnatory exudate is also sponges in rats. decreased. BW-75%, but not indanethacin, reduces the size of a myocardial infarct produced in dogs by coronary occlusion followed by reperfusion. The mechanism may involve inhibition of lipoxygenase to reduce the production of LTC and LTDq which constrict coronary arteries and of LTB4 which is c h m t a h for inflamnatory cells.
Me
r
3
-5
At concentrations lower than those needed to inhibit either lipoxygenase or cyclooxygenase, BW-75% enhances lymphocyte activation by BW-755C and phenidone, applied topically to mouse skin, mitogens. prevent the induction of epidermal ornithine decarhxylase caused by application of the tumor p m t e r 12-0-tetra-decanoyl phorphol-13acetate. Nafazatrm (2) inhibits 5- and 12-lipoxygenases but not the cyclooxygenase of B16a tumor cells and also inhibits tumor Other studies have shown nafazatran increases FG12 proliferation. synthesis by aortic strips and has antithrclmbotic activity.138
’
Benoxaprofen (5) has antiinflmtory activity and also inhibits the release of SRS-A.139 At a concentration of 100 pM, benoxaprofen inhibits a 5-lipoxygenase in guinea pig peritoneal cells and HL-60 cells with no effect on the 12-lipoxygenase of h m n platelets or a soybean 15-lipoxygenase.l’+O In rat Rulhis, benoxaprofen is a better inhibitor of cyclooxygenase than 5-lipoxygenase. Its in vivo activity in animal models is consistent with these results, because inhibition of edema occurs at lower doses than inhibition of cellular influx to inflamed sites. Benoxaprofen is claimed to be ineffective clinically to inhibit aspirin-induced bronchospasm 1 4 1 but has shown beneficial activity in the treatment of psoriasis,142c14 a disease associated with the presence of increased levels of lipoxygenase products in epidermal lesions.
Biology of Leukotrienes
Chap. 24
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OH
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U-60,257 (7) is an inhibitor of glutathione-S-transferase 1 4 4 but its principal action seems to be inhibition of a 5-lipoxygenase. 1 4 5 U-60,257 inhibits SRS, but not histamine, release from human lung, competitively antagonizes the contactile effects of LTC4 and LTD on guinea pig ileum and inhibits lysosamal enzyme release frcm human F d N s . In Ascaris sensitive mnkeys, U-60,257 given by aerosol (0.05-1%) or intravenously (0.01-5 q/kg) inhibits antigen-induced bronchoconstriction. l 4 2) Leukotriene Antaqonists - FPL-55712 (8)consistently inhibits contractile responses to LTD4, but its effect on responses to LTC4 are et al. claim that FPL-55712 does not antagonize LTC4 on less clear. Krell -guinea pig parenchymal strips. llr7 On trachea and bronchus, FPL-55712 antagonizes responses to low concentrations of LTC , but potentiates responses to high concentrations of LIT4. Krell afso showed that antigen-induced bronchospasn in dogs is not inhibited by i.v. or aerosol FPL-55712. 0
0
-8 Limited studies with FPL-55712 have been conducted in humans. Inhaled FPL-55712 in 4 allergic astbtics gave inconclusive results. 1 4 9 'Jho of the four patients s h m d improved FEV1. Tracheal mucous velocity is decreased in asthtics challenged with antigen. 5 0 Inhalation of 0.5-1% FPL-55712 prevents the decrease in mucous velocity but does not inhibit the bronchospasn. Inhalation of LTC4 or LTD by normal subjects induces coughing which is blocked by aerosol FPL-557f2. 1 5 1 In BasenjiGreyhound dogs, the bronchospasn to inhaled citric acid is associated with increased plasma levels of SRS (but not histamine) and is partly blocked by FPL-55712. 1 5 2 Much of the pharmacology of FPL-55712 is ascribed to its antagonism of SRS-A; however, at concentrations only slightly higher than those needed to inhibit SRS-A, FPL-55712 also inhibits thrmboxane synthetase' and lipoxygenase in broken cell, but not intact cell preparations. FPL-55712 also inhibits the extensive necrosis of liver parenchymal cells and death in animals injected with endotoxin and D-galactosamine. 4R,%,62-2-nor LTD (4R-hydroxyl-5S-cysteinylglycyl-6Z-nonadecenoic acid) is an analog of L h 4 which at a concentration of 100 ~ J M , antagonizes
Sect. V
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the contractile response to LTD4, LTC and LTE4 on guinea pig airways. 1 s 6 * 1 5 7 The effects of histdine are not blocked. The vasoconstrictor effects of LTD4 on guinea pig pulmnary artery are also blocked. In vivo, an intravenous dose of 5 mg/kg inhibits the bronchoconstrictor response to LTD4 given 1 min later. 4R,5S,6Z-nor LTE also has antagonist activity while 4R, 5S,62-nor LTCl and 4S, 5R,6E-nor L d l are agonists. Summary - The leukotrienes are a group of biologically active mediators derived from arachidonic acid. While LTB is a potent chemotactic agent and may mediate inflamnatory reactions, L'k and LTD are predaninantly snooth muscle contractile agents with an &lied r o d in allergic diseases. The availability of pharmacological agents with in vivo activity that inhibit the biosynthesis or actions of leukotrienes (Chapter 11, hlmonary and Antiallergy Agents) will greatly facilitate our understanding of the role of leukotrienes in pathological conditions. Steroids with antiinflatmatory activity prevent the release of arachidonic acid and, thereby, inhibit the subsequent generation of both cyclooxygenase and lipoxygenase products. Same of the effects of steroids not shared @ aspirin-like drugs may, therefore, be due to an inhibition of leukotriene formation. References 1. 2. 3. 4.
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Chapter 25.
Endogenous N a t r i u r e t i c Agents
Mary Anna N a p i e r and Edward H. B l a i n e Merck Sharp and Dohme Research L a b o r a t o r i e s Rahway, NJ 07065 and West P o i n t , PA 19486
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Introduction Endogenous n a t r i u r e t i c f a c t o r s , which a r e i m p o r t a n t i n t h e maintenance of t h e e x t r a c e l l u l a r f l u i d volume, have been d e t e c t e d i n plasma and u r i n e and have been e x t r a c t e d from b r a i n t i s s u e and f r o m c a r d i a c a t r i a . Expansion o f t h e e x t r a c e l l u l a r f l u i d volume l e a d s t o i n c r e a s e d sodium i o n e x c r e t i o n ( n a t r i u r e s i s ) by t h e kidney, independent o f changes i n g l o m e r u l a r f i l t r a t i o n r a t e (GFR) and o f t h e r e n i n - a n g i o t e n s i n - a l d o s t e r o n e system.l Considerable evidence i n d i c a t e s t h a t volume expansion (VE) i n l a b o r a t o r y animals and i n man causes t h e r e l e a s e o f a humoral n a t r i u r e t i c f a c t o r , which i s e x c r e t e d i n t h e u r i n e . One such agent t h a t appears t o be an endogenous i n h i b i t o r o f t h e sodium t r a n s p o r t system has been r e f e r r e d t o as n a t r i u r e t i c hormone (NH). Many s t u d i e s have i m p l i c a t e d NH i n t h e pathogenesis o f h y p e r t e n s i o n i n man and e x p e r i mental animals.2-6 A second substance, i s o l a t e d from c a r d i a c a t r i a l t i s s u e and d e s i g n a t e d a t r i a l n a t r i u r e t i c f a c t o r (ANF), does n o t a f f e c t Na+,K+-ATPase and i s s t r u c t u r a l l y d i f f e r e n t f r o m NH. The r o l e o f ANF i n b l o o d p r e s s u r e c o n t r o l o r h y p e r t e n s i o n may r e l a t e t o c a r d i a c a t r i a l r e c e p t o r s t h a t a r e presumed t o be s e n s i t i v e t o changes i n i n t r a a t r i a l p r e s s u r e , p o s s i b l y r e s u l t i n g i n r e l e a s e o f ANF.' This chapter w i l l r e v i e w t h e i s o l a t i o n , t h e c h a r a c t e r i z a t i o n o f t h e chemical and b i o l o g i c a l p r o p e r t i e s , and t h e p o s s i b l e r o l e s o f t h e s e agents i n t h e c o n t r o l o f e x t r a c e l l u l a r f l u i d volume and development o f h y p e r t e n s i o n . More d e t a i l e d reviews o f t h e n a t r i u r e t i c hormone have appeared r e c e n t l y . 2 - 6 N a t r i u r e t i c Hormone - A humoral f a c t o r which causes n a t r i u r e s i s when c r o s s c i r c u l a t e d t o r e c i p i e n t animals has been demonstrated i.n VE donor a n i r n a l ~ . ~ , E ~ x~t r ~a c*t s~ o~f b l o o d o r u r i n e f r o m VE s u b j e c t s a l s o causes n a t r i u r e s i s i n assay animals, i n d i c a t i n g a t r a n s f e r a b l e n a t r i u r e t i c sub~ t a n c e . ~T h i s agent i s e i t h e r l e s s a c t i v e o r u n d e t e c t a b l e i n nonexpanded s u b j e c t s . 4 A n a t r i u r e t i c substance has been e x t r a c t e d f r o m k i d n e t i s s u e o f VE animals, b u t n o t from k i d n e y s o f hydropenic a n i m a l s . l l The evidence suggests t h a t NH i n c r e a s e s sodium i o n e x c r e t i o n by i n h i b i t i n g r e a b s o r p t i o n as a r e s u l t o f d i r e c t i n h i b i t i o n o f t h e r e n a l Nat,K+-ATPase, t h e e n e r g y - r e q u i r i n g sodium i o n t r a n s p o r t pump.12 A h i g h c o n c e n t r a t i o n o f t h e Na+,Kt-ATPase i n h i b i t o r , d i g o x i n , produces n a t r i u r e s i s b y i m p a i r i n g t h e c e l l u l a r pump o f t h e r e n a l t u b u l e . 1 2 An unknown p r e s s o r agent has been observed i n t h e b l o o d o f animals and man w i t h VE, l o w r e n i n , h y p e r t e n ~ i 0 n . l ~Suppressed Na+,K+-ATPase a c t i v i t y i n c a r d i o v a s c u l a r t i s s u e s o f a n i m a l s w i t h e i t h e r one-kidney r e n a l , d e o x y c o r t i c o s t e r o n e a c e t a t e o r reduced r e n a l mass h y p e r t e n s i o n suggests t h a t t h i s p r e s s o r substance i s an i n h i b i t o r o f t h e Na+,Kt-ATPase. By i n h i b i t i n g t h e Nat,K+-ATPase o f v a s c u l a r smooth muscle, i t i s p o s s i b l e t h a t such an agent c o u l d c o n t r i b u t e t o i n c r e a s e d v a s c u l a r r e s i s t a n c e o r
ANNUAL REPORTS IN MEDICINAL CHEMISTRY-I9
Copyright 0 1984 by Academic Press, Inc All rights of reproduction in any form reserved. ISBN 0-12-040519-9'
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r e a c t i v i t y a s s o c i a t e d w i t h hypertension.14 A s u b s t a n t i a l i n c r e a s e i n a r t e r i a1 p r e s s u r e has been observed f o l l owi ng in j e c t i o n o f d i g i t a l is p r e p a r a t i o n s i n i n t a c t animals15 as w e l l as i n e x c i s e d a r t e r i a l 1 6 , 1 7 and venous16 t i ssues , A t h e o r e t i c a l r o l e f o r a s a l t - e x c r e t i n g hormone i n h y p e r t e n s i o n was proposed by Dahl e t a1.l8,lg and expanded by Blaustein,20 Haddy e t a1.,14 and deWardener.21-B=60d pressure i s a f u n c t i o n o f both t o t a l v o l u m e o f t h e b l o o d and t h e r e s i s t a n c e t o i t s f l o w t h r o u g h t h e c i r c u l a t o r y system. The r e s i s t a n c e t o f l o w i s determined by t h e degree of c o n s t r i c t i o n o f t h e a r t e r i o l e s . A p r i m a r y d e f e c t i n t h e k i d n e y t h a t reduces i t s a b i l i t y t o e x c r e t e sodium r e s u l t s i n i n c r e a s e d b l o o d volume, r e l e a s e o f n a t r i u r e t i c hormone, t h e i n h i b i t i o n o f t h e r e n a l Nat,Kt-ATPase and i n c r e a s e d u r i n a r y sodium e x c r e t i o n . A t t h e same t i m e , however, t h e v a s c u l a r t i s s u e s would be exposed t o t h e same Nat,Kt-ATPase i n h i b i t o r , causing increased nons p e c i f i c s e n s i t i v i t y t o c o n s t r i c t o r agents such as a n g i o t e n s i n , vasop r e s s i n o r n 0 r e p i n e p h r i n e . 2 2 9 2 ~ I n h i b i t i o n o f t h e v a s c u l a r Nat,Kt-ATPase leads t o i n c r e a s e d l e v e l s o f i n t r a c e l l u l a r Cat' i n b l o o d vessels, e i t h e r by a Nat-Cat+ exchange mechanism20 o r by p a r t i a l d e p o l a r i z a t i o n o f t h e c e l l membranes i n c r e a s i n g t h e p e r m e a b i l i t y t o ~ a l c i u r n . 1 ~T h i s increases t h e s e n s i t i v i t y t o c i r c u l a t i n g p r e s s o r agents and r e s u l t s i n enhanced c o n t r a c t i o n and increased b l o o d pressure. Another mechanism proposed by Buckalew and Gruber i n v o l v e s a more d i r e c t r o l e o f t h e sympathetic nervous ~ y s t e m . ~I n nonhypertensive i n d i v i d u a l s , NH i n c o n j u n c t i o n w i t h o t h e r n a t r i u r e t i c f o r c e s , i n c r e a s e s r e n a l sodium e x c r e t i o n and m a i n t a i n s normal plasma volume. Since NH can cause n a t r i u r e s i s w i t h no e f f e c t on b l o o d pressure, volume can be regul a t e d i n normal i n d i v i d u a l s w i t h o u t causing hypertension24. I n hypert e n s i v e s u b j e c t s , r e n a l response t o NH i s b l u n t e d , causing NH l e v e l s t o become h i g h e r t h a n i n normotensive i n d i v i d u a l s . P a t h o l o g i c a l l y e l e v a t e d NH l e v e l s l e a d t o i n c r e a s e d b l o o d p r e s s u r e e i t h e r by a c t i v a t i n g t h e sympathetic nervous system, by i n c r e a s i n g v a s c u l a r r e a c t i v i t y , o r both. The i n c r e a s e d b l o o d pressure adds another n a t r i u r e t i c f o r c e h e l p i n g t o overcome t h e d e f e c t i n r e n a l sodium e x c r e t i o n . Thus, h y p e r t e n s i o n i s a r e s u l t o f t h e need t o r e g u l a t e volume i n t h e presence o f a d e f e c t i n r e n a l sodium e x c r e t i o n . C o n t r o l o f NH r e l e a s e by a c e n t r a l s i t e was suggested by i m p a i r e d n a t r i u r e s i s and i m p a i r e d s e c r e t i o n o f NH i n r a t s w i t h l e s i o n s o f t h e a n t e r o v e n t r a l t h i r d v e n t r i c l e (AV3V) r e g i o n o f t h e brain.25 Evidence t h a t NH may be an endogenous Nat ,Kt-ATPase i n h i b i t o r i n c l u d e s t h e i n h i b i t i o n o f sodium t r a n s p o r t i n an anuran membrane, a model o f t h e r e n a l d i s t a l t ~ b u l e ,and ~ i n isolated rabbit collecting tubule.Z6 N a t r i u r e t i c u r i n e e x t r a c t s a l s o d i s p l a c e 3H-ouabain from r e n a l N ~ ' , K + - A T P ~ s ~ . * ~ D i r e c t i n h i b i t i o n o f Nat,Kt-ATPase i n v i t r o has a l s o been r e p o r t e d w i t h e x t r a c t s o f p l a ~ m a and ~ ~ u, r ~i n e~. 4 6 m y n e t a l . show a s i g n i f i c a n t c o r r e l a t i o n between t h e Nat,Kt-ATPase i n h i b i t o r y a c t i v i t y i n t h e plasma and t h e mean a r t e r i a l p r e s s u r e o f t h e donor p a t i e n t . 2 9 A cytochemical assay f o r i n h i b i t i o n o f guinea p i g r e n a l Nat,Kt-ATPase30 has been used t o show t h e presence o f a c i r c u l a t i n g i n h i b i t o r y a c t i v i t y t h a t i s 25-times g r e a t e r i n t h e plasma o f s u b j e c t s on h i g h s a l t t h a n t h o s e on l o w s a l t diets.31 Plasma from h y p e r t e n s i v e p a t i e n t s , plasma w i t h r e n i n values below normal, and plasma from o l d e r s u b j e c t s e x h i b i t e d h i g h e r l e v e l s o f Nat,Kt-ATPase i n h i b i t o r y a c t i v i t y . 3 2 A n a t r i u r e t i c e x t r a c t o f u r i n e from normal o r ~ a l t - l o a d e d 3 3 9 3 and ~ uremic35 humans was a l s o shown t o i n h i b i t t h e Nat,Kt-ATPase i n p i g r e n a l
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Endogenous Natriuretic Factors
Napier, Blaine
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t u b u l a r membranes. The i n h i b i t i o n i s g r e a t e s t i n t h e p r o x i m a l and d i s t a l t u b u l e s and i n t h e t h i c k ascending l i m b o f t h e l o o p o f Henle,30 and t h e e x t r a c t i s a n t i n a t r i f e r i c on t h e s e r o s a l s u r f a c e o f f r o g s k i n . 3 1 Plasma induced i n h i b i t i o n i s s i m i l a r l y d i s t r i b u t e d w i t h i n t h e renal tubule. U r i n e f r o m VE dogs and uremic p a t i e n t s has a l s o been r e p o r t e d t o c o n t a i n an N H - l i k e a ~ t i v i t y , 3 ~ - 3 8and a n a t r i u r e t i c f a c t o r has been d e s c r i b e d i n t h e serum o f p a t i e n t s w i t h c h r o n i c ~ r e m i a . ~ ~ - ~ ~ Buckalew and Gruber h y p o t h e s i z e d t h a t a n t i b o d i e s a g a i n s t a Na+,K+ATPase i n h i b i t o r , such as d i g o x i n , c o u l d be used as probes f o r n a t r i u r e t i c hormone on t h e b a s i s t h a t a n t i b o d i e s t o drugs t h a t b i n d t o s e c i f i c r e c e p t o r s m i g h t r e c o g n i z e and b i n d t o t h e endogenous l i g a n d . $ 8 I n two o f t h e i r s t u d i e s , c r o s s r e a c t i v i t y o f t h e i n h i b i t o r o f Nat,Kt-ATPase w i t h a l . showed t h a t t h e r e a n t i - d i g o x i n a n t i b o d i e s was o b ~ e r v e d . ~ Rudd ~ , ~ _ ~e t _ was s i g n i f i c a n t l y more Na+,K+-ATPase i n h i b i t i o n and d i g o x i n immunor e a c t i v i t y i n t h e plasma o f VE dogs compared t o hydropenic dogs.42 The plasma e x t r a c t w i t h d i g o x i n i m m u n o r e a c t i v i t y f r o m VE dogs i s a l s o n a t r i u r e t i c i n r a t s . D i g o x i n i m m u n o r e a c t i v i t y i s found i n t h e plasma o f Rhesus and A f r i c a n Green monkeys w i t h 2 - k i d n e y Y 1 - c l i p G o l d b l a t t hypert e n s i o n . The h y p e r t e n s i v e monkeys have two- t o t h r e e - f o l d h i g h e r l e v e l s o f d i g o x i n i m m u n o r e a c t i v i t y t h a n t h e normotensive c o n t r o l s . 4 3 Increased e x c r e t i o n o f a d i g o x i n - l i k e hormone i n r a t s d u r i n g s a l t - l o a d i n g , which i s f u r t h e r enhanced d u r i n g t h e development o f h y p e r t e n s i o n and adaptat i o n t o c h r o n i c r e n a l f a i l u r e , has been r e p o r t e d r e c e n t l y . 4 4 I n c o n t r a s t t o t h e s e s t u d i e s , Hamlyn _ et _ a1 d i d n o t observe any a n t i - d i g o x i n immunor e a c t i v i t y i n plasma samples from normal o r h y p e r t e n s i v e p a t i e n t s . 2 9
.
I m p a i r e d sodium e f f l u x was measured i n l e u k o c y t e s from p a t i e n t s w i t h e s s e n t i a l h y p e r t e n s i o n . I n c u b a t i o n o f normal l e u k o c y t e s w i t h plasma from h y p e r t e n s i v e p a t i e n t s caused i m p a i r e d sodium t r a n s p o r t . 4 5 F u r t h e r more, p a r t i a l l y p u r i f i e d f r a c t i o n s from plasma, as w e l l as u r i n e , i n h i b i t sodium e f f l u x f r o m p e r i p h e r a l b l o o d l e ~ k o c y t e s . ~The ~ severity o f the d e f e c t i n l e u k o c y t e c a t i o n t r a n s p o r t i s i n v e r s e l y r e l a t e d t o t h e plasma r e n i n a c t i v i t y and i s g r e a t e s t i n p a t i e n t s w i t h e s s e n t i a l h e r t e n s i o n i n whom t h e r e n i n response t o sodium r e s t r i c t i o n was a t y p i c a l Although t h e s e s t u d i e s s u p p o r t t h e h y p o t h e s i s t h a t NH i s a d i g i t a l i s - l i k e substance which i n h i b i t s Na+,K+-ATPase i n numerous t i s s u e s i n c l u d i n g t h e k i d n e y , i t remains t o be determined whether t h e n a t r i u r e t i c e f f e c t o f NH i s due e x c l u s i v e l y t o i n h i b i t i o n o f r e n a l Na+,K+-ATPase.
.$?
It has been proposed t h a t t h e hypothalamus m i g h t s e c r e t e a subs t a n c e which c o n t r o l s sodium e x c r e t i o n . 4 8 The AV3V r e g i o n o f t h e b r a i n i s important f o r r e g u l a t i n g blood pressure, since i t s d e s t r u c t i o n prevents s e v e r a l forms o f h y p e r t e n ~ i o n . ~Furthermore, ~ l e s s Na+ i s e x c r e t e d , t h e b l o o d l e v e l o f Na+ i s e l e v a t e d , and NH i s absent i n animals w i t h AV3V 1e s i o n s Consequently, s e v e r a l groups have prepared hypothalamic o r b r a i n e x t r a c t s and s t u d i e d t h e i r a n t i n a t r i f e r i c o r Na+,Kt-ATPase i n h i b i t o r y p r o p e r t i e s , b u t n a t r i u r e t i c a c t i v i t y was n o t r e p o r t e d . On t h e o t h e r hand, an acetone e x t r a c t o f t h e r a t hypothalamus ( b u t n o t c e r e b r a l c o r t e x , p i t u i t a r y , o r o t h e r t i s s u e s ) possessed v e r y p o t e n t Na',K+-ATPase inhibit o r y a c t i v i t y which was i n c r e a s e d 1 5 0 - f o l d i n animals on a h i g h sodium d i e t . 5 2 The plasma from t h e s e animals a l s o c o n t a i n e d Na+,K+-ATPase i n h i b i t o r y a c t i v i t y , w i t h s e v e n - f o l d more a c t i v i t y i n t h e plasma o f t h e r a t s on t h e h i g h sodium d i e t . Aqueous-acetone e x t r a c t i o n o f r a t b r a i n , i n t h e presence o f n i t r o g e n , r e s u l t e d i n a s p e c i f i c Na+,K+-ATPase i n h i b i t o r . 5 3 A l o w m o l e c u l a r w e i g h t " o u a b a i n - l i k e " f a c t o r , which i n h i b i t s i n v i t r o Na+,Kt-ATPase a c t i v i t y , b l o c k s 3H-ouabain b i n d i n g t o b r a i n m i c r o soma1 Na+,Kt-ATPase o r i n h i b i t s 86Rb u p t a k e i n human e r y t h r o c y t e s , has
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been p a r t i a l l y p u r i f i e d from b o v i n e h y p ~ t h a l a m u s , ~ guinea ~ , ~ ~ p i g 5 6 and rat5' brain. The chemical s t r u c t u r e o f NH has n o t been determined and t h e r e i s no assurance t h a t t h e a c t i v i t i e s a s c r i b e d t o t h e v a r i o u s e x t r a c t s a r e caused by t h e same substance. Furthermore, t h e r e i s no s t a n d a r d i z e d assay which a l l t h e i n v e s t i g a t o r s agree i s b e s t f o r d e t e c t i n g NH. However, t h e r e i s general agreement t h a t t h e substance i s low m o l e c u l a r w e i g h t (< 1000 d a l t o n s ) , a c i d i c , w a t e r s o l u b l e and h e a t s t a b l e . 4 The presence o f two n a t r i u r e t i c f a c t o r s i n t h e plasma and u r i n e o f volume expanded s u b j e c t s has been r e p o r t e d ,58 One f a c t o r causes n a t r i u r e s i s i n r a t s a f t e r a 20 min d e l a y and appears t o be l a r g e r t h a n t h e second f a c t o r which produces immediate n a t r i u r e s i s . The l o w m o l e c u l a r w e i g h t f a c t o r i s a n t i n a t r i f e r i c ( i n h i b i t s i o n f l u x i n i s o l a t e d f r o g s k i n o r toad b l a d d e r membrane p r e p a r a t i o n s ) w h i l e t h e h i g h e r m o l e c u l a r w e i g h t f a c t o r i s n o t . These r e s u l t s and t h e f i n d i n g t h a t NH a c t i v i t y i n c r e a s e s i n plasma f o l l o w i n g i n c u b a t i o n f o r 30 minutes a t room temperature supports t h e presence o f an NH precursor.59 Evidence f o r t h e pe t i d e n a t u r e o f NH i s i n d i c a t e d by i t s s e n s i t i v i t y t o enzymatic d i g e s t i o n 3 9 ~ 3 9 , 4 0 ~ 6 0 , 6 1 and i t s b e h a v i o r d u r i n chromatography by t e c h n i q u e s used f o r t h e i s o l a t i o n o f small peptides.t2 Buckalew and coworkers have s t u d i e d a h e p t a p e p t i d e w i t h sequence homology t o a fragment o f ACTH/a MSH (Met-G1 u-Hi s-Phe-ArgTrp-Gly [ o r Asp]), which may be r e l a t e d t o an endogenous NH s i n c e i t s b i o l o g i c a l e f f e c t s mimic t h o s e o f NH.63-66 The p e p t i d e i n h i b i t s Na+,K+ATPase i s n a t r i u r e t i c a t low doses, and i s h y p e r t e n s i v e a t h i g h d 0 s e s . 6 2 , ~ ~ Others, however, do n o t b e l i e v e t h e f a c t o r t h e y i s o l a t e from u r i n e o f salt-expanded dogs i s a p e p t i d e . 4 ~ ~ 2 ~ 6I 0n s p i t e o f t h e r e p o r t e d c r o s s r e a c t i v i t y o f NH with a n t i - d i g o x i n a n t i b o d i e s , NH does n o t appear t o be a s t e r o i d , s i n c e i t i s i s o l a t e d i n aqueous s o l u t i o n s and i s s u s c e p t i b l e t o a c i d h y d r o l y s i s .4 Expansion o f t h e e x t r a c e l l u l a r f l u i d volume, r e s u l t i n i n a n a t r i u r e t i c response a f f e c t s proximal t u b u l e sodium resorption.6yP68 However, i n h i b i t i o n o f sodium r e s o r p t i o n a t a more d i s t a l nephron s i t e such as t h e c o l l e c t i n g d u c t , a l s o occurs d u r i n g expansion o f t h e e x t r a c e l l u l a r f l u i d volume and i s a major determinant o f t h e magnitude o f n a t r i u r e ~ i s . ~ g ,R~e c~o l l e c t i o n m i c r o p u n c t u r e t e c h n i q u e s were used t o show a s i g n i f i c a n t decrease i n f r a c t i o n a l sodium r e s o r p t i o n i n t h e p r o x i m a l t u b u l e s o f t h e s u p e r f i c i a l nephrons o f uremic r a t s when a n a t r i u r e t i c response occurred f o l l o w i n g t h e a d m i n i s t r a t i o n o f a serum d e r i v e d NH.71 I n r a b b i t , t h e r e n a l c o l l e c t i n g d u c t ( t u b u l e ) has been i m p l i c a t e d as an i m p o r t a n t s i t e f o r t h e r e g u l a t i o n o f a c t i v e sodium t r a n s p o r t by t h e n a t r i u r e t i c f a c t o r . A n a t r i u r e t i c sample a p p l i e d t o t h e p e r i t u b u l a r surface o f t h e i s o l a t e d perfused c o r t i c a l c o l l e c t i n g tubule i n h i b i t e d t h e p o t e n t i a l d i f f e r e n c e and decreased t h e n e t sodium f l u x from t h e lumen t o t h e p e r i t u b u l a r surface.26 T h i s i s c o n s i s t e n t w i t h r e s u l t s from s t u d i e s on t h e i s o l a t e d t o a d b l a d d e r ( a s t r u c t u r e w i t h many s i m i l a r i t i e s t o t h e d i s t a l nephron) which have shown t h a t NH a c t s a t t h e s e r o s a l s u r f a c e and i n h i b i t s t r a n s e p i t h e l i a l t r a n s p o r t by r e d u c i n g sodium movement across t h e s e r o s a l b a r r i e r . 44
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A t r i a l N a t r i u r e t i c Factor Evidence has accumulated which suggests t h a t t h e c a r d i a c a t r i a m i g h t f u n c t i o n as a sensor f o r d e t e c t i n g changes i n Indeed , numerous experimental procedures e x t r a c e l 1u l a r f 1u i d v o l ume which a l t e r i n t r a a t r i a l pressure o r s t r e t c h t h e a t r i a l w a l l r e s u l t i n s i g n i f i c a n t changes i n water and e l e c t r o l y t e e x c r e t i o n . Such a l t e r a t i o n s i n i n t r a a t r i a l p r e s s u r e should r e f l e c t changes i n and p r o v i d e a means f o r r e g u l a t i ng e x t r a c e l 1u l a r f 1u i d v o l ume . 7 ,72
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Changes i n w a t e r e x c r e t i o n t h a t a r e secondary t o a l t e r e d i n t r a a t r i a l p r e s s u r e can be a t t r i b u t e d l a r g e l y t o changes i n t h e s e c r e t i o n o f v a s o p r e s s i n by t h e p o s t e r i o r p i t u i t a r y gland. T h i s r e f l e x has been documented b o t h i n e x p e r i m e n t a l animals and i n man, and t h e r e l e v a n t d a t a has been reviewed r e c e n t l y by Bie.73 On t h e o t h e r hand, i t has n o t been p o s s i b l e , u n t i l r e c e n t l y , t o account f o r t h e i n c r e a s e d u r i n a r y Nat e x c r e t i o n a s s o c i a t e d w i t h changes i n a t r i a l pressure. et a l . have e x t r a c t e d a substance from t h e c a r d i a c a t r i a de B o l d o f r a t s which, when i n j e c t e d i n t o o t h e r r a t s , r e s u l t s i n a 3 0 - f o l d i n c r e a s e i n u r i n a r y Na+ e x c r e t i o n and a 1 0 - f o l d i n c r e a s e i n u r i n e S i m i l a r e x t r a c t s o f c a r d i a c v e n t r i c l e were w i t h o u t e f f e c t . volume.74 Numerous independent l a b o r a t o r i e s have now c o n f i r m e d t h e i n i t i a l observ a t i o n o f a t r i a l n a t r i u r e t i c f a c t o r (ANF).75-88 T h i s a c t i v i t y has been observed i n t h e a t r i u m o f a l l mammalian s p e c i e s s t u d i e d , i n c l u d i n g man.83~89,90 The s p e c i f i c a c t i v i t y appears t o be h i g h e s t i n r a t a t r i a . The d i s t i n c t i o n between t h i s m a t e r i a l and NH r e s t s on s e v e r a l c r i t e r i a : 1 ) t h e minimum m o l e c u l a r w e i g h t f o r ANF i s a t l e a s t 3000 d a l t 0 n s 8 ~ w h i l e NH i s p r o p o r t e d t o be l e s s t h a n 1000,12 2 NH appears t o i n h i b i t Nat,Kt-ATPase w h i l e ANF has no such a c t i v i t y , 4 ~ and 3) NH i s t h o u g h t t o be h y p e r t e n s i v e w h i l e ANF c l e a r l y l o w e r s b l o o d p r e s ~ u r e . ~ , 7 4
I
" S p e c i f i c granules"ql have been d e s c r i b e d i n t h e myocytes o f t h e c a r d i a c a t r i a which c l o s e l y resemble t h e s t o r a g e g r a n u l e s o f o t h e r p e p t i d e hormone s e c r e t i n g t i s s u e s . These g r a n u l e s c o - p u r i f y w i t h ANF92393 and immunocytochemical s t u d i e s c o n f i r m t h e a s s o c i a t i o n between t h e s p e c i f i c a t r i a l g r a n u l e s and ANF.94995 Furthermore, t h e highest concentrations o f antigenic material are l o c a l i z e d i n t h e s u b p e r i c a r d i a l a r e a o f t h e a t r i a l a ~ p e n d a g e s . 9 ~ D i r e c t bioassay o f d i s s e c t e d a t r i a c o n f i r m t h e s e h i g h l e v e l s o f ANF a ~ t i v i t y . 9 ~ It i s n o t understood what f a c t o r s c o n t r o l t h e s y n t h e s i s , s t o r a g e and s e c r e t i o n o f ANF, b u t a l t e r a t i o n s i n body f l u i d volume appear t o change g r a n u l e d e n s i t y and e x t r a c t a b l e a c t i v i t y . G r a n u l a r i t y i n c r e a s e s markedly i n response t o s a l t o r w a t e r d e p r i v a t i o n and decreases when e x t r a c e l l u l a r f l u i d volume i s expanded secondary t o d e s o x y c o r t i c o s t e r o n e However, t h e r e i s no c o r r e l a t i o n between t h e g r a n u l a r i t y treatment.97 It i s p o s s i b l e t h a t o f t h e a t r i a and t h e amount o f e x t r a c t a b l e ANF.g8 t h e s t o r a g e f o r m o f ANF under t h e s e c o n d i t i o n s may n o t be a c t i v e , and f u r t h e r p r o c e s s i n g i s r e q u i r e d b e f o r e b i o l o g i c a l a c t i v i t y can be expressed. Although e x t r a c t a b l e ANF i s reduced i n t h e a t r i a o f spontane o u s l y h y p e r t e n s i v e r a t s , i t i s n o t known whether t h i s change i s r e l a t e d t o t h e e l e v a t e d b l o o d pressure.99
P o t e n t n a t r i u r e t i c a c t i v i t y was e v i d e n t i n a s i m p l e phosphate b u f f e r e d s a l i n e e x t r a c t o f whole a t r i a . 7 4 Subsequently, t h e m a t e r i a l was While t h e e x t r a c t i s b o t h a c i d found t o be a c i d stable.75,87,88,100,1*1 and heat759101 s t a b l e , t r y p ~ i n , ~ ~ , 8 9 , 9as 3 w e l l as o t h e r p r o t e o l y t i c e n z y m e ~ 7such ~ as chymotrypsi n, ami nopept i d a s e A and carboxypept i d a s e s However, t r e a t m e n t w i t h carboxyB and C d e s t r o y n a t r i u r e t i c a c t i v i t p e p t i d a s e A o n l y b l u n t e d a c t i v i t y , 7 g and c o n c a n a v a l i n A had no e f f e ~ t . 7 ~ These o b s e r v a t i o n s a r e c o n s i s t e n t w i t h ANF b e i n g a s m a l l p e p t i d e .
.
P u r i f i c a t i o n o f ANF from r a t a t r i a by s e v e r a l independent l a b o r a t o r i e s u t i l i z i n d i f f e r e n t i s o l a t i o n procedures c o n f i r m e d i t s p e p t i d i c n a t u r e . 8 5 ~ 1 0 2 ~ 1 ~ 3 , 1 0 4An amino a c i d c o m p o s i t i o n w i t h o u t sequence was r e p o r t e d from two l a b o r a t o r i e s 8 5 ~ 1 0 2 b u t t h e t o t a l number o f r e s i d u e s
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d i f f e r e d c o n s i d e r a b l y (49 and 36) and t h e r e were d i f f e r e n c e s i n composit i o n . An amino a c i d sequenc f o r r a t ANF was r e p o r t e d e s s e n t i a l l y s i m u l C u r r i e e t a1 ,lo5 Kangawa and Matsuo,1o6 t a n e o u s l y by F1 nn e t a l . , loS et a1 .loJ and Napier e t a1 .87,* T h e l o n g e s t p e p t i d e r e p o r t e d has Seidah t h e 33 amino a c i d sequence shownbelow, 1, which c o n t a i n s t h e C s12-Cys28 d i s u l f i d e b r i d g e , and i s t h e Tyr a c i d r a F h e r t h a n t h e amide.87,{8,107
.
- -
H- Leu -A1 a- G1y- Pro-Arg5- Se r-Leu- A r g Arg Se r 10- Se r-Cys- Phe- 61yGly15-Arg- Ile-Asp-Arg-Ile20-Gly-Al a-G1 n-Ser-Gly25-Leu-Gly-CysA~n-Ser3~-Phe-Arg-Tyr-OH
-1 Other sequences a r e N-terminal t r u n c a t e d v e r s i o n s o f t h i s p e p t i d e (22,28, 31,32 residues).87,88,103,107 C u r r i e e t a l . have p u r i f i e d two a t r i a l p e p t i d e s ( a t r i o p e p t i n s I and 11) o f 2 1 a n T 2 3 amino a c i d s . l o 5 The l o n g e r o f t h e s e i s i d e n t i c a l t o t h e 10-32 fragment o f Kangawa and Matsuo have i s o l a t e d ANF from human a t r i a and determined t h e amino a c i d sequence o f a 28 amino a c i d p e p t i d e which d i f f e r s from t h e r a t ANF sequence o n l y a t p o s i t i o n 17 where I l e i s r e l a c e d by Met.1o6 Based on t h e sequence d i s c o v e r e d by Seidah e t a1.,lug s y n t h e t i c m a t e r i a l was made and t h e i m p o r t a n t c o n f i r m a t i o n o f t h e n a t u r a l m a t e r i a l was obtained. The synt h e t i c p e p t i d e composed o f t h e 8-33 sequence o f Seidah e t a l . has f u l l b i o l o g i c a l a c t iv i ty.lU7
1.
I n i t i a l l y , t h e p r i n c i p a l b i o l o g i c a l a c t i v i t y o f ANF was t h o u g h t t o be n a t r i u r e s i s . Most i n v e s t i g a t o r s r e p o r t e d 30 t o 4 0 - f o l d i n c r e a s e s i n u r i n a r y sodium e x c r e t i o n w i t h l e s s e r e f f e c t s on u r i n a r y K+ e x c r e t i o n . Although t h e mechanism o f t h e n a t r i u r e s i s has n o t been s t u d i e d e x t e n s i v e l y , r e p o r t s u s i n g m i c r o p u n c t u r e and m i c r o c a t h e t e r i z a t i on t e c h n i q u e s suggest t h a t i n h i b i t i o n o f Na+ t r a n s p o r t i n t h e f a r d i s t a l nephron ( i . e . c o l l e c t i n g d u c t ) i s t h e p r i n c i p a l mechanism o f a c t i o n . 7 7 ~ 1 0 8 These d a t a must be i n t e r p r e t e d c a u t i o u s l y , however, because t h e magnitude o f t h e n a t r i u r e t i c response would argue f o r a more p r o x i m a l s i t e o f a c t i o n . Indeed, a r e c e n t r e p o r t by Seymour e t al., i n which s y n t h e t i c ANF was i n f u s e d d i r e c t l y i n t o t h e r e n a l a r t e r y o f a n e s t h e t i z e d dogs, i n d i c a t e d t h a t f r a c t i o n a l sodium e x c r e t i o n exceeded 10% d u r i n g maximal n a t r i u r e sis.109 Based on what i s c u r r e n t l y known about sodium d e l i v e r y t o t h e v a r i o u s nephron segments, t h i s would argue f o r a s i t e o f a c t i o n a t l e a s t i n t h e c o r t i c a l d i l u t i n g segment (a s i t e s i m i l a r t o t h i a z i d e d i u r e t i c s ) . Nevertheless, t h e d a t a do not exclude m u l t i p l e s i t e s o f a c t i o n , o r changes i n r e n a l hemodynamics o r nephron h e t e r o g e n e i t y . Some evidence a l s o suggests t h a t changes i n g l o m e r u l a r f i l t r a t i o n a r e i m p o r t a n t i n t h e n a t r i u r e t i c r e s onse, b u t t h e s e s t u d i e s were conducted i n i s o l a t e d p e r Other s t u d i e s i n i n t a c t animals have n o t demonstrated fused k i dneys lP0 m a j o r changes i n e i t h e r g l o m e r u l a r f i l t r a t i o n o r r e n a l b l o o d flow.74,78~79
.
I n a d d i t i o n t o t h e p o t e n t n a t r i u r e t i c a c t i v i t y , ANF has s i g n i f i c a n t r e l a x a n t e f f e c t s on v a s c u l a r and perhaps o t h e r smooth muscle t i s ~ u e s . C8u r r i e~ ~a l . observed ~ ~ et r~e l a x a~ t i o n~ o f r~a b b i~t a o ~r t a ~ and c h i c k rectum, which had r e v i o u s l y been c o n t r a c t e d w i t h e p i n e p h r i n e and carbachol, r e s p e ~ t i v e l y . ~They ~ use t h i s s p a s m o l y t i c a c t i o n t o m o n i t o r ANF a c t i v i t y and a r e t h e f i r s t group t o separate smooth muscle r e l a x a n t a c t i v i t y o f s i m i l a r p e p t i d e s i s o l a t e d from c a r d i a c a t r i a , a l though b o t h p e p t i d e s a r e n a t r i u r e t i c . l o 5 A r e c e n t r e p o r t by Winquist -e t a l . has d e t a i l e d t h e v a s c u l a r smooth muscle r e l a x i n g e f f e c t o f synthet i c ANF u s i n g a v a r i e t y o f a g o n i s t s and d i f f e r e n t v a s c u l a r smooth muscle p r e p a r a t i o n s . l 1 4 Based on t h e s e o b s e r v a t i o n s , ANF appears t o have spasmolytic p r o p e r t i e s s i m i l a r t o sodium n i t r o p r u s s i d e .
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ANF has p o t e n t e f f e c t s on c y c l i c GMP (cGMP) l e v e l s . I n v i t r o , a d d i t i o n o f a t r i a l e x t r a c t s t o minced k i d n e y t i s s u e o r t o p r i m a r y i d n e y c e l l c u l t u r e s r e s u l t s i n i n c r e a s e d l e v e l s o f ~ G M p . 1 1 ~I n j e c t i o n i n t o a n e s t h e t i z e d r a t s r e s u l t e d i n a 4 - f o l d i n c r e a s e i n plasma cGMP l e v e l s and a 2 8 - f o l d i n c r e a s e i n u r i n a r y e ~ c r e t i 0 n . l ~Sodium ~ nitroprusside also causes i n c r e a s e d cGMP l e v e l s i n t r e a t e d t i s s u e s , b u t p r o b a b l y a c t s d i r e c t l y t h r o u g h s t i m u l a t i o n o f g u a n y l a t e c y c l a s e . F u r t h e r s t u d i e s must be done t o c l a r i f y t h e r o l e o f cGMP i n t h e v a s o r e l a x a n t and t h e n a t r i u r e t i c a c t i o n s o f ANF. The p o t e n t n a t r i u r e t i c a c t i v i t y o f ANF has i n e v i t a b l y l e d t o comp a r i s o n s w i t h known d i u r e t i c s . One r e p o r t by Sonnenberg e t a l . i n d i c a t e d t h a t probenecid b l u n t e d t h e n a t r i u r e t i c a c t i v i t y o f ANF.lm B e c a u s e p r o b e n e c i d i n t e r f e r e s w i t h t h e n a t r i u r e t i c a c t i v i t y o f most o t h e r d i u r e t i c s b y competing w i t h t h e i r s e c r e t o r y t r a n s p o r t i n t o t h e nephron lumen, t h e s e a u t h o r s suggest t h a t ANF m i g h t be a c t i n g as an endogenous d i u r e t i c . Furosemide i s t h e d i u r e t i c most f r e q u e n t l y c i t e d as s i m i l a r t o ANF, w i t h r e g a r d t o e l e c t r o l y t e e x c r e t i o n . ANF promotes sodium, potassium and c a l c i u m e x c r e t i o n w i t h c h l o r i d e as t h e p r i n c i p a l anion. Although f u r o s e mide has been a r e f e r e n c e s t a n d a r d i n a t l e a s t one paper,89 i t s h o u l d be p o i n t e d o u t t h a t r i g o r o u s dose-response s t u d i e s comparing ANF t o o t h e r d i u r e t i c s have n o t been conducted. The most d e f i n i t i v e s t u d i e s suggested a c e i l i n g s i m i l a r t o t h e t h i a z i d e d i u r e t i c s , a maximum f r a c t i o n a l sodium Also, l o o p d i u r e t i c s such as furosemide e x c r e t i o n o f about lU%.109 i n h i b i t NaCl t r a n s p o r t i n b u l l f r o g cornea, an analog o f t h e m e d u l l a r y t h i c k ascending l i m b o f t h e l o o o f Henle whereas a t r i a l e x t r a c t s a r e i n a c t i v e i n t h i s preparation.ll! Because o f t h e p o t e n t i a l f o r ANF t o be i n v o l v e d i n b l o o d p r e s s u r e c o n t r o l , s e v e r a l i n v e s t i g a t o r s have s t u d i e d i t s e f f e c t s on t h e c a r d i o v a s c u l a r system o f normal and h y p e r t e n s i v e animals. I n a n e s t h e t i z e d normotensi ve and spontaneously h y p e r t e n s i v e r a t s , a t r i a l e x t r a c t decreased a r t e r i a l b l o o d p r e s s u r e i n a s s o c i a t i o n w i t h a decrease i n b o t h t o t a l p e r i p h e r a l r e s i s t a n c e and c a r d i a c c o n t r a c t i 1 i t y . l l 6 Also, a n e g a t i v e c h r o n o t r o p i c e f f e c t has been r e p o r t e d i n r a t s . l 1 7 Dahl s a l t s e n s i t i v e r a t s appear t o have g r e a t e r amounts o f ANF i n t h e i r a t r i a , b u t a r e hyporesponsive when exogenous m a t e r i a l i s i n j e c t e d . 9 0 R e s u l t s from t h i s s t u d y a l s o suggest an i n c r e a s e i n r e n a l p a p i l l a r y plasma f l o w and a washout o f t h e m e d u l l a r y osmotic g r a d i e n t , f a c t o r s which c o u l d c o n t r i b u t e s i g n i f i c a n t l y t o t h e a s s o c i a t e d n a t r i u r e s i s and d i u r e s i s . Increased m e d u l l a r y and i n n e r c o r t i c a l b l o o d f l o w a l s o were r e p o r t e d i n normotensive r a t s .I18
___ Summary - I n t h e case o f n a t r i u r e t i c hormone, t w e n t y - f i v e y e a r s o f r e search have f a i l e d t o c h a r a c t e r i z e a p u r e substance t h a t has n a t r i u r e t i c a c t i v i t y and i s an i n h i b i t o r o f Nat,Kt-ATPase, although, a v a s t body o f c i r c u m s t a n t i a l evidence does f a v o r t h e e x i s t e n c e o f such a substance. A t r i a l n a t r i u r e t i c f a c t o r , on t h e o t h e r hand, i s a r e c e n t d i s c o v e r y which y i e l d e d r e a d i l y t o chemical c h a r a c t e r i z a t i o n and s y n t h e s i s . I t s b i o l o g i c a l p r o f i l e suggests t h a t i t may be a component o f t h e v a r i o u s c o n t r o l systems s u b s e r v i n g f l u i d volume and p r e s s u r e r e g u l a t i o n . However, no c o n v i n c i n g evidence has y e t been o f f e r e d t h a t a t r i a l n a t r i u r e t i c f a c t o r p l a y s an i m p o r t a n t p h y s i o l o g i c a l r o l e i n p r e s s u r e o r volume homeostasis.
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G.A. MacGregor and H.E. dewardener, B r i t . Med. J., 3,847 (1981). L. Poston, S. W i l k i n s o n , R.B. Sewell and R. W i l l i a m s , C l i n . S c i . , 63, 243 ( 1 9 8 2 ) . R.P.S. Edmondson and G.A. MacGregor, B r i t . Med. J., 282, 1267 ( 1 9 8 n . H.W. Smith, Amer. J. Med., 23, 623 (1957). M. Brody and A.K. Johnson i n " F r o n t i e r s i n Neuroendocrinology," L. M a r t i n i and W.F. Ganong, Eds., Raven Press, New York, Vol. 6, 1980. A.K. Johnson i n " P e r s p e c t i v e s i n Nephrology and Hypertension," H. S c h m i t t and P. Meyer, Eds., Wiley, New York, 1976, p 106. S . B e a l e r , J.R. Haywood, A.K. Johnson, K.A. Gruber, V.M. Buckalew and M.J. Brody, Fed. Proc., 38, 1232 (1979). J. Alaghband-Zadeh, S. Fenton, K . Hancock, J . M i l l e t t and H.E. dewardener, J. E n d o c r i n o l . , 98, 221 (1983). K.R. Whitmer, E.T. W a l l i c k , D.E. Epps, L.K. Lane, J.H. C o l l i n s and A. Schwartz, L i f e Sci 30, 2261 (1982). G.T. H a u p e r c Jr., L. Kormos, L. Anderson, S. Ray and L.C. Cantley, A b s t r a c t s , 1 6 t h Ann. Mtg. Amer. SOC. Nephrology, p 192A (1983). G.T. Haupert, Jr. and J.M. Sancho, Proc. N a t l . Acad. Sci., 4658 (1979). M.C. Fishman, Proc. N a t l . Acad. Sci., 2, 4661 (1979). D. L i c h t s t e i n and S. Samuelov, Biochem. Biophys. Res. Comm., 96, 1518 ( 1 9 8 0 ) . H.E. dewardener, C l i n . Sci. Mol. Med., 53, 1 (1977). K.A. Gruber and V.M. Buckalew, Jr., Proc. SOC. Exp. B i o l . Med., 463 ( 1 9 7 8 ) . V.M. Buckalew, Jr. and K.A. Gruber i n "The Kidney i n L i v e r Disease," 2nd ed., M. E p s t e i n , Ed., E l s e v i e r , New York, N.Y., 1983. p 479. K.A. Gruber and V.M. Buckalew, Jr. i n "Hormonal R e g u l a t i o n o f Sodium E x c r e t i o n , " 6. L i c h a r d u s , R.W. S c h r i e r and J. Ponec, Eds., E l s e v i e r , New York, N.Y., 1980, p 349. A.N. Radhakrishnan. 5 . S t e i n . A. L i c h t . K.A. Gruber and S. Udenfriend. J. Chromatography, 5 5 2 - ( 1977). . J.F. Hennessy, V.M. Buckalew, Jr., J.R. Lymanqrover and K.A. Gruber, C l i n . Res., 30, 848A (1982). M.C. K l e i n , P.M. Hutchins, V.M. Buckalew, J r . , J.F. Hennessy, J.R. Lymangrover and K.A. Gruber, Fed. Proc., 42, 1020 (1983). K.A. Gruber, J.F. Hennessy, J.R. Lymangrover, M.C. K l e i n , P.M. H u t c h i n s and V.M. Buckalew, Jr., Fed. Proc., 42, 1174 (1983). J.R. Lymangrover, J.F. Hennessy, M.C. K l e i n , P.M. H u t c h i n s , V.M. Buckalew, Jr. and K.A. Gruber, Fed. Proc., 42, 1174 (1983). B.M. Brenner, K.H. Falchuk, R . I . Keimowitz and R.W. B e r l i n e r , J. C l i n . I n v e s t . , 48, 1519 (1969). J.H. D i r k s , W.J. Cirksena and R.W. B e r l i n e r , J. C l i n . I n v e s t . , 1160 ( 1 9 6 5 ) . H. Sonnenberg, Amer. J. Physiol., 223, 916 (1972). J.H. S t e i n , R.W. Osgood, 5 . B o o n j a r e r n and T.F. F e r r i s , J. C l i n . Invest., 52, 2313 (1973). H. Weber, J.J. B o u r g o i g n i e and N.S. B r i c k e r , Amer. J . P h y s i o l . , 226, 419 ( 1 9 7 4 ) . K.L. Goetz, G.C. Bond and D.D. Bloxham, P h y s i o l . Rev., 55. 157 (1975). P. B i e , P h y s i o l . Rev., 60, 961 (1980). A.J. de Bold, H.B. B o r e n s t e i n , A.T. Veress and H. Sonnenberg, L i f e Sci., 89 (1981). N.C. T r i m o d o . A.A. MacPhee. F.E. Cole and H.L. B l a k e s l e -v .. Proc. SOC. EXD. B i o l . Med., 502 (1982). G. T h i b a u l t , R. Garcia, M. C a n t i n and J. Genest. H y p e r t e n s i o n 5 (Suppl. I ) , 1-75 (1983). ,J.P. B r i g g s , B. S t e i p e , G. Schubert and J. Schnermann, P f l u e g e r s Arch., 395, 271 (1982). R. K e e l e r , Can. J. P h y s i o l . Pharm., 3, 1078 (1982). D.M. P o l l o c k and R.O. Banks, C l i n . Sci., 65, 47 (1983). A. Bonham, K. Barron, M. A l l e n , 0. P o l l o c k , R. Banks and M.J. Brody, Fed. Proc., 42, 494 (1983). Hathaway and S. Solomon, Fed. Proc., 42, 475 ( 1 9 8 3 ) . M.J.F. Camargo, H.D. K l e i n e r t , J.E. S e a l z , J.H. Laragh and T. Maack, Fed. Proc., 42, 475 ( 1 9 8 3 ) . M.N. Nemeh and J.P. Gilmore, C i r c . Res., 53, 420 ( 1 9 8 3 ) . M.G. C u r r i e , D.M. G e l l e r , B.R. Cole, J.G.Boylan, W. YuSheng, S.W. Holmberg and P. Needleman, Science, 221, 7 1 (1983). R.T. Grammer, H. Fukumi, T. Inagami and K.S. Misono, Biochem. Biophys. Res. Commun., 696 (1983). I. K i t o r , D.J. Levenson, O.C. Ohuoha and V.J. Dzau, A b s t r a c t s , 1 6 t h Ann. Mtg. Amer. SOC. Nephrology, p 167A (1983). M.A. N a p i e r , R.S. Dewey, G. Albers-Schonberg, C.D. Bennett, J.A. Rodkey, E.A. Marsh, M. Whinnery, A.A. Seymour and E.H. B l a i n e , Fed. Proc., 43, 501 (1984). M.A. N a p i e r , R.S. Dewey, G. Albers-Schonberg, C.D. Bennett, 5 . K Rodkey, E.A. Marsh, M. Whinnery, A.A. Seymour and E.H. B l a i n e , Biochem. Biophys. Res. Commun. ( i n press).
.,
76,
159,
132,
2,
28,
a,
> -
77.
Endogenous N a t r i u r e t i c Factors
116,
262 89. 90. 91. 92. 93. 94. 95. 96. 97. 98. 99. 100. 101. 102. 103. 104. 105. 106. 107. 108. 109. 110. 111. 112. 113. 114. 115. 116. 117. 118.
Sect. V
- Topics in Biology
Egan, Ed.
5
N.C. Trippodo, A.A. MacPhee and F.E. Cole, Hypertension (Supp. I ) , 1-81 (1983). M. Ganquli, Y. H i r a t a and L. Tobian, A b s t r a c t s , 1 6 t h Ann. Mtg. h e r . SOC. Nephro-
l o g y , p 191A (1983). Jamieson and G.E. Palade, J. C e l l B i o l . , 23, 151 (1964). A.J. de Bold, Can. J. P h y s i o l . Pharm., 60, 32471982). R. Garcia, M. Cantin, G. T h i b a u l t , H. Ong and J. Genest, E x p e r i e n t i a , 1071 (1982). M. Cantin, J. Gutkowski, G. T h i b a u l t , R.W. M i l n e , S. Ledoux. S. M i n l i , R. Garcia, P . Hamet and J. Genest, Histochem. ( i n press). M. Cantin, J. Gutkowski, R.W. M i l n e , G. T h i b a u l t , R. Garcia and J. Genest, 37th Ann. Mtg. Council f o r High Blood Pressure Research, 1983, abst. 39 (1983). J. B r i g g s , W. K r i z and J. Schnermann, C i r c u l a t i o n 68 (Suppl. 1 1 ) , abst. 172 (1983). A.J. de Bold, Proc. SOC. Exp. B i o l . Med., 50871979). G. T h i b a u l t , R. Garcia, M. C a n t i n and J. Genest, Fed. Proc., 42, 611 (1983). H. Sonnenberg, S . M i l o j e v i c , C.K. Chong and A.T. Veress, Hypertension, 5. 672 (1983). A.J. de Bold, Fed. Proc., 40, 554 (1981). A.J. de Bold, Proc. SOC. Exp. B i o l . Med.. 133 (1982). A.J. de B o l d and T.G. Flynn, L i f e Sci., 2,297 (1983). T.J. Flynn, M.L. de Bold and A.J. de Bold, Biochem. Biophys. Res. Comm., 859 (1983). G. T h i b a u l t , R. Garcia, N.G. Seidah, C. Lazure, M. Cantin, M. C h r e t i e n and J. Genest, FEBS L e t t . , 286 (1983). M.G. C u r r i e , D.M. G e l l e r , B.R. Cole. N.R. S i e g e l , K.F. Fok, S.P. Adams, S.R. Eubanks, G.R. G a l l u p p i and P. Needleman, Science, 223, 67 (1984). K. Kangawa and H. Matsuo, Biochem. Biophys. Res. Comm., 131 (1984). N.G. Seidah, C. Lazure. M. C h r e t i e n , G. T h i b a u l t , R. Garcia, M. Cantin, J . Genest, R.F. N u t t , S.F. Brady, T.A. L y l e , W.J. Paleveda, C.D. Colton, T.M. Ciccarone and D.F. Veber, Proc. N a t l . Acad. Sci. ( i n press). H. Sonnenberg, W.A. Cupples, A.J. de B o l d and A.T. Veress, Can. J. P h y s i o l . Pharm., 60, 1149 (1982). A.A. Seymour, R. N u t t , E. Mazack and E.H. B l a i n e , Fed. Proc., 43, 502 (1984). H.D. K l e i n e r t , J.E. Sealey, J.H. Laragh and T. Maack, P h y s i o l o g s t , 26, A-59 (1983). H. Sonnenberg, C.K. Chong and A.T. Veress, Can. J. P h y s i o l . Pharm., 59. 1278 (1981). D.C. Throckmorton and J.P. Gilmore, Fed. Proc., 42, 475 (1983). R.C. Deth, K. Wong, S. Fukozawa, R . Rocco, J.L. Smart, C.J. Lynch and R. Awad, Fed. Proc., 3, 983 (1982). R.J. Winquist, E.P. Faison and R.F. N u t t , Europ. J. Pharmacol. ( i n p r e s s ) . P. Hamet, J. Tremblay, G. T h i b a u l t , R. Garcia, M. C a n t i n and J. Genest, A b s t r a c t s , 6 5 t h Ann. Mtg. Endocrine S o c i e t y , p 289 (1983). U. Ackermann, T. I r i z a w a and H. Sonnenberg, Fed. Proc., 3, 1353 (1982). U. Ackermann, T.G. I r i z a w a , S . M i l o j e v i c and H. Sonnenberg, Fed. Proc., 2, 1096 (1983). H.B. Borenstein, W.A. Cupples, H. Sonnenberg and A.T. Veress, J. Physiol., 133 (1983). J.D.
38,
161, 170,
117,
164,
118,
334,
Section VI
- Topics in Chemistry and
Drug Design
Editor: Richard C. Allen, Hoechst-Roussel Pharmaceuticals Inc., Somerville, New Jersey 08876 Chapter 26. Enzymic Methods in Organic Synthesis Mark A. Findeis and George M. Whitesides, Department of Chemistry, Harvard University, Cambridge, Massachusetts 02138 Introduction - Enzyme-based synthetic chemistry has continued to grow rapidly in recent Technical problems,which have inhibited the widespread use of enzymes as catalysts, have been much reduced by the introduction of new procedures for enzyme immobilization and stabilization and for in situ cofactor recycling. A widespread interest in asymmetric synthesis has focussed attention on the demonstrated utility of enzymic catalysis in producing chiral fragments. In fact, the major hindrance to the widespread use of enzymic catalysis is the residual unfamiliarity of many classically trained synthetic chemists in the techniques of enzyme isolation, manipulation, and assay. This last barrier is disappearing as biochemistry and enzymology become an accepted part of the education of an organic chemist. This review emphasizes procedures which use partially or highly purified enzymes to catalyze organic reactions potentially useful in medicinal chemistry. Biochemical procedures using microbiological transformations or cell culture are not discussed. General Techniques - More than two thousand enzymes are known,' and several hundred can be obtained commercially. Many other enzymic activities are available through straightforward isolations or small-scale fermentations. Increasing attention is being paid to large-scale production of enzymes;8 Kula has developed efficient methods based on liquid-liquid extractions. Given sufficient demand, many enzymes can be produced in quantity by recombinant DNA techniques. We note that highly purified enzyme preparations are not always necessary in synthetic applications since contaminating enzymes may have no effect on the reactants and products present in the reaction mixture. If an enzyme to be used in synthesis has intrinsically low specific activity (units of catalytic activity per weight of protein), crude preparations can cause practical problems by requiring large volumes of immobilization medium and correspondingly large reactor volumes. The use of microbial cells in enzyme-catalyzed synthesis represents a limiting case in purification; since no purification is involved, their manipulation is straightforward. The activity of these preparations may be low. In favorable cases, however, they represent
ANNUAL REPORTS IN MEDICINAL CHEMISTRY- 19
Copyright 0 1984 by Academic Press. Inc. All rights of reproduction in any forni reserved. ISBN 0- 12-040519-5,
264
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the simplest basis for enzyme-catalyzed reactions, and several successful industrial processes have been developed using immobilized whole cells. 12 Enzyme Immobilization - Enzymes used in synthetic applications are commonly immobilized in or on insoluble materials because immobilization enhances their stability and allows their recovery and reuse. While bench-scale experiments are most conveniently carried out as batch processes, industrial processes often depend on long-lived immobilized enzymes in continuous processes. Many immobilization methods have been developed. 12-14 Only a few of these techniques deserve explicit comment. Glutaraldehyde is the most commonly used bifunctional reagent in immobilization,l2 forming covalent linkages of still incompletely defined nature between amino groups. It is used to bind enzymes to solid supports or cross-link enzymes adsorbed on a support, and to cross-link enzymes with carrier proteins or with themselves to form insoluble aggregates. Immobilization procedures for industrial applications based on functionalized ceramics cross-linked with enzyme via glutaraldehyde have been developed. 1 4 , l5 Wood et al. have developed an immobilization procedure using Whitesides et al. have developed an polyurethane-based membranes. l6 immobilization method based on polyacrylamide-E-N-acryloxysuccinimide cross-linked with triethylene tetramine in the presence of enzyme. 17 This method is particularly useful with the relatively delicate enzymes useful in‘ organic synthesis. Kula et al. have used membrane reactors containing soluble enzymes. l8 The enzymes are not immobilized, but reactor performance has been good.
-
Enzyme Stabilization Enzyme stabilization is a concern before, during, and after immobilization. Immobilization often greatly increases the stability of enzyme^.^'-^^ Reasons for this stabilization are not clearly understood. Enzyme deactivation during the immobilization process is often troublesome. Addition of substrates or inhibitors of the enzyme during immobilization helps to occupy and protect the active site, and increases yields on immobi1i~ation.l~ A number of strategies are useful in maintaining activity in soluble and immobilized enzymes during use. 23-26 Thiol reagents (dithiothreitol, D-mercaptoethanol, 1,3-dithiopropan-2-01) maintain the reduced state of catalytically essential thiols in the enzyme. Chelating agents inhibit metal ion catalyzed oxidations of en~ymes.~’ The stability of soluble enzymes can be enhanced by the addition of 01 ols, salts, and certain polymers, and by chemical modification. 23,2zp-3H Thermally inactivated immobilized enzymes have been reactivated by thiol reagents and reversible heat treatment. 33
-
Cofactor Regeneration About 70% of enzymes use nucleoside triphosphates, nicotinamide derivatives [NAD(P)(H)], or CoA as cofactors. These enzymes include many of those of greatest interest in the synthesis of fine chemicals. Since these cofactors are too expensive to be used stoichiometrically, it has been necessary to develop recycling systems for them. The problem of recycling the nucleoside triphosphates
Chap. 26
Enzymic Methods
Findeis, Whitesides
265
is essentially solved at the level of laboratory-scale synthesis,34-38 and that of recycling NAD derivatives is well-advanced toward solution. 39-46 None of these schemes has been tested on a production scale, although they work satisfactorily for syntheses of several moles of products. Recycling of ATP from ADP or AMP rests on the development of practical syntheses of the phosphate donors acetyl phosphate37 and phosphoen~lpyruvate~~ to be used with the enzymes acetate kinase and pyruvate kinase, respectively. Acetate kinase is also applicable to recycling of GTP, UTP, CTP and the corresponding 2’-deoxy-nucleoside triphosphates.’ ,34 Regeneration of these species from nucleoside monophosphates is not truly practical since adenylate kinase is specific for AMP, but relatively few reactions generate nucleoside monophosphate. CoA recycling has not been explored. Recycling of S-adenosyl-l-methionine, a cofactor in enzyme-catalyzed transmethylations, is currently difficult.47 The conversion of reduced nicotinamide cofactors [NAD(P)H] to the oxidized form [NAD(P)], developed by Jones et al., is the most widely used procedure for this transformation, but suffers from the need for large amounts of flavin and from slow reaction rates.39 Oxidative regeneration based on the conversion of a-ketoglutarate to lutamic acid works well and does not require the presence of oxygen. The best system for reductive regeneration of NADH from NAD is based on formate dehydrogenase. 18,46*48 This procedure works very well, although the enzyme does not accept NADP as a substrate and is relatively expensive. The most practical procedure for regeneration of NADPH uses glucose-6-phosphate dehydrogenase. 42
“
Equilibrium Manipulation - Enzymes are catalysts and therefore serve only to accelerate attainment of equilibrium. In many instances the equilibrium constant for a given reaction in water does not adequately favor the desired product or water acts as an undesired reactant. Sometimes product can be favored using excess starting material, or by reacting the product in an irreversible manner.49 Occasionally precipitation of product will drive an unfavorable reaction, but precipitation can foul immobilized enzymes. Kinetic control of a reaction can sometimes be used to maximize yields.49 In cases where less polar materials are being manipulated, water miscible organic cosolvents have been used, but this technique can reduce enzyme activity. 50-53 Careful selection of cosolvent can, however, maintain enzyme activity and dramatically shift equilibrium.54 A more general approach for working with less polar compounds is the use of a two-phase system incorporating a waterimmiscible organic solvent. This approach has been discussed extensively by Martinek and coworkers. 55-59 Enzyme Mediated Processes
-
Simple Hydrolases and Isomerases These are enzymes requiring no added cofactors, and which catalyze hydrolyses, isomerizations, some condensations, and related reactions. Such enzymes are among the simplest to use and are the most widely used in industry (Table 1). A valuable introduction to processes using these enzymes has been published. 12
266 -
Sect. VI Table 1.
- Topics in Chemistry and Drug Design
Allen, Ed.
Selected Industrial Applications of Enzymes.
-
ref.
6-aminopenicillanic acid penicillin-G fumaric acid -L-aspartic acid ~ L - m a l i acid c fumaric acid starch glucose glucose fructose N-acetyl-D,L-amino acids L-amino acids L-arginine L-citrulline
12,60,61 12,62 12,63 30,64 30,65,66 12 12
Amidases - Acylase is used in continuous production of L-amino It has been used to resolve D,L-phenylalanine6' and a-formylacids." e -acyl-D ,L-lysine. Penicillins have been synthesized from 6-aminopenicillanic acid with penicillinase. 69
-
Esterases Esterases from several sources have been used in stereoselective and regioselective hydrolysis of esters in the preparation of chiral esters, acids, and alcohols. Sih and coworkers have developed a valuable theoretical treatment for quantitative analysis of such biochemical kinetic resolutions relating the extent of conversion (c) of racemic substrate, the optical purity (ee) of the starting material and products, and the enantiomeric selectivity of the enzyme.70 Recycling of enantiomerically enriched substrate can greatly increase ee. Racemic threo ester L w a s hydrolyzed with pig liver esterase (PLE) to (-)-acid 1 (64% ee). Reesterification and incubation to 80% c resulted in 1_ with >90% ee. Erythro ester 2 was treated with Gliocladium roseum to hydrolyze the 2&,32isomer. At 70% c the remaining ester had 95% ee.
/l\rC02R OH
(4 I , R=CH, 1-1 2 , R = H
/+
(4 3
CO,CH,
OH
PLE was used in the enantiotopically selective hydrolysis of diethyl-@. Reduction, derivatization and methylglutarate to the half-ester 2 resolution raised the ee of 2 from 69% to 91%. 70
R02C
C02Et
4,RzEt; 5 , R = H
6(69 %eel
6 (91% eel
Substituent effects have been studied in PLE-catalyzed hydrolysis of a range of symmetrical dicarboxylic acids and a model has been developed to determine absolute configuration in the monoester product^.^' Esterases have successfully resolved compounds 7-2. Imidazolone Dimeth 1-2,4-dimethylglutarates 2 and r~seum.~' Dimethyl-b-aminoglutarate 2 was used as starting material for syntheses of &- and S=4-[(methoxycarbonyl)methy1]-2-azetidinone~,~~useful nuclei for carbapenem p-lactam antibiotic synthesis. &-Esters of 3-hydroxy-3methylalkanoic acids 11
-7 was converted to (+)-biotin.72 -9 were resolved with PLE and 5.
Chap. 26
Enzymic Methods
Findeis Whitesides
267
were prepared for testing as inhibitors of 3-hydroxy-3lnethylglutarylCoA reductase and in compactin analogue syntheses.75 Schneider and coworkers have used PLE-catalyzed hydrolyses in asymmetric syntheses of (1R 3&)-chrysanthemic 12,permethrinic 13 and caronic acid 1 4 derivatives,7 6 and in syntheses of disubstituted monoalkyl malonates7/nd kane dicarboxylate monomethyl esters. 78 0
yy
'
PhbNKNAPh
MeO,C
H++i nPrOzC
Me0,C
C0,Me
8
C0,nPr
CO,Me
1 2-cycloal-
i;'
Me0,C
Cope
10
9
7 Me OH
R
&Cop II
R'
12, R = H , R ' s CH=CMe2 13, R = H, R'= CH=CCI, 14, R = Me, R'. CO,H
6% cOzR
Cambou and Klibanov7' have used transesterifications catalyzed by PLE and yeast lipase to resolve a variety of alcohols with great effectiveness. Their novel approach employed a biphasic system of aqueous enzyme solution absorbed in a porous solid phase placed in a mixture of "matrix ester'' (methyl propionate or tributyrin) and racemic alcohol (Scheme I). 0 Scheme I. 0 &O-(-)-R chiral ester t esterase
matrix ester
r a cemic alcohol
chiral alcohol
+ Me OH
The low water content of this system simplified subsequent purification and effectively suppressed ester hydrolysis. Lipases have also been applied to resolutions of chloroglycerol derivatives,80181 3-acetylthiocycloheptene,82 and 2-chloromethyl-1-propyl propionate .83 Cholinesterases have been used to resolve D,L-carnitine. 84 Proteases ~-
-
Proteases have been applied to several synthetic purposes the most important of which are peptide bond synthesis and protein semisynthesis. Recent extensive reviews cover this area.85,86 Proteases have been used in ester synthesis87 and resolution.88 Semisynthesis of human insulin has been achieved by enzymic removal and replacement of one amino acid in porcine i n ~ u l i n . ~ ~ , ~ 'All peptide bonds in the N-terminal octapeptide of dynorphin [H-Tyr-Gly-Gly-Phe-LeuArg-Arg-Ile-OH] have been formed using proteases. 91 A precursor of aspartame has been made by themolysin-catalyzed condensation of benzyloxycarbonyl-L-aspartic acid and L-phenylalanine methyl ester. 92 Aldolases - Aldolases catalyze reversible aldol condensations of sugars.y' A well-studied enzyme is fructose-lY6-diphosphate aldolase from rabbit muscle. This enzyme exhibits a high specificity for dihydroxyacetone phosphate as the nucleophile, but tolerates a range of aldehydes as electrophiles (Scheme 11) .94 This broad specificity allows synthesis of sugars such as 6-deoxyfructoseg5 and isotopically labeled glucose
268
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- Topics in Chemistry and Drug Design
Allen, Ed.
derivatives.94 Other aldolases exist with different substrate specificities for possible application to preparative sugar synthesis. Scheme 11.
Other - Farnesylpyrophos hate synthetase has been used in asymmetric synthesis of isoprenoids.g6 Potato acid phosphatase has been applied to mild hydrolysis of polyprenyl pyrophosphates. 97 Sulfatase-catalyzed hydrolysis of &naptho1 sulfate has been used to separate a- and B-napthol~.~~ NADg9 and flavin adenine dinucleotide“’ have been made by enzymic coupling reactions. Cofactor Requiring Enzymes Phosphorylation - Enzymic phosphorylation with coupled in situ ATP regeneration has been used to prepare g l u c o s e - 6 - p h o s p h a t e ~ ~ - g l y cerol-3-phosphate,lo* creatine-phosphate,lo3 and 5-phosphoribosyl-lpyrophosphate. 104
-
Chiral Redox Chemistry Nicotinamide cofactor dependent oxidoreductases have been applied in chiral synthesis. Enantioselectivity is often only moderate, but in certain cases excellent results have been obtained. Horse liver alcohol dehydrogenase (HLADH) is the most widely explored enzyme for these purposes with most of the work in this area having been done by Jones and coworkers. lo5 HLADH has been studied sufficiently Cis-3-( 2-hydroxythoroughly to allow modeling of the active site. ethy1)cyclopentanol 2 was oxidized by HLADH to (+)35-(2-hydroxyethyl) cyclopentanone 16with 97% ee; the remaining diol had 70% ee.lo7
-
2-Substituted tetrahydropyran-4-ones were reduced to (-)trans alcohols with 100% ee. lo8 Remaining (+)ketones had 51-86% ee. HLADH-catalyzed oxidation of monocyclic mesodiols x p r o c e e d s to bicyclic lactones g i n
high yield with complete stereotopic selectivity. log Essentially no selectivity is seen in oxidation of the trans diols. =-decalin-2,719 is reduced specifically to (-)(7S-,92,10&)-7-hydroxy-&-decdione alin-2-one 20llo*lll which can be converted to (+)4g-twistanone 21 in 51% yield from the dione. D- and L-lactic dehydrogenases reduce chloropyruvic acid Z t o D- and L-chforolactic acids 23; these can be
Enzymic Methods
Chap. 26
3
H
H
0
Findeis, Whitesides
OH
converted to epoxyacrylic acids 24. 'I2 Progesterone has been reduced to ZO-B-hydroxy-pregn-4-ene-3-one with 20-P-hydroxysteroid dehydrogenase using reversed micelles in organic solvent and H2 as ultimate reductant Microbial reductions have been used in intermediate steps in syntheses of natural brefeldin-A, ' 1 4 (+)compactin 'I5 and L-carniL-leucine dehydrogenase has been used in the reductive tine amination of a-ketoisocaproate to L-leucine18 in a membrane reactor. NAD was covalently modified by attachment of polyethylene glycol to make it unable to cross the membrane.
. .
Multi-Enzyme Cofactor Requiring Processes - A more complex level of applied enzymology is reached in the use of multi-enzyme schemes to synthesize complex molecules. Examples of such syntheses include ribu10se-l,5-diphosphate,~~ im ortant in the study of ribulose-diphosphate carboxylase; lacto~amine,'~~ from the first use of the Leloir pathway enzymes in synthesis; and S-adenosyl-L-methionine. 47 Oxidations - Enzymes which functionalize inactivated carbon are often difficult to obtain and handle. Many examples exist of oxidations using microbial e.g. for steroids, and recently in olefin oxidation. Such preparative transformations have not been achieved with purified enzymes and are unlikely to be amenable to large-scale 2 vitro approaches because of instability and complexity of the enzyme systems. Klibanov and coworkers, however, have developed systems with horseradish peroxida~e'~~,~~' and xanthine oxidaset2' for oxidation of aromatic alcohols and amines, for use in syntheses and waste water treatment. Hydroxyphenyl compounds can be oxidized to dihydroxy derivatives. L-DOPA has been made from L-tyrosine in this manner. l Z 3 c clohexanone was oxidized to t-caprolactone with a bacterial oxygenase. 1 2 4 The Future - Enzymic synthetic methods will see increased use in research and industry. Numerous examples exist of preparations of useful quantities of chiral compounds for use in synthesis, and the use of hydrolytic enzymes for simple synthesis. More importantly, enzymes will allow the facile synthesis of complex molecules important in biological research. Immunology, neurobiology, endocrinology, molecular genetics, membrane biology, and plant and insect biology are areas becoming more molecular in scope. Research in such fields will increasingly depend on biologically active compounds not readily accessible by more conventional chemistry. Molecules that are water soluble, or highly functional-
270
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ized such as carbohydrates, nucleic acids, lipids, and proteins may prove available by enzymic synthetic methods. Enzymology will be useful in modifications of poly- and oligosaccharides and proteins. Enzymes will also see growth in applications in medical diagnostics and treatment, and in food chemistry. 125 Recombinant DNA and RNA methods rely on enzymes, and as these methods are developed,the op ortunity for enzyme engineering of synthetic catalysts will grow. 126*p27 Enzyme-based synthetic methods will be an important part of future organic synthesis, especially in synthesis of new pharmaceutical products. Acknowledgement - We acknowledge financial support from the National Institutes of Health, Grant GM 30367. References 1. 2.
3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38.
G.M. Whitesides and C.-H. Wong, Aldrichimica Acta, g, 27 (1983). J.B. Jones in "Asymmetric Synthesis," J.D. Morrison, Ed., Academic Press, New York, N.Y., 1983. C.J. Suckling and H.C.S. Wood, Chem. Brit., 2,243 (1979). J.B. Jones, C.J. Sih and D. Perlman, Eds., "Applications of Biochemical Systems in Organic Chemistry," Tech. Chem., lo,Wiley, New York, N.Y., 1976. J.B. Jones, Method. Enzymol., 831 (1976). C.J. Sih, E. Abushanab and J.B. Jones, Annu. Repts. Med. Chem., 12, 298 (1977). International Union of Biochemistry. Nomenclature Committee. "Fzyme Nomenclature, 1978," Academic Press, New York, N.Y. (1979). B.S. Hartley, T. Atkinson and M.D. Lilly, Eds., Phil. Trans. R. SOC. Lond., 237 (1983). M.R. Kula, K.H. Kroner and H. Hustedt. Adv. Biochem. Eng., 2, 73 (1982). P.-A. Albertsson, Adv. Protein Chem., 2, 309 (1970). P.-A. Albertsson, "Partition of Cell Particles and Macromolecules," 2nd ed., 1971. Almquist and Wiksell, Stockholm, Wiley-Interscience, N.Y., I. Chibata, "Immobilized Enzymes--Research and Development." Halsted Press, New York, N.Y., 1978. K. Mosbach, Ed., "Immobilized Enzymes," Method. Enzymol., 4ft (1976). A.M. Klibanov, Science, 219, 722 (1983). R.P. Rohrbach, U.S. 4,268,419 (1979). L.L. Wood, F.J. Hartdegen, P.A. Hahn, U.S. 4,312,946 (1982). A. Pollak, H. Blumenfeld, M. Wax, R.L. Baughn and G.M. Whitesides, J. Am. Chem. SOC., 102, 6324 (1980). C. Wandrey, A.F. Buclanann and M.R. Kula, Biotechnol. Bioeng., 2,2789 (1981). A.M. Klibanov, Anal. Biochem., 1 (1979). K. Martinek, A.M. Klibanov, V.S. Goldmacher and I.V. Berezin, Biochim. Biophys. Acta, 485, 1 (1977). K. Martinek, A.M. Klibanov, V.S. Goldmacher, A.V. Tchernysheva, V.V. Mozhaev, I.V. Berezin and B.O. Glotov, Biochim. Biophys. Acta, 485, 13 (1977). A.M. Klibanov, N.O. Kaplan and M.D. Kamen, Arch. Biochem. Biophys., 199, 545 (1980). R.D. Schmid, Adv. Biochem. Eng., &, 41 (1979). A.M. Klibanov, Biochem. SOC. Trans., 2. 19 (1983). A.M. Klibanov, N.O. Kaplan and M.D. Kamen, Enzyme Eng., 2, 135 (1980). A.M. Klibanov, N.O. Kaplan and M.D. Kamen, Proc. Nat. Acad. Sci. U.S.A., 15, 3640 (1978). A.M. Klibanov, N.O. Kaplan and M.D. Kamen, Biochim. Biophys. Acta, 547, 411 (1979). J.B. Jones and D.R. Dodds, Can. J. Chem., 2533 (1979). S. Branner-Jdrgensen, Biochem. SOC. Trans., 2, 20 (1983). C. Bucke, Biochem. SOC. Trans., &, 13 (1983). K. Gekko and S.N. Timasheff, Biochem., 2,4667; 4677 (1981). T. Arakawa and S.N. Timasheff, Biochem., 2, 6536; 6545 (1982). A.M. Klibanov and V.V. Mozhaev, Biochem. Biophys. Res. Comm., 2, 1012 (1978). C.-H. Wong, S.L. Haynie and G.M. Whitesides, J. Am. Chem. SOC., 105, 115 (1983). B.L. Hirschbein, F.P. Mazenod and G.M. Whitesides, J. Org. Chem., 2, 3765 (1982). C.-H. Wong, A. Pollak, S.D. McCurry, 'J.M. Sue, J.R. Knowles and G.M. Whitesides, Method. Enzymol., g,I08 (1982). D. Crans and G.M. Whitesides, J. Org. Chem., 3130 (1983). R.L. Baugh, 0. Adalsteinsson, and G.M. Whitesides, J. Am Chem. SOC., 100, 304 (1978).
*,
a,
z,
x,
z,
Chap. 26 39. 40. 41. 42. 43. 44. 45. 46. 47. 48. 49. 50. 51. 52. 53. 54. 55. 56. 57. 58. 59. 60. 61. 62. 63. 64. 65. 66. 67. 68. 69. 70.
71. 72. 73. 74. 75. 76. 77. 78. 79. 80. 81. 82. 83. 84. 85. 86. 87. 88. 89. 90. 91. 92.
Enzymic Methods
F i n d e i s , Whitesides
271
J.B. Jones and K.E. Taylor, Can. J. Chem., 15, 2969 (1976). C.-H. Wong and G.M. Whitesides, J. Am. Chem. S O C . , 104,3542 (1982). C.-H. Wong and G.M. Whitesides, J. Org. Chem., 2816 (1982). C.-H. Wong and G.M. Whitesides, J. Am. Chem. SOC., 103,4890 (1981). C. Burstein, H. Ounissi, M.D. Legoy, G. Gellf and D. Thomas, Appl. Biochem. Biotechnol., L, 329 (1981). E. Chave, E. Adamowicz and C. Burstein, Appl. Biochem. Biotechnol., L. 431 (1982). C.-H. Wong and G.M. Whitesides, J. Am. Chem. SOC., 105,5012 (1983). Z. Shaked and G.M. Whitesides, J. Am. Chem. SOC., 102, 7104 (1980). A. Gross, S. Geresh and G.M. Whitesides, Appl. Biochem. Biotechnol., g, 415 (1982). H. Schutte, M.R. Kula and H. Sahm, Method. Enzymol., 2,527 (1982). K. Martinek and A.N. Semenov, J. Appl. Biochem., 2, 93 (1981). L.G. Butler, Enzyme Microb. Technol., L, 253 (1979). J.B. Jones and H.M. Schwartz, Can. J. Chem., g,335 (1982). J.B. Jones and D.H. Pliura, Can. J. Chem., 2, 2921 (1981). J.B. Jones and M.M. Mehes, Can. J. Chem., 2245 (1979). G.A. Homandberg, J.A. Mattis and M. Laskowski, Jr., Biochem., 17,5220 (1978). K. Martinek, A.N. Semenov and I.V. Berezin, Biochim. Biophys. Acta, 658, 76 (1981). K. Martinek and A.N. Semenov, Biochim. Biophys. Acta, 658, 90 : ) 1 8 9 ( K. Martinek, A.N. Semenov and I.V. Berezin, Dokl. Akad. Nauk. SSSR, 253, 358 (1980). K. Martinek, A.N. Semenov and I.V. Berezin, Dokl. Akad. Nauk. SSSR, 252, 394 (1980). A.M. Klibanov, G.P. Samokhin, K. Martinek and I.V. Berezin, Biotechnol. Bioeng., E, 1351 (1977). B.J. Abbott, Adv. Appl. Microbiol., 20, 203 (1976). E. Lagerlof, L. Nathorst-Westfelt, B. Ekstrom and B. Sjoberg, Method. Enzpol., 3, 759 (1976). I. Chibata, T. Tosa and T. Sato, Method. Enzymol., 739 (1976). I. Chibata, T. Tosa and I. Takata, Trends. Biotechnol., 1 ,9 (1983). M.K. Weibel, W.H. McMullen and C.A. Starace, Dev. Ind. Microbiol., 19, 103 (1977). N.B. Havewala and W.H. Pitcher, Jr., Enzyme Eng., L, 315 (1974). N.H. Mermelstein, Food Technol., 2,20 (1975). T. Kitahara and S. Asai, Agric. Biol. Chem., 7, 991 (1983). Y. Minematsu. Y. Shimohigashi, M. Waki and N. Izumiya, Bull. Chem. SOC. Jap., 1899 (1978). A.N. Semenov, K. Martinek, V. Yu.-K. Shvyadas. A.L. Margolin and I.V. Berezin, Dokl. Akad. Nauk. SSSR, 258, 1124 (1981). C.-S. Chen, Y. Fujimoto, G. Girdaukas and C.J. Sih, J. Am. Chem. SOC., 104 7294 (1982). P. Mohr. N. Waesue-Sarcevic, C. Tamm, K. Gawronska and J.K. Gawronska, Helv. Chim. Acta, 2501 (i983). S . Iriuchijima, K. Hasegawa and G. Tsuchihashi, Agric. Biol. Chem., 66, 1907 ( 982). C.-S. Chen, Y . Fujimoto, C.J. Sih, J. Am. Chem. SOC., 103,3580 (1981). M. Ohno, S. Kobayashi, T. Iimori, Y.-F. Wang and T. Izawa, J. Am. Chem. SOC., 103. 2405 (1981). W.K. Wilson, S.B. Baca, Y.J. Barber, T.J. Scallen and C.J. Morrow, J. Org. Chem., 48, 3960 (1983). M. Schneider, N. Engel, and H. Boensmann, Angew. Chem. Int. Ed. Engl., G,64 (1984). M. Schneider, N. Engel, and H. Boensmann, Angew. Chem. Int. Ed. Engl., 2, 66 (1984). M. Schneider, N. Engel, P. Honicke, G. Heineman and H. Gorisch, Angew. Chem. Int. Ed. Engl., 2, 67 (1984). B. Cambou and A.M. Klibanov, J. Am. Chem. SOC., 106,in press (1984). S . Iriuchijima and N. Kojima, Agric. Biol. Chem., 1153 (1982). 1593 (1982). S . Iriuchijima, A. Keiyu and N. Kojima, Agric. B i o l . Chem., 3, S. Iriuchijima and N. Kojima, J.C.S. Chem. Corn., 185 (1981). J. Lavayre, J. Verrier and J. Baratti, Biotechnol. Bioeng., 24 2175 (1982). E.P. Dropsy and A.M. Klibanov, Biotechnol. Bioeng. 2, in press (1984). J.S. Fruton, Adv. Enzymol., 2, 239 (1982). I.M. Chaiken, A. Komoriya, M. Ohno and F. Widmer, Appl. Biochem. Biotechnol. 1,385 (1982). D. Tarquis, P. Monsan and C. Durand, Bull. SOC. Chim. Fr. II., 76 (1980). Y. Nishida, H. Ohrui and H. Meguro, Agric. Biol. Chem., 7, 2123 (1983). K. MOKihaKa, T. Oka, H. Tsuzuki, Y. Tochino and T. Kanaya, Biochem. Biophys. Res. Comn., 396 (1980). K. Inouye, K. Watanabe, K. Morihara, Y. Tochino, T. Kanaya, J. Emura and S . Sakakibara, J. Am. Chem. SOC., 101,751 (1979). W. Kullman, J. Org. Chem., 67,5300 (1982). K. Oyama, S. Nishimura, Y. Nonaka, K. Kihara and T. Hashimoto, J. Org. Chem., 5, 5241 (1981).
x,
x,
c,
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&,
s,
z,
272 93. 94. 95. 96. 97. 98. 99. 100.
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- Topics
i n Chemistry and Drug Design
P.D. Boyer, Ed., "The Enzymes," 7-, Chapters 6-9 (1972). C.-H. Wong and G.M. Whitesides, J. Org. Chem., 2, 3199 (1983). C.-H. Wong, F.P. Mazenod and G.M. Whitesides, J. Org. Chem. 3493 (1983). T. Koyama, A. Saito, K. Ogura and S. Seto, J. Am. Chem. Soc., 102, 3614 (1980). H. F u j i i , T. Koyama, K. Ogura, Biochem. Biophys. Acta, 712. 716 (1982). G. Pelsy, A.M. Klibanov, Biotechnol. Bioeng., 3. 919 (1983). D.R. Walt, M.A. Findeis. V.M. Rios-Mercadillo, J. Auge and G.M. Whitesides, J. Am. Chen. SOC., 234 (1984). S. Shimizu, K. Yamane, Y. Tan1 and H. Yamada, Appl. Biochem, Biotechnol., 237
ss
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(1983). 101. 102. 103. 104. 105. 106. 107. 108. 109.
A l l e n , Ed.
z,
A. Pollak, R.L. Baughn and G.M. Whitesides, J. Am. Chem. SOC., 2366 (1977). V.M. Rios-Mercadillo and G.M. Whitesides, J. Am. Chem. SOC., 5828 (1979). Y.-S. Shih and G.M. Whitesides, J. Org. Chem., 2, 4165 (1977). A. Gross, 0. Abril, J.M. Lewis, S. Geresh and G.M. Whitesides, J. Am. Chem. SOC.,
101,
105, 7428 (1983). T. Takemura and J.B. J.B. A.J. J.A.
Jones, J. Org. Chem., 5. 791 (1983). and previous papers. Jakovac, Can. J. Chem., 9 19 (1982). Jones, J. Am. Chem. SOC., 1625 (1977). Haslegrave and J.B. Jones, J. Am. Chem. SOC., 106. 4666 (1982). Jacovac, H.B. Goodbrand, K.P. Lok and J.B. Jones, J. Am. Chem. SOC., 106, 4659 Jones and I.J. Irwin and J.B.
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I.J. (1982).
110. D.R. Dodds and J.B. Jones, J.C.S. Chem. C m . , 1080 (1982). 111. M. Nakazaki, 8. Chikamatsu and M. Taniguchi, Chem. L e t t . , J l 1761 (1982). 112. B.L. Hirschbein and G.M. Whitesides, J. Am. Chem. SOC., 104,4458 (1982). 113. R. Hilhorst, C. Laane and C. Veeger, FEBS Lett., 159, 225 (1983). 114. C. LeDrian and A.E. Greene, J. Am. Chem. SOC., 2,5473 (1982). 115. C.T. Hsu, N.Y. Wang, L.H. Latimer and C.J. Sih, J. Am. Chem. SOC., 105, 593 (1983). 116. B. Zhou, A.S. Gopalan, F. Vanmiddlesworth, W.-R. Shieh and C.J. Sih, J. Am. Chem. SOC., 105,5925 (1983). 117. C.-H. Wong, S.L. Haynie and G.M. Whitesides, J. Org. Chem., 5416 (1982). 118. K. Kieslich, "Microbial Transformations of Non-Steroid Cyclic Compounds," John Wiley h Sons, New York, N.Y., 1976. Chem. Corn., 849 (1978). 119. H. Ohta and H. Tetsukawa, J.C.S. 120. A.M. Klibanov, B.N. A l b e r t i , E.D. Morris and L.M. Felshin, J. Appl. Biochem., 1,414 (1980). 121. A.M. Klibanov and E.D. Morris, Enzyme Microb. Technol., 2, 119 (1981). 122. G. Pelsy and A.M. Klibanov, Biochim. Biophys. Acta, 762, 352 (1983). 123. A.M. Klibanov, 2. Berman and B.N. A l b e r t i , J. Am. Chem. SOC., 103,6263 (1981). 124. J.M. Schwab, J. Am. Chem. SOC., 103,1876 (1981). 125. H. Samejima, K. Kimura and Y. Ado, Biochemie, g , 299 (1980). 126. W.H. Rastetter, Appl. Blochem. Biotechnol., 3 423 (1983). 127. T.H. Maugh, 11, Science, 223, 154 (1984).
zs
27 3 -
Chapter 27.
Stable Isotopes in Drug Metabolism and Disposition
Thomas A . Baillie and Albert W. Rettenmeier, Department of Medicinal Chemistry, University of Washington, Seattle, WA 93195 Lisa A . Peterson and Neal Castagnoli, Jr., Department of Pharmaceutical Chemistry, University of California, San Francisco, CA 94143 Since the 1977 review in Annual Reports,' the applications of stableisotope-labeled compounds to biomedical research have increased dramatically. Three international conferences devoted to the use of stable isotopes in the life and one to synthesis and general applicat i o n ~ have ~ been held. Review articles have focused on mass spectrometric-based analytical methods, 13'' on applications of stable isotopes in clinical8-'' and pharmacological research , "-' and on more specific studies concerning groblems in drug metabolism, l r '15 pharrnacokinetics16' and synthesis. "' The present review covers mainly the literature published since 1981 and focuses on recent developments in the use of stable isotopes for investigations of drug metabolism and disposition.
'
Metabolite Structure Elucidation Isotope Clusters - The isotope cluster (isotope doublet, twin ion) technique has proved to be a powerful method for the detection and structural identification of drug metabolites in complex biological matrices. An equimolar mixture of [8-13C;1,3-15N~]caffeineand unlabeled caffeine was used to establish that the acetyl group of the metabolite, 4-acetyl-6amino-3-methyluracil, did not contain the original C-8 carbon atom since it was labeled with two 15N atoms only." In this and a related studyYZL [ 2-14C]caffeine was co-administered to aid in monitoring the excretion of drug metabolites. Although a useful adjunct to the stable isotope cluster technique, the administration of radiotracers in human research is limited on ethical grounds, particularly in pediatric and obstetric populations. Studies on the biotransformation of theophylline in premature newbornsz2 and on the placental transfer and fetal metabolism of this compound in humansz3 relied exclusively on the use of [ 2-1 3C;1,3-15N2 Itheophylline as tracer. The stable isotopes of carbon and nitrogen also have been successfully applied to ion cluster metabolism studies of [ 13C, "N~]hexobarbital. " Although the use of deuterium in drug metabolism involving the isotope cluster technique may be complicated by isotope effects,2 5 both cost and convenience have led most workers t o rely on this isotope. Recent examphencyclidine, ples include reports on arninopyrine ,z 6 pencycuron , fentanyl, 3 0 and diethylstilbestrol. 3 1 In the case of steroids and their derivatives, ion cluster studies have been performed exclusively with deuterium-labeled substrates. Examples include investigations with [ 'H5 Iethynylestradiol,3 z [ 'H2 ]4-hydroxyandrost-4-ene-3, 17-dione, [ 'H2 ]ursodeoxycholic acid, 3 y and [ 'H~lbudesonide.3 5
''
ANNUAL REPORTS IN MEDICINAL CHEMISTRY-19
''"'
Copyright 0 1984 by Academic Prebs, Inc. All rights of reproduction in any form reserved. ISBN 0-12-040519-9
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Allen, Ed.
The isotope cluster technique has proved valuable in the identification of metabolites similar or identical to endogenous compounds present in the biological extracts. Use of a hexadeutero variant of valproic acid, a short-chain fatty acid, led to the identification of a new biotransformation product, 2-g-propyl-4-oxopentanoic acid. 3 6 A similar approach was used to reveal a novel pathway of benzoic acid metabolism in the horse. 3 7 - 3 9 Metabolites of rutin, a flavinol glycoside, were identified in the urine of rats" and humans'l given [2',5',6'-2H3]rutin. Deuteriumlabeled phenylephrineW2 has been used in a similar fashion. Derivatization of samples with an equimolar mixture of labeled and unlabeled reagents to produce ion clusters has been applied recently to the characterization of the bleomycins by FD and FAB mass ~pectrometry.'~ A variation of this technique utilizes a mixture of labeled and unlabeled reagent gases { pyridine/[ 2Hs]pyridine ," trimethylchlorosilane (TMCS)/ [ 2H9]TMCS, tetramethylsilane (TMS)/[ 2H12 ]TMS," and NH3/N2H3" ' r 7 1 in conjunction with CI mass spectral analyses. 'lhe isotope cluster technique also may be exploited with the newer ionization methods of SIMS48 and FAB" through the addition of appropriate counter-ions (for example ,
''
Ag'"),
Work continues on the development of computer software for automatic screening of large amounts of mass spectral data for specified isotope clusters. O ' ' The Shift Technique - The stable isotope shift technique refers to a procedure in which the mass spectrum of an unlabeled compound is compared with that of a stable-isotope-labeled counterpart. The observed differences in masses between corresponding ions in the two spectra provide valuable information on ion composition and hence on molecular structure. This technique may be used in two distinct ways. The drug may be labeled at a specific site(s) and the mass spectra of metabolites derived from the labeled compound compared with those obtained from the unlabeled drug. Recent examples of this application include reports on the metabolism of oxybutynin,5 2 tocainide, and ketamine. 54 Alternatively , a heavy isotope (usually 'H) is introduced into the metabolite of interest by reaction with a labeled derivatizing reagent. For example, perdeuteromethylation results in a mass difference of 3 daltons between labeled and unlabeled derivatives per methyl group. Structures of metabolites obtained from caffeine" and phenytoin5' have been confirmed by this technique. Tri(deuteromethy1)silylation has been employed in the identification of metabolites of carbama~epine~'and afloqualone.
- The applications of stable isotopes coupled with NMR analysis have multiplied as higher field magnets and sophisticated computer techniques have increased sensitivity. A recent review which discussed NMR studies employing stable isotopes is available. 5 9 NMR Studies
Natural abundance 13C-NMR spectroscopy has been applied to the structure analysis of several metabolites derived from acetaminophen." The structures of metabolites of prenaltero16 and trans-sobrerol" have been studied. 13C-NMR analyses of metabolites derived from I3C-enriched amitriptyline,6 3 BHT," propachl~r'~ and aminopyrine66'67 have been reported.
Chap. 27
Stable Isotopes
Bailie et al. 275
Mechanistic Studies Cytochrome P-450 Catalyzed Reactions - Studies with "02 have established that the cvtochrome P-450 mediated hvdroxvlation of camDhor bv the bacterial enzymk68 and the enzyme purified from rat liver6' reshts in the incorporation of atmospheric oxygen in the 5-*-hydroxylation product. Cumene [ "02]hydroperoxide will transfer its peripheral oxygen atom to a variety of compounds which serve as substrates for mammalian cytochrome P450." AS expected, " 0 2 served as the oxygen source for the bacterial cytochrome P-450 catalyzed epoxidation of 5,6-dehydrocamphor .7 1 N-Hydroxymethylcarbazole, formed by the cytochrome P-450 catalyzed oxidation of Nmethylcarbazole, incorporates " 0 exclusively from dioxygen. 7 z ' Under anaerobic conditions cytochrome P-450 may catalyze the intramolecular transfer of oxygen present in tertiary amine N-oxides. a Mechanistic studies on S-dealkylation and S-oxidation reactions also have used " 0 tracer methods.7' The oxidation of 1,2-dideuterocyclohexene by both cytochrome P-450 and model chemical systems gave cyclohexenol with various degrees of allylic rearrangement of the deuterium atoms, suggesting a mechanism involving caged radical intermediates. The bioactivation of terminal olefins by cytochrome P-450 leads to the alkylation of the heme prosthetic group. Mechanistic studies using trans-1-[ 'Hloctene as substrate demonstrated that the trans-stereochemistry is retained in both the heme alkylation product and the epoxide. This result is inconsistent with heme alkylation occurring by reaction with octene oxide itself. 76 The structures of heme adducts produced from several olefins now have been determined and it has been established, using oxygen-18, that the adducts contain an atom of oxygen derived from the atmosphere." Mechanistic studies with deuterium-labeled arenes have provided evidence against a proton abstraction or direct insertion mechanism for the hydroxylation of certain benzenoid systems at the meta p~sition.~' Finally, "N-labeling has been employed in an investigation of the process through which metabolism of 3,5-diethoxycarbonyl-l,4-dihydrocollidine by cytochrome P-450 leads to self-destruction of the enzyme and formation of N-methylprotoporphyrin IX. 7 9 Metabolic Pathways - Compounds labeled at specific sites with stable isotopes have been employed in the identification of short-lived, potentially toxic metabolites. The metabolic formation of reactive episulfonium ions from the conjugation of 1,2-dihaloethanes with glutathione has been studied with deuterium-labeled substrates."' 8 2 The detection of iminocyclophosphamide from cyclophosphamide was aided by deuterium and oxygen18 labeling techniq~es.'~ Quantitative studies on the a-hydroxylation bioactivation pathway of carcinogenic nitrosamines were approached by measuring 'N2 evolution from doubly "N-labeled N-nitrosodimethylamine and N-nitrosomethylaniline from rat liver homogenates. " The evolution of I3CO from l3Ccl4 could be distinguished from unlabeled CO derived from the destruction of heme and lipid per~xidation.'~ The two isotopes of chlorine (35Cl and 37Cl) have been used t o probe the origin of electrophilic chlorine species generated during the metabolism of CClt, in hepatic microsoma1 preparations. 8 6 Reductive dehalogenation of Cc11, to the trichloromethylperoxy radical has been proposed as the initial event which leads to the electrophilic chlorine specie^.'^ In a study using oxygen-18 as a metabolic tracer, the porphyrinogenic agent allylisopropylacetamide was shown to undergo biotransformation to three different chemically reactive intermediates which appear to alkylate different cellular constituents
."
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Allen, Ed.
Using ''02 Anderson et al. showed that the two major phenolic metabolites of N-phenyl-2-naphthylamine produced by liver enzymes are likely to be derived via arene oxide intermediate^.'^ The mechanism by which bromobenzene is converted to 4-bromocatechol in isolated rat hepatocytes was shown with " O z to proceed via dehydrogenation of a dihydrodiol intermediate rather than by two successive hydroxylation reactions of the substrate. Similar studies have been performed on chlorpromazine. 9 1 Using 'H-labeled drugs, arene oxides have been implicated in the metabolism of phenytoing2 and propranolol. Deuterium-labeled variants of propranolol also have been used to study stereoselective aspects of its metabolism and disposition. 9 3 '" Spontaneous decomposition of anti-neoplastic agents such as the 2-haloethylnitrosoureas to give reactive alkylating and carbamoylating intermediates has been examined with deuterium and oxygen-18 labeled compounds.9 5 - 9 7 Studies of the enterohepatic recycling of 3C- and 2H-labeled mercapturic acid conjugates of propachlor have been reported. 6 s '
'
'-'
Deuterium Kinetic Isotope Effects - The analysis of deuterium kinetic isotope effects provides a useful tool for the elucidation of mechanisms Recent studies of deuterium isoof drug metabolism and toxicity. l o '-lo tope effects on cytochrome P-450 catalyzed reactions have led to a general consensus that these types of oxidations proceed by hydrogen radical abstraction, with the transient formation of a carbon radical prior to recombination with a hydroxyl radical to form the final product. '4-1 Cytochrome P-450 catalyzed oxidative N-dealkylations tend to proceed with modest isotope effects at best.107-'og Studies with 5-['H~]phenyl-5phenylhydantoin showed that no isotope effect was observed for both paraand s-hydroxylations with rat liver microsomes. Analogous results were obtained with deuterium-labeled ~arfarin,~'suggesting that these biotransformations occur by an addition-rearrangement mechanism. In vitro kinetic isotope studies have been reported with monoamine oxidas7" "12 and epoxide hydrolase.ll3 The introduction of deuterium at C-6 of penicillanic acid resulted in a significant increase in its a-lactamase inhibiting properties.
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vivo alterations in rates of metabolism and excretion of deuterIn ium-labeled versus unlabeled drugs have been discussed. I' Such changes in metabolic patterns can influence the pharmacological properties of some drugs such as the increased potency of the hexadeuterated analog of the gastric antisecretory agent N,N-dimethyl-"-[ 2-(diisopropylamino)ethyl]N'-(4,6-dimethyl-2-pyridyl)urea. l6 Substitution of deuterium in the Nmethyl groups of imipramine caused a decrease in systemic clearance, increase in half life and, upon oral administration, an increased bioavailability. ' 1 7 The uptake, clearance and brain levels of the hallucinogen N,N-dimethyltryptamine were potentiated upon deuterium substitution at the ct and f3 positions of the side chain. 'I8 The in vivo rate of degradation of the antidepressant phenelzine was found to be retarded in a deuterium-labeled analog,' which apparently was responsible for alterations in the observed biochemical , behavioral , " and spontaneous motor activity effects of the drug."' Kinetic isotope effects also have been used to aid in the elucidation of the in vitro and in viva metabolic pathways of the anticancer agent 6-mercaptopurine. 12' A variety of isotope effect studies have been conducted in an effort to characterize. critical bioactivation steps which may be responsible for the toxicity of xenobiotics. Examples include investi ations on the metabolism of ally1 alcohol,'23 methoxyflurane, BHT,12' 1,2-dibromoethane9l Z 6 chloroform 12 7 9 1 2 8 and carcinogenic nitrosamines. 1 2 9 - 1 3 4
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Chap. 27
Stable Isotopes
B a i l i e , et al. 277
Pharmacokinetic Investigations Quantitative Applications - The use of stable-isotope-labeled compounds as internal standards for the quantitation of drugs and metabolites in biological fluids offers a unique combination of sensitivity and selectivity of detection for pharmacokinetic studies. The principles of the technique have been outlined, and applications up to 1981 have been compiled.135 Specific aspects of the isotope dilution method, e.g. its utility as a reference technique' and associated procedures for handling the data generated from the use of multiple isotope tracers simultaneously,l 3 'have been discussed. Examples of isotope dilution methods used in clinical psychopharmacology have also been reviewed. 3 8 Deuterium continues to be the most widely used isotope for preparation of labeled standards and appears to be well suited for this purpose. However , certain 'H-labeled compounds have been found to become diluted with the protio forms upon storage, presumably due to isotope exchange phenomena. A case in point is [ 3,3,4 ,4-2H4 ]6-oxoprostaglandin Fla,1.59 Stable isotopes other than deuterium have been employed with increasing frequency for internal standards. A sensitive assay for glyceryl trinitrate in human plasma by GC-negative ion CIMS made use of two multiply15N-labeled analogs of the drug. The [l5N3] variant was employed to minimize absorption of glyceryl trinitrate on the GC column and a [2H5,15N3] s ecies served as internal standard for quantitative purposes. l Y 0 [ 3C 15N3]Guanfacine has been used as an internal standard for [ 2H3,13C6]dexamethasone for the corresponding unlaguanfacine14' "'*'and beled corticosteroid. 1 4 3
P
In certain situations, "0 may be suitable for labeling purp o s e ~ ~ ~ ~and ~ ' some " ~ exploratory work has been carried out with polychlorinated compounds enriched at > 90% excess with 37C1.146y127In the latter case, greatly simplified molecular ion clusters were evident in the mass spectra of 37C1-labeled compounds such as heptachlor and related organochlorine insecticides. Although ' S is available commercially , compounds enriched in this isotope do not appear to have been used as yet in the field of drug metab~lism."~ Lithium is important in the treatment of manic depressive illness and a stable isotope dilution assay for 6Li, using 'Li as internal standard , has been published. l 4 Where preparation of stable-isotope-labeled drug metabolites is required, an economical approach is to administer the labeled drug to a suitable animal species and to isolate the corresponding labeled metabolites from urine samples. Deuterium-labeled metabolites of antipyrine were obtained in this manner , while deuterated metabolites of 6-0x0PGFla were prepared by incubation of the labeled parent prostaglandin with Mycobacterium rhodochrous.
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The "soft" ionization MS techniques of FD15* and FAB"3-155 have been employed successfully for quantitative applications with stable-isotopelabeled internal standards and interesting developments in the use of microwave plasma discharges, either alone or in combination with mass spectrometic detection, promise to extend the utility of stable isotope labeling methods in quantitative studies of drug dis New methods have been reported for the measurement of lgN enrichment in ammonia, based on conversion into hexamethylenetetramine, 5 8 and for the ' analysis of 3C0 by combustion to "C$.
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278
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Pharmacokinetics Determined under "Steady-State" Conditions - Administration of a single "pulse dose" of a stable-isotope-labeled analog is an effective method for defining the kinetics of a drug which is being given chronically, as there is no need to interrupt the dosage regimen to conduct the study.11'16 The value of this technique for use in clinical pharmacology is illustrated convincingly by work on the changes in valproic acid pharmacokinetics which take place during pregnancy. These parameters were determined by following the disappearance of a bolus dose of [ 1,2-13C2]valproic acid given to a pregnant epileptic patient. 16' For ethical reasons, this type of study could not have been carried out by alternative methodology using radioactive isotopes. A similar investigation of valproic acid pharmacokinetics in non-pregnant humans was performed using a deuterium-labeled variant of the drug. l b 2 It is essential that the labeled species chosen for administration be demonstrated to be pharmacokinetically equivalent to its unlabeled counterpart. The likelihood for non-equivalence is remote when 13C or 15N is employed. Re orts have appeared in which investigators have validated the use of [ 13C,P5NN]phenobarbital'63-16s and [ 13C,'5N2]phenytoin166 for such applications. Concern over apparent differences in vivo between phenytoin and a deuterium-labeled analog led Poupaert et &. to synthesize [ 3C6]phenytoin for pulse dose studies. Nevertheless, when properly validated, drugs labeled with deuterium can be used for administration in Recent examples of the latter have involved studies kinetic experiments. on the influence of long-term infusions of lidocaine on the kinetics of this agent 16' to investigations of methadone kinetics during maintenance treatment,l6 17' and to stereoselective aspects of the disposition of methadone enantiomers in humans given the racemic drug. 17' The singledose kinetics of A'-tetrahydrocannabinol were studied in light and heavy cannibis users with the aid of a 'H3-1abeled variant of the drug.172
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Bioavailability Studies - The use of stable isotopes in studies of absolute or relative bioavailability offers a number of important advantages over the conventional "cross-over" approach in which different (unlabeled) formulations are administered on separate occasions. l 1 " ' l7 "7 4 This is especially true for drugs which are subject to extensive first-pass elimination, since these compounds usually exhibit the greatest variation in bioavailability as a function of time. l7 The absolute bioavailabilities of timol01~'~''~~and bepridil17' have been studied by the simultaneous oral and intravenous administration of unlabeled and 13C-labeled forms, respectively. The bioavailability of a standard formulation of clovoxamine fumarate was compared with that of a slow-release preparation using [ '3C]clovoxamine as the reference dosage form. 17' The absolute bioavailabilities of verapamillso and methadone'" have been investigated with the aid of specifically deuterated analogs, and the results of a ilot study on the oral bioavailability of captopril have been published. 1 P 2 Additional examples of the use of stable isotopes in measurements drug bioavailability include the study of stereoselective increases propranolol bioavailability during chronic dosinglB3 and the influence urinyq: pH and route of administration on meperidine disposition This technique also can be employed to quantify the conversion man. prodrugs into the active species in vivo, as illustrated in a study prodrugs of indomethacin. l E 5
of in of in of
on
Chap. 27
S t a b l e Isotopes
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e t al.
279
Conclusions S t a b l e i s o t o p e t e c h n i q u e s o f f e r a number o f s i g n i f i c a n t a d v a n t a g e s o v e r a l t e r n a t i v e methods f o r i n v e s t i g a t i o n s of d r u g m e t a b o l i s m and d i s T h e s e a d v a n t a g e s , some of w h i c h h a v e b e e n h i g h l i g h t e d i n t h i s position. b r i e f r e v i e w , a r e becoming more w i d e l y a p p r e c i a t e a a n d w i t h t h e growing a v a i l a b i l i t y of mass s p e c t r o m e t e r s s u i t a b l e f o r t h e measurement of i s o t o p i c a l l y - e n r i c h e d m o l e c u l e s , t h e u s e o f s t a b l e i s o t o p e s i n m e t a b o l i c and p h a r m a c o k i n e t i c s t u d i e s may b e e x p e c t e d t o i n c r e a s e s t e a d i l y i n t h e coming years. Although s t a b l e i s o t o p e s w i l l n e v e r r e p l a c e t h e i r r a d i o a c t i v e c o u n t e r p a r t s f o r many t y p e s of a p p l i c a t i o n , t h e y have become w e l l estab l i s h e d as i n d i s p e n s i b l e t o o l s f o r u s e i n m e t a b o l i c i n v e s t i g a t i o n s .
Aden~icPress, N&r Ymk, 1Y% Smidt. H. F'c;rsbl erd K. HRinzirger, Ms., Wable Isotopes. h c e d h g s of the 4th Internatioral ccnferencs," i&&.€r,k m t d a n , 1982. 5. W.P. Dncm and A.B. susul, Ms., " S y n ~ aand A M c a U m of hu@caUylabaled ' h p U X l s , f f Elsevier,
L. H A .
Apstcndam, 1933. 6. A.L. B~.llngem,T.A. W e , P.J. Denick ard O.S. hizhov, A d . h., 2,214R (193). E-, A. Dell and D.H. b d ,Ad.. o l e m a , 2, 363R (1$82)7. A.L. B 8. D. IktllidayandKJ. Reonie, CUn. sci., 63, 485 (1982). 9. R.M. Capriau and D.M. Ber, in f W & d ApplcatiOnS of hSpsCkxmhy. First &pphrmtmy Voluue," NRJ Ymkp 1W,p. 8%. C.R. WBUar and 0.C. Darnar, M S . 8 10. J, Sjb;all, in flAdvanm in h s Spctraretry," 8B, A. W l e , M., byden and Sen, Ladcn, lW,p. .@l 11. T.A. m e , Pnanmml. REV., 2,81 (1961). 12. T.A. Paillie, H. Ibgbs, ard D.S. &vies, in fqstahleI S O ~ ~H.-L.S , ~W ~ t , H. Fb;gtsl, and K. Heinzirgsr, Ms., Elsevler, Amstsrdam, 1482, p. 187. 13. N.J. Hasldns, fftomed. h s SpsCtrau., 9, 26q (1%?). d g 8 and I.L. Ims, Ms., ?he 14. D.R. Haddns, in %otapjs: Essential Ctmistty ard ApplicationS," I.A. W olenrical Society, kdn,lW,p.232. 15. W.J.A. V m u v d , in "Synthesis and A@icaticm of IsotCpblJy Iateled Ccmpnarls," W.P. Rncan erd A.B. 3Ms., Mw, Amdeadam, 1933, pa 77. 16. P.J. M.u-&v and H.R. Eullivan, Antn~.Rev. €lmmcol. T d m l . , 20, 0 (1933). 17. M. Bcheltaum, G.E. MI bruh and A. Sopngyi, C&. Ruumscaldn., I,4 9 (1982). 18. D.G. Gtt, YiynVeems w i t h Stable Isotopes of Cartcn, Nitrogen and Wgm," J& Mley and Scns, Nev York,
193.
19. A.P. Tulloch, Rog. Lipid Res., 22, 235 (1963). 20. A.R. Branfbn, M.F. b h t i s h , R.J. M, M.M. CaUahm, R. Fbtertmn and D.W. Y e s a i r , h g b h b . DispJs.,
2, 2% (1981). 21. M.M. callahan, R.S. b b e r b n , A.R. hnhn,M.F. IkCudsh a d D.W. Yesair, hg &tab. Disp3s., 2, 2ll (1963). 22. J.L. kdm,B. M h , M. Desage and B. selle, Ebsd. h s SpsCtrcm., 2, 169 (1W). 23. J.L. M, B. ~ibcn,M. ~esageand B. salle, in t1mw-b in MWS spectranew in iaochemistry, Mdicine and FZwhmena Research," 7, A. -0, M., Elsevier, A m s t d a ~ , 1981, p. 27. 2.4. M. Demge, J.L. M e r erd R. Int. J. hss Spectrcm. Ion ms., &, 85 (1583). 25. K. Mmna and S. Rib, @em. W. W. (Tolg.o), 2, P-43(1961). 26. T. C o r a m ~ ~T., ma.uta, S. Rib, A. W a n d s . Iguchi, olem. h. Bull. (T&o),a, 17&(1%1). 27. I. Ileyana, S. h q c c h i , I.Kotcal,T.H&iw,Y. I s h i i a n i I . T a b e , J.Pgric.FwdO1Em.,3l, 1061 (1982). 28. A.K. b,R.C. Ihmmmr and L. Ate, Life sci., 3,1075 (191). 23. G. Fa&&, R.C. lhnmrer, C.H. wan, D.A. W t z , E.W. Di %fen, and A.K. &, hg b b b . DispDs., 3 47 (1987). b h b . Msp3e;., 33. T. Ganmaru, H. ktamm, T. Fbuta, S. Rib, N. Y a , T. M p d i i , T. Semsshimn, 10, 542 (1462). 31. rblddmfeld, P. Carter, V . N . R a i n h o l d , S . B . l s n n e r I V a n d L . L . ~ , ~ . ~ s S F e c t r c m . , 5 , 5 8 7 (1978). 32. B.4. W k g , M. Axalscn, D.J. cdllins and J. Sf&lJ., J. bum-., 277, 453 (1961). 33. D.A. k&, L. Ropamfp, K.I.H. WFWams, H.J. Bndie and A.M.H. M e , Bocbm. hrmml., 2,7M (1982) 2. M.Tahm,Y.N&nta,H. hut&, T.Kurosaka, I.MddmandS.kkagaka,@em. Rrurm. W. TWO),^, 137 (1481).
w,
280 35.
36.
37.
3. 39. 40.
41
.
42. 43. M.
45.
16. L7. @. 49.
50. 51 * 52.
53.
54.
55. 56. 57. 58. 59. 60. 61. 62. 63.
66. 65. 63. 67.
68. CN. 73.
n. n. 73. 74.
75. 76.
n. 78.
79. 8).
01. 82.
83.
86. 85.
ab. 87. 88. €9. 9. 91.
Sect. V I
- T o p i c s i n Chemistry and Drug Design
A l l e n , Ed.
Bailie
Stable Isotopes
Chap. 27
e t al.
281
W t a f a , M. C l a e s a , J. Adline, D. V a n d m o r s t ard J.H. Finpie&, Dng kth D i p . , 2, 574 (1983). ktab. bpx., lo, 122 (1932). 93. T. hhlle, J.E. Oatis, Jr., U.K. W& ard D.R. Krapp, %. T. Wle, M.J. wilscn, U.K. k l l e ard S.A. Bai, h g &tab. Dlspss., 2, 54.4 (1933). (1981). 95. J.W. Lam ard S3.S. &uhn, J. Q'g. m.,&, $6. J.W. Lam ard S.M.S. U , J. (kg. Qlecn., Q, 851 (1982). 9. J.W. Lam, R.R. K c g e n e ard A.V. J C ~ WJ., Org. M.,Q, 2M7 ( 1 9 ) . 9. J.E. W, J. Rafter, G.L. Iarsen, J.A. htafsson ard B.E. htafssan, ktab. D i p . , 2, 525 (1931). 9. G.L. knrsan ard R. ftyhage, Xembiotioa, 2, 855 (1%). 103. G.L. Iarssn ard J.E. W, Xenobiotiar, 13, 115 (1907). 101. J.P. KUnmn, Ariv. byuol., &, L15 (14785. l a . D.B. Northup, Arm. Rev. Biochem., 103 (1981). 103. W.W. clelani, m &it. ~ e v . ~tiochem., 2,385 (1982). l a . J.T. Groves, S. Kriehmn, G.E. A v a r i a , T.E. h, in W a u i m t i c QamLshy,"D. C. Wnnh, Y. k a k a m i , I. 'Igt*lshi, Ms., American G-mical Society, bhingtm, D.C., 1583, p. 277. 105. M.H. Celb, D.C. Heimfnwk, P. &&&nard S.C. sllgar, W s t r y , 21, 370 ( 1 9 ) . 105. T. Sam, Y. Mzu, T. Tcda and N. oshino, Emg &tab. 2, 4$(1$81). 107. G.T. M-a, W.A.carlerd, B.J. Hcdson ard A.Y.H. Lu, J. Ebl. M., a 9 (199). l a . G.T. H-a, J.S. Wsh, G.L. Weris ard P.F. H o l l e n b e r g , J. Blol. W., 258, 1&5 (1983). ( & 2633 , (192). l@. T. &no, T. T d a ard N. Oshjm, J. Am. a m . Soc., I 110. M. CLeesen, M.A.A. h t a f a , J. Adline, D. Vezxlervorst ard J.H. pouFaert, fhvg kth Msps., l o ,667 (1932). 111. M. I6lsain, D.E. EMumdsm ard T.P. singer, Biochemistry, Zl, 595 (1W). 112. P.H. Yu, S. kclay, B. hvis ardA.A. Boultcn, B i h m . p)uurmcol., 30, 339 (191). XB, 195 113. R.B. kstkaempr ard K.P. FanzUk, Arch. B i h m . Bio*s., 114. D.C. and J.R. W l e s , loche em is try, 0, 3683 ( ~ ) 7 115. A.B. Fcater, in "Advances i n I h l g Research," B. T e s t a , Fd., A c a d m L c Press, I.d.m, 1984, in p s s . HofM,C.N. Kakker, A.M. Pietruszkiewicz, W.A. blhofer, E.J. CraCJoe, Jr., M. Lxl T c a r h i a r a ard 116. J.M. R. Kirschnenn J. M b m . , 26, 1650 (1983). 117. I.W. Taylor, C. Icamides, P. %ma, J.C. Tuner ard D.V. parke, Biochem. pharmncol., 32, 6!,1 (1933). 118. S.A. Barker, J.M. h t m , S.T. Qlristian, J.A. hti and P.E. Elcaris, Blcchm. Eharmocal., 31, 2513 (1982). 119. L.E. Q&, D.A. ~sden,P.H. YU, B.A. mvis and A.A. ~oulton,mochem. pharmncol., 3, 151971983). 120. C.T. h r i s h , K.M. W, L.E. Qck ard A.A. Boultcn, PsychopuumLcol.,8l, 122 ( 1 9 3 . 1. Ricchd. W v . , l9, 471 (1583). 121. C.T. M s h , A.J. Cmenshu ard A.A. Bmltcn, 122. M. Jamn, J.H. Kihuis, C.B. E m , V.C. Knick, G. Lamb, D.J. Nelscn ard R.L. Tuttle, in I I S t a h l e Isotqe~,~~ H.-L. Schmidt, H. F'drstel ard K. €k&jngm, Ells., Elsevier, Amsterdam, 1982, p. 217. 123. J.M. patel, W.P. Coldcn,.S.D. N e b ard K.C. Isitam, h g Metab. Disps., 2, 164 (1933). 103 (1982). 124. J.M. hien, S.A. Rice ard R.I. Wze, Anesthesiolag, 125. T. Hzutani, K. yamamoto ard K. Tajim, Tcudcol. A@. Ruumpml., 283 (1983). Ruunacol., 63, 170 (1933). 1%. R.D. Wnite, A.J. cendolfi, G.T. Bxkn ard I.C. Sips, Toxiwl. 127. R.V. hnchfhm ard L.R. Pohl, Taxlwl. A M . Pluumsool, 61, w)7 (1931). 128. M. hmdimkh, C . 4 . Kuo and J.B. h k , J. Toldcol. hvlrca. Wth, 8, 105 (191). 5019 (1981). 129. S.S. Hecht ard R. Yourg, Cancar Res., 1 3 . M.S. %a, D.G. SarpUi ard W. LijinsQ, &rdccgenssis, 2, 731 (1981). 131. W. LijW ard M.D. Reutar, Cancer Lett., & 273 (199). 132. W. Lijinsky, M.D. R m k , T.S. hvies and C Y . Pigps, J. ktl. Cancar Inst., 1127 (192). i j w ,Center Res., &, 2105 (1982). 133. G. Farrally, M.L. S W , J.E. SBaVedra and W. L 134. S.S. Mt, D. Lin ard A. Cas-, carcinogenesis, 4, 305 (1933). h m m w . Sci., 2, 392 (1931). 135. W.A. G s r l a r x i a r d M . L . hell., J. C 136. I. B j & k h , in "stable Isotopes," H.-L. W d t , H. Fmtel and K. Heinzinger, Ells., Elsevier, Ansterdam,
9. M.A.A.
so,
blm,
m.,
255,
k) .
,
2,
9,
m.
g,
9,
19% p.
593.
k b y , K. Nakanua, M.J. Kreek and P.D. &in, in "stable Isotops," €I.-L. S h i d t , H. F k t e l and K. kimirga, Ms., Elsevier, Amsterdnm, 1982, p.235. L. BEo-tilssan, i n V M c a l FhmmcoogY in Psychiaq," E. usdin, Fa., Elsavier, New York, 1981, p. 59. A.I. Millet, Prcateglaniins leuko~enesM., t3, 181 (1982). G. Idzu, M. Ishiard H. Mpmki, J. Qrromstogr., g,327 (1%). ,C.- J C. Lap, D. Iavene, J.R. Kiechel and J.J. Bassaller, Int. J. h s Spectrom. Ian @s.,
137. D.L. 138. 119. 140. 1Ll.
&, m
(1983).
kk ard J s L Raid, J. h. -., 5,114 (1983). Ye J A M t h s , J.P. FreemEln ard R.K. Nt&m, in ''Syntbda arrl A @ h t i ~ of Is~topicaUy IakLed cupnds," W.P. k c a n ard A.B. Susan, Ms., Elsevier, Amsterdam, 1983, p. 369. W.C. Plckett ard R.C. Anal. Bicchem., lll,115 (1931). D.M. anl M. ~ a i ,~ ~ o m e db~ . spcb., 2, Ln (1933). D.H.T. Cnien ard J.R. k y s , J. Agric. F a d &an., 30, 3% (1982). N. H. b h b ard L.A. W f , €Lo&. Miss Spctrcin., 2, l+5 (1%). R.H. White, Anal. Biochem., l & 369 (1931). J.R. Lbyl ard F.H. Field, B l d . Miss Spctmm., a, 19 (1931).
142. N.D.
14.3.
14.
145. 146. 147.
148. 1L9.
w,
28 2 1%. 151.
152. 153. 1%.
155. 156.
157.
158. 159. 160. 161.
162. 163. 1 Q.
165. 1%.
167. 163. I@. 170. 171. 172.
173. 174. 175. 1%.
177.
178. 179. 183. 181. 1 8 .
183.
14. 185.
Sect. V I
- Topics i n Chemistry and Drug Design
Allen, Ed.
Chapter 28. Drug Discovery at the Molecular Level: A Decade of Radioligand Binding in Retrospect Michael Williams and David C. U'Prichard Nova Pharmaceutical Corporation P.O. Box 21204, Wade Avenue Baltimore, MD 21228 Introduction - In the design of new therapeutic entities, the medicinal chemist works from information Erom known active compounds or from the structure of the putative neuroeffector agent thought to be involved in the etiology of the disease state under study.' The search for novel chemical entities which have therapeutic potential has become increasingly more complex;2 thus any rational strategy that will increase the probability O E finding a new drug is of considerable interest to those involved in drug research. One such approach is that utilizing radioligand binding assays , 3 which permits the evaluation of compounds for their direct interaction with cell surface recognition sites (receptors) independent of events distal to the actual recognition process. Structure-activity relationships generated using radioligand binding assays theoretically reflect the actual physicochemical properties required for receptor recognition, and thus provide information directly pertinent to the improvement of specificity and potency. In addition, receptor assays require only small amounts of compound and animal tissue, and are relatively rapid to perform. However, like classical pharmacological screens, these assays have certain drawbacks. They take no account of compounds that require metabolic activation, and generally measure only relative potency rather than efficacy, so that it is difficult to assess whether a compound interacting with a ligand binding site is an agonist or antagonist. These issues, and the advances and insights gained through the technique of radioligand binding, are the subject of this review. The Binding Technique - The radioligand binding assays developed to date are based on the fact that compounds known to interact with given receptors, when labeled to high specific radioactivity (10 Ci/mmole or H or 1251,bind to such sites in membrane or intact cell greater) with preparations from mammalian tissue with high affinity (Kd 'lo-' M) in a reversible, saturable manner. The difference between total and nonspecific binding, the specific binding, can then be used to measure compound interactions with the receptor. Ideally, specific binding should represent 70% or greater of the radioactivity bound. However, 50% specific binding, if not ideal, is acceptable. Binding assays where specific binding is only 20-30% of the total, present problems in 'signal-to-noise' reliability for routine screening purposes. In general for screening purposes, compounds with Icso values greater than M can be deemed 'inactive,' especially if at other recognition sites, they have I C s o values in the lo-' M range. The large majority o f radioligand binding assays make use of brain tissue because of its ease in preparation, and i t s richness in receptors
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on a weight per weight basis.3 Other tissues which have known physiological responses to a given agonist tend not to be good targets for radioligand binding because of the low density of receptors present as compared to CNS tissue.3 The ease with which CNS tissue can be utilized should not, however, preclude examination of peripheral tissue radioligand binding, since it is highly likely that subtle pharmacological and biochemical nuances exist among receptors of a given type as a function of their tissue source. For instance, despite considerable effort, the nicotinic antagonist, mecamylamine, which is exceedingly potent in autonomic ganglia, has no demonstrable binding in the CNS. 4 Furthermore, ligands specific for dopaminergic receptor subtypes in the CNS have not proven suitable for labeling the subtypes present in peri phera 1 tissues
.
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Validation of Receptor'Binding Assays There has been considerable concern that because of the non-physiological conditions used to examine radioligand binding, the sites labeled may represent in vitro artifacts. Such conditions include the use of hypoosmotic buffers; long incubation periods, which are inconsistent with the millisecond time courses related to the proposed physiological actions of neurotransmitter analogues and antagonists; and the use of non-physiological assay temperatures. These basic criticisms have some validity, but in general, like many in vitro test paradigms, reflect the interface between the complexity of nature and mankind's necessarily simplistic approaches. Several criteria have been proposed5 to validate a binding assay. These are saturability, indicating a finite number of binding sites; reversibility, consistent with a biological process involved in information transfer; correlation of binding affinity with the demonstrated concentration of the neurotransmitter or drug under physiological conditions; tissue and subcellular distribution of binding consistent with the known localization or target site of the ligand; agonist and antagonist pharmacology of the binding site corresponding to the known properties of the compound from other pharmacological tests; and finally, correlation of binding data with biological dose-response curves in identical tissue preparations. Most binding assays currently used fulfill a good many of these criteria. With these guidelines in mind, the status of the radiollgand binding assays currently in use will be considered. In many cases the initial observations of relatively simple recognition sites has progressed to the delineation of receptor subtypes. While offering exciting possibilities in terms of drug development, many of the assays for receptor subtypes have a weak pharmacological basis and in some instances do not fulfill many, if not all, of the criteria listed above. Binding and Receptor Subtypes
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a) Dopamine receptors Recognition sites for this monoamine, which is implicated i n the etiology of both schizophrenia and Parkinsonism, have been labeled with more than 20 iigands.6 Five or more distinct receptor subtypes have been described; of these, only those designated D-1 and D-2, which activate and inhibit adenylate 7 cyclase activity, respectively, are considered tangible entities. Apomorphine, spipelone, and domperidone label both D-1 and D-2 recognition sites. While newer antagonists such as S$H 23390 (1) appear to be selective for the D-1 receptor in vitro, in vivo this
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distinction is not apparent.” Newer agonists, such as RDS127’l (2) and PHNO (?), appear to be selective D-2 ligands. Putative dopamine autoreceptor agonists, such as 3-PPP and TL-99, have generated considerable interest Unfortunately, neither binding nor i,n viva pharmacological procedures have provided definitive evidence that either of these compounds has absolute selectivity for presynaptic dopamine Whether pre- and post-synaptic receptors represent distinct pharmacological, as opposed to anatomical, entities is a matter f o r further investigation.
I -
2
3 -
b) Alpha-adrenoceptors - A stronger association has been established between radioligand binding sites and functional receptor entities for epinephrine and norepinephrine. Alpha-1 and alpha-2 receptors were originally discriminated by location, being post- and pre-junctional respectively.16 Subsequently, alpha-2 receptors were shown to be negatively coupled to adenyiate cyclase, while alpha-1 receptors stimulate PI turnover and Ca2 entry.17 Among antagonist radioligands, peptide ergots (e.g. dihydroergocryptine) and phentolamine label both receptors with equal affinity,” while prazosin and the are highly potent and selective aminotetralone, HEAT (RE 2254)” alpha-1 ligands; WB-4101 i s partially selective for the alpha-1 receptor,’l while yohimbine and its isomer, rauwolscine, are somewhat Alpha-2 antagonists appear to accelerate alpha-2 receptor selective.” antidepressant- induced down-regulation of beta- and 5HT-2 receptors, and are currently the subject of intense intere~t.’~ The selective alpha-2 antagonist, the imidazoline, RX 781094, has been successfully used as a radi~ligand.’~ Several catecholamines and imidazolines selectively label high affinity states of the alpha-2 receptor.25 Preand postjunctional components of alpha-2 receptor binding have been identified, but no differential pharmacological characteristics have yet been observed.
c) Beta-adrenoceptors - These have been readily labeled with antagonists such as dihydroalprenolol, carazolol, iodohydroxybenzylpindolol ,26,27 cyanopindolol and pindolol.” Beta receptors were originally subdivided into beta-1 and beta-2 types on the basis of differential agonist and antagonist pharmacology and tissue location. Ligand binding studies corroborate the pharmacological subclassification quite precisely. Since the above mentioned antagonists are not subtypeselective, estimates of subtype relative abundance and competitor preference have relied on the generation of biphasic competition curves, with computer-assisted curve-fitting. These have not, however, been without problems; IPS 339 (4-1, a beta-1 selective antagonist as judged by computer assisted binding data3’,has little subtype selectivity in other tissue receptor assays. 3 1
’’
28 6 -
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OH
..
-4 I
5
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The biochemical characterization of beta receptors and their coupling to adenylate cyclase has been intensively studied 32 using agonist radioligands (epinephrine, hydroxybenzylisoproterenol) to label high affinity receptor conformations, especially of the beta-2 receptori3 more hydrophilic antagonists (e.g. CGP 12177 (5))to label cell surface, but not internal, receptor populations (especially useful for examining regulation of beta receptors in cell culture); 34and photoactivated, irreversibly binding antagonists such as p-azidobenzylcarazolol, to tag the receptor during purification to apparent homogeneity.35
-
d) Serotonin receptors These have been labeled with serotonin itself and LSD,36 spiperoneg7using cortical tissue, and more recently, ketanserin.38 Serotonin receptors in brain tissue appear to exist in two distinct forms termed 5HT-1 and 5HT-2. The former, which can be labeled with serotonin, have been further subdivided Into 5-HT-1A and 5-NT-1B sites.39 Both spiperone and ketanserin label 5-HT-2 receptors in cortical and other brain regions. Interestingly, the pharmacological actions of ketanserin are mediated via alpha-1 receptor blockade. 4 0 The 5-HT-2 receptor has been implicated in depression, chronic treatment with the tricyclic, amitriptyline, reducing the number of 5-HT-2 binding sites in rat frontal cortex.41 The physiological relevance of the receptor has, however, been questioned since atypical antidepressants such as mianserin can reduce the density of serotonin-2 sites when given acutely.42 The relationship of central serotonin binding sites to those designated as M (excitatory; blocked by morphine) and D (inhibitory; blocked by dibenyline) is unclear at the present time.
e) Muscarinic cholinergic receptors - Many highly potent and selective antagonlsts have been used to label muscarinic receptors in central and peripheral tissues, including N-methylscopolamine, 3-quinuclidinyl benzilate (QNB), N-methyl-4-piperidinyl benzilate, and the covalent ligand, benzylcholine mustard. Excellent correlations have been obtained between affinities at these binding sites and pA values in classical organ preparations such as guinea pig ileum.43 Agonist competitors at antagonist binding sites exhibit complex, heterogeneous interactions manifested by very "shallow" competition curves; these can be partitioned into "super- high" (SH), "high" (H) and "low" (L) affinity states. Agonist ligands (*-methyldilvasene, acetylcholine, oxotremorine-M) preferentially label, and improve resolution of, the SH state of the receptor. 4 4 The relative proportion of these three "states" was found to differ markedly in different tissues and brain areas. 4 4 I t has become apparent, however, that there exist two pharmacologically distinct types of muscarinic receptor, 4 5 and antagonist designated M-1 and M-2. The agonist McN-A-343 are selective for M-1 receptors; no M-2 receptor pirenzepine selective agents are known. Given the apparent tissue specificity for the two receptor types (M-1 in most brain regions and autonomic ganglia;
(L),46
(a),
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M-2 in peripheral effector organs such as heart), there is considerable interest in the development of subtype-selective agents.
(CH,),~CH~C=CCH,OCONH
-Q \
6
CI 7
8
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f ) Nicotinic cholinergic receptors Acetylcholine (ACh) receptors sensitive to the alkaloid nicotine have been labeled in Torpedo tissue and in autonomic and central nervous system using radiolabeled ACh,47 nicotine 4aand alpha-bungarotoxin (BTX).49 BTX is, however, not a good ACh antagonist in mammalian nervous tissue. 5 0 The classical ganglionic blockers, hexamethonium and mecamylamine do not displace these ligands from CNS tissue,5 0 a finding which may indicate that agonist and antagonist binding sites are distinct entities. Mecamylamine shows no speci€ic binding in brain tissue. The neuromuscular blocker, dihydro-beta-erythroidine (DBE; (S)) does label central nicotinic recognition sites. DBE binding is insensitive to ganglionic blockers, suggesting that the nicotine recognition site in mammalian brain is more selective for neuromuscular type than ganglionic receptor blockers.4
g) GABA receptors - These have been labeled with tritiated GABA51 and muscimol.’L Electrophysiological and binding studies in mammalian tissue have led to the discovery of two distinct types of GABA re~eptor:~ the GABA-A site, which is labeled by GABA and muscimol and which is sensitive to blockade by bicuculline; and the GABA-B site,which can be selectively labeled by baclo€en and is insensitive to blockade by bicuculline. The GABA-B receptor may be linked in a modulatory manner to a membrane bound adenylate ~ y c l a s e . ~ ~ h) Excitatory amino acids - As many as four excitatory amino acid receptor subtypes have been described. These are sensitive to the agonists, N-methyl-D-aspartate (NMDA), kainic acid, quisqualic acid and APB (2-amino-4-phosphonobutyric acid) .5 Excitatory amino acids have been termed ‘excitotoxins’and can cause cell death in a manner similar to that seen in Huntington’s disease.55 In general, the affinities of the various ligands used to label excitatory amino acid recognition sites in mammalian brain tissue are low compared with other putative neurotransmitter recognition sites, with Kd values around M.55
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i) Purinergic receptors Receptors for the purines may be divided into P-1 and P-2 subtypes.’b P-1 receptors are adenosine-sensitive and cyclase-linked, while P-2 receptors are ATP-sensitive, affect prostaglandin synthesis, and have no effect on cyclic AMP production. Two subtypes of the P-1 receptor exist; 57’5%he A-1 or Ri and A-2 or Ra, which, respectively, inhibit o r activate adenylate cyclase. Both A-1 and A-2 receptors are sensitive to blockade by xanthines such as caffeine and theophylline. To date, ligand binding assays have only been described for the A-157’5%nd P-2 5 9 receptors. Binding studies have led to the description of further subtypes which show species 60 dependence. Functional differences in A-1 receptor function have
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also been reported.61'62 The 5'-N-ethylcarboxamide. of adenosine, NECA, may show preferential selectivity for A-2 sites. 6 3
-
j) Histamine receptors Mepyramine has been extensively used to label H-1 receptors,ss while tiotidine has been shown to demonstrate specific binding consistent with the labeling of an H-2 receptor. 6 5 The recently described irreversible H-2 blocker, L-643441 may be an ideal ligand for this receptor subclass.66 In view of the profound effects of histaminergics on CNS tissue function and the search for second generation antiulcer agents, i t seems highly appropriate that a reliable H-2 binding assay should be available for drug screening.
(z),
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k) Opiate receptors Binding assays for opiates have contributed significantly to the discovery and characterization of the enkephalins and endorphins. At least four, and perhaps five opiate receptor subtypes are amenable to study by radioligand binding techniques. 6 7 The mu-receptor can be labeled with dihydromorphine (DHM) and by the enkephalin analog, DAGO. The delta receptor, which is involvedsin spinal analgesia, is labeled with the enkephalin analog, DADLE. The kappa receptor, thought to be involved in the psychotomimetic effects of some opiate partial agonists, es6 Becially benzomorphans,70can be labeled with ethylketocyclazocine (EKC) or bremazociyf (10).The sigma opiate receptor can be labeled with SKF 10,047. The epsilon receptor can be labeled with beta-endorphin, although this is controversial at a newly introduced analgesic agent, has this time. Meptazinol been reported to interact with a subtype of the mu receptor, mu-1 which 72 is responsible for the central analgesic actions of the opiates. BY inference, the mu-2 receptor is responsible for the respiratory depression and inhibition of gastric motility associated with opiate action. Radiolabeled meptazinol has not proven to be a satisfactory ligand because of its lipid solubility.
(c),
Hd 9
0
lo
1) Benzodiazepine (BZ) receptors - Using 311-BZs, up to three subclasses of BZ receptors have been described;73 two central receptors, BZ-1 and BZ-2, which have been reported to mediate the anxiolytlc and sedative/ataxic actions of the BZ's, respectively,74 can be labeled with clonazepam; a third peripheral type receptor, which has low affinity for clonaeepam, can be labeled with the BZ, Ro 5-4864.75 Whether the two central receptors are distinct entities or different forms of a single receptor remains to be determined.76 Ro 15-1788, the BZ antagonist or 'inverse agonist' can be used to label central BZ receptors.77 Using the central BZ binding assay, several non-BZs with anxiolytic potential have been reported. These include the pyrazoloquinoline, CGS 9896 78and the triazolopyridine, CL 218872,74 which displace BZ binding; and the pyrazolopyridine, etazolate,79 which enhances binding. The BZ receptor is somewhat unique in that it exists as a receptor complex involving a GABA recognition site and chloride ionophore, the latter being responsible for the physiological actions of the B Z S . ~ ~
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m) Peptide receptors - Many neuromodulatory peptides in addition to the enkephalins and endorphins have been discovered in both the gut and brain which have a common embryological,origin. Radioligands have been used to identify the cell surface recognition sites for peptides. The classical peptides first studied were insulin 5 and glucagon; assays for Substance P, cholecystekinin, neurotensin, bombesin, angiotensin, and arginine vasopressin have been described. 8 0 Although some progress has been made in delineating substance P receptor subtypes using binding criteria, radioligand binding assays for peptides and other related entities such as the leukotrienes 8 2 should at the present time be described as recognition site, rather than receptor assays. Nonetheless they are exceedingly useful screening tools.
!2
!3
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n) Receptors-for calcium agonists and antagonisAs- While calcium antagonists bind to ion channels and cannot be considered as receptor antagonists per se, the binding of three classes of such agents has been described: the dihydropyridines 8 3 (DHP) (e.g. nifedipine and nicardepine); the nitrilesE4 (e.g. verapamil and D-600); and the benzothiazepines (e.g. diltiazem). Binding assays for the DHPs have been characterized in brain and visceral tissue. Verapamil, D-600 and diltiazem are weak, partial displacers of DHP binding, which may imply the existence of either distinct calcium channels or a 'receptor' complex with different recognition sites. A binding assay for verapamil has been described re~ently.'~ Diltiazem can stimulate DHP binding and can reverse the inhibitior, of binding seen with tiapamil. 8 6 More recently DHPs have been described which facilitate calcium entry including Bay K 8644 8 7 (2) and CGP 28392 (2). 8 8 While calcium antagonist binding sites are present in brain, their pharmacological significance is unclear since they do not appear to affect calcium fluxes in synaptosomal preparation^.^^
-
Potpourri A variety of ligand binding sites are unassociated with defined receptors but are useful in the drug screening process. These include the proteins, labeled by antidepressants, which are associated with reuptake processes for 5-HT ( H-imipramir~e~,~H-paroxetine 9 9 and norepinephrine ( H-desmethylimipramine 1. Other potentially important sites are those for the psychotomimetic phencyclidine (PCP) 9 3 and the atypical antidepressant, trazodone. 9 4 The potential significance and usefulness of these and other such sites must remain in limbo until a relevant biochemical or cellular function has been attributed to them. Specific 'drug' recognition sites need not, however, be invoked for each and every novel compound with therapeutic potential. The search for unique binding sites using radiolabeled
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probes and tee extrapolation to a search for the "endogenous ligand" may lead to unnecessary complications. A potentially useful therapeutic agent may have a distinct 'non-receptor,' but still locus specific, site of action. An example of this in the anticonvulsant/central sympathomimetic/anxiolytic MK 801g5 which has in vivo activity but has no appreciable activity in several ligand binding assays. The pharmacological activity of a compound may also be dependent on activity at more than one recognition site. The example of ketanserin, an in vitro ligand for the 5HT-2 receptor but a alpha-1 antagonist in vivo has been cited.40 The is another instance where the binding serotoninomimetic, MK 212 (g), profile of the compound and its in vivo activity do not c ~ r r e l a t e . ~ ~ ' ~ ~
(E),
Ligand binding methods have facilitated the study of receptor alterations in disease states, whether secondary or presumptively etiological examples incude human postmortem brain tissue obtained from patients with neurodegenerative (Parkinsonism, Huntington's Chorea, Alzheimer's Disease) or psychiatric (Schizophrenia) disorders and in formed blood elements obtained from patients before, or during, drug treatment (e.g. platelet alpha-2 receptors and H-imipramine binding sites; white cell beta receptors). 9 9 Especially in the realm of neuropsychiatric disorders, a variety o f receptor data has been amassed that is at this stage primarily phenomenological and as yet of little value with regard to etiological issues or rational therapy.
,'*
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Conclusion The future of radioligand binding methods for research on receptors and their function can be predicted to follow several directions: (a) the increased use of receptor autoradiography with computer-assisted quantitative imaging to combine biochemical precision with high morphological and spatial resolution;100 and (b) an increased emphasis on isolation and purification of receptors, facilitated by the use of covalent affinity probes, to allow controlled reconstitution of receptor-effector systems, and to facilitate development of receptor antibodies which in turn will be used as probes to determine specific receptor RNAs and DNAs. This will ultimately lead to a better understanding of receptor expression, and the effects of drugs thereon, and to cloning of receptors in host cells on a scale large enough to provide sufficient material to study in depth the physico-chemical properties of the ligand-binding "active site" of receptor proteins, allowing for truly rational drug design.
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292 56. 57. 58,
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i n Chemistry and Drug Design
A l l e n , Ed.
G. Burnstock in "Regulatory Function of Adenosine," R . H . Berne, T.W. Rall and
R. Rubio, Eds., Nijhoff, Boston, 1983, p. 49. J.W. Daly, J. Med. Chem., 3 . 1 9 7 (1982). M. Williams, Handbook Neurochem., a.1 (1984). R.M. Levin, R. Jacoby and A.J. Wein, Mol. Pharmacol.,23,1 (1982). K.M.M. Murphy and S.H. Snyder, Mol. Pharmacol., 22,250 (1982). T.V. Dunwiddie and T. Worth, J. Pharmacol. Exp. Ther., =,70 (1982). J.J. Katims. 2. Annau and S.H. Snyder. J. Pharmacol. Exp. Ther., 227.167 (1983). S-M. Yeung and R.D. Green, PharmacoloRist, 23.184 (1981). R.S.L. Chang. V. Van Tran and S.H. Snyder. EUK. J. Pharmacol.. 3 . 4 6 3 (1978). G.A. Gajtkowski, D.B. Norris, T.J. Rising and T.P. Wood, Nature. 3 2 , 6 5 (1983). M.L. Torchiana. R.J. Pendleton. P.C. Cook, C.A. Hanson and B.V. Clineschmidt. J. Pharmacol. Exp. Ther., 224,514 (1983). M.C.C. Cillan and H.W. Kosterlitz, Brit. J. Pharmacol., 77,461 (1982). L.E. Robson and H.W. KosteKlitZ, Proc. Roy. SOC. London. Series B. 205.425 (1979). G.W. Pasternak. Proc. Aat. Acad. Sci. U.S.A.. 77. 3691 (1980). R.W. Foote and R. MauKeK. EUK. J. Pharmacol., E.99 (1982). G.W. Pasternak, M. CaKKOll-BUatti and K. Spiegel, J. Pharmacol. Exp. Ther.. 2 , 1 9 2 (1981). K. Spiegel and C.W. Pasternak, J. Pharmacol. Exp. Ther.. 228.414 (1984). M. Williams, J. M&d. Chern.. g.619 (1983). A.S. Lippa, D.J. Critchett, M.C. Sano, C.A. Klepner, D.W. Creenblatt, J. Cooper and B. Beer, Pharmacol. Biochem. Behav., 10.831 (1979). P.J. Marangos, J. Patel. J-P. Boulenger and Clark-Rosenberg, Mol. Pharmacol., 22.26 (1982). 1.L. Martin, C.L. B r o w and A. Doble. Life Sci..L2.1925 (1983). C. Braestrup, R. Schmiechen, G. Neef. M. Nielsen and E.N. Petersen, Science. 216,1241 (1982). =okiyama and T. Glenn in "Abstracts of Papers;' 184th National Meeting of the American Chemical Society, Kansas City, MO.,Sept. 12-17, 1982. Abstr. MEDI 58. M. Willlams and E.A. Risley. Life S c i . , 24,833 (1979). K-J. Chang and P. CuatreCaSaS i n "Brain Peptides," D.T. Krieger. M.J. Brownstein and J.B. Martin, Eds., Wiley Interscience, New York, 1983, p. 565. C-M. Lee, L.L. Iversen. M.R. Hanley and B.E.B. Sandberg. Naunvn- Schmiedeberg's Arch. Pharmacol., 318,281 (1982). R.F. Bruns, W.J. Thomsen and T.A. Pugsley. Life Sci., 33,645 (1983). R.A. Janis and D.J. Triggle, J. Med. Chem., 2 , 7 7 5 (1985). R.J. Miller, Life Sci., in press (1984). J-P. Calizzi, M. Fosset and M. Lazdunskl, Biochem. Biophys. Res. Commun.. 118.239 (1984). R.J. Could, K.M.M. Murphy and S.H. Snyder. Proc. Nat. Acad. Sci. U.S.A.,=3656 (1983). M. Schramm, G. Thomas, R. Towart and C. Franckoniak, Nature, 303.535 (1983). P. Erne, E. Burgissen, P.R. Buhler, B. Dubache, H. Kuhnis, M. Meier and H. Rogg, Biochem. Blophys. Res. Commun., 118,842 (1984). I. Shallaby, S.A. Freedman and R.J. Miller, SOC. Neurosci. Abstr.. 2,565 (1983). R. Raisman, M.S. Briley and S.Z. Langer, Nature, 281,148 (1979). E.T. Mellerup, P. Plenge and M. Engelstoft, Eur. J. Pharmacol., 9 6 , 3 0 3 (1983). C.M. Lee, J.A. Javitch and S.H. Snyder, J. Neurosci., 2.1515 (1982). R. Quirion and C.B. Pert, Eur. J. Pharmacol., E , I 5 5 (1982). D.A. Kendall, D.P. Taylor and S.J. Enna, Mol. Pharmacol., 2 , 5 9 4 (1983). B.V. Clineschmidt, M. Williams, J.J. Witoslawski, P.R. Bunting, E.A. Risley and J.A. Totaro, Drug Development Res., 2,147 (1982). B.V. Clineschmidt, Gen. Pharmacol.. 10.287 (1979). M. Williams and E.A. Risley, Fed. P r z . , 2 . 1 3 3 0 (1982). R.W. Olsen. P. Van Ness. C. Napias, M. Bergman, and W.W. Tourtelotte, Adv. Biochem. Psychopharmacol., 21.451 (1980). M.S. Briley, S.Z. Langer, R. Raisman, D. Sechter and E. Zarifan, Science, =,30(198G) W.S. Young and M.J. Kuhar. Brain Res., 179,255 (1979).
Chapter 29. Forskolin and Adenylate Cyclase: New Opportunities in Drug Design Kenneth B. Seamon, National Center for Drugs and Biologics, FDA Bethesda, MD 20205 Cyclic AMP is synthesized by the membrane-bound enzyme adenylate cyclase which is present in almost all mammalian tissues. Hormonal regulation of adenylate cyclase is initiated by the hormone binding to cell surface receptors. The resulting hormone-receptor complex interacts with either stimulatory regulatory proteins, (Ns), or inhibitory regulatory proteins, (Ni), which bind guanine nucleotides and interact with the catalytic subunit (C) of adenylate cyclase to modulate the enzyme activity. ATP
CAMP
-FO
RS KO LI N
/ /
GTP > 40nm
INHIBITION
STIMULATION
Hormone receptor agonists and antagonists have been used for years as therapeutic agents which affect cyclic AMP levels by acting at receptors. Although there has been much progress in the isolation and purification of the hormone receptors and the regulatory proteins, Ns and Ni, there has been less progress in the purification and characterization of the catalytic subunit of adenylate cyclase. Forskolin (l), the major pharmacologically active diterpene isolated from the roTts of Coleus for~kohlii,l-~ can directly activate the
-1
0
H3C
15
CH3 OH
catalytic subunit of adenylate cyclase, and is offering new insights into the regulation of this complex enzyme system. This review will discuss the unique properties of forskolin as an activator of adenylate cyclase which are of potential interest to the medicinal chemist.
ANNUAL REPORTS IN MEDICINAL CHEMISTKY- 19
Copyright 0 1984 by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0-12-0405 19-9
29 4
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Drug Design
Allen, Ed.
Activation of Adenylate Cyclase - Metzger and Lindner first demonstrated that forskolin activates adenylate cyclase in rabbit heart Subsequent studies demonstrated that forskolin and liver membranes .4 9 could activate adenylate cyclase in rat brain membranes,6 and in membranes from most mammalian tissue^.^ The EC50 for forskolin activation is generally between 211M and 101~M. The maximal extent of activation varies among different tissues and cells. Adenylate cyclase in membranes from rat liver is activated 20-f0ld,~while adenylate cyclase in turkey erythrocyte membranes is activated only 3-fold over basal level^.^ Forskolin activation of adenylate cyclase is observed in the presence of cyclic AMP phosphodiesterase inhibitors and does not require the presence of calcium or calmodulin.6 Forskolin activates solubilized adenylate cyclase with similar EC50's and maximal activations as observed for membrane-bound enzyme .l0-12 Forskolin increases the Vmax of adenylate cyclase with little or no effect on the Km for the substrate, MgATP or MIIATP.~~Forskolin has no effect on the Ka for MgC12 in activating liver or platelet adenylate cyclase.*r13 Forskolin can protect adenylate cyclase in membranes from human platelets, 549 lymphoma cells, and rat brain against thermal denaturation, proteolytic inactivation, and inactivation with N-ethylmaleimide (NEM) .I3 Solubilized preparations of adenylate cyclase are also protected against thermal denaturation by forskolin.l1 These include preparations which do not contain functional Ns protein. Aden late cyclase from sperm or testis is not stimulated by f~ r s k o l i n . ~ ~ These enzymes are not hormonally responsive and are not associated with Ns or Ni proteins. Forskolin can activate sperm membrane adenylate cyclase when extracts from human erythrocyte membranes are added to sperm membranes; however, it is not clear if the reconstitution is revealing adenylate cyclase activity in the human erythrocyte membrane or adding back a factor required for forskolin stimulation of the sperm membrane adenylate ~yclase.~5,16 Aden late cyclase from invertebrates such as mollusks 17-20 and insects21sH2 is stimulated by forskolin. Adenylate cyclases from 5. Pertussis,23 E. C~liN , e~u r o ~ p o r a ,and ~~ D. Discoidium7 are not stimulated by forskolin or Ns. Interaction with the Catalytic Subunit - Forskolin stimulates adenylate cyclase in membranes from the S49 c c- 1 phoma cell about 20-fold with an EC50 of about 25 micromolar.10~23,25-~ As this mutant of the S49 lymphoma cell line does not contain a functional Ns subunit, it was proposed that forskolin is activating adenylate cyclase by an interaction at the catalytic subunit or other closely associated protein.1° Forskolin can activate other adenylate cyclase preparations which are unresponsive to hormones or guanine nucleotides and do not contain functional Ns protein. These include detergent solubilized preparations of aden late cyclase from rat striatum,ll cortex,12 heart,12 and liver.3B In the absence of a functional Ns protein, it is necessary to include Mn2+ in the assay to measure the activity of the catalytic subunit. Forskolin does not require Mn2+ to stimulate the catalytic subunit.
Chap. 29
Forskolin and Adenylate Cyclase
Seamon
295
The 7-0-hemisuccinate-7-desacetyl derivative of forskolin has been coupled to Sepharose to form an affinity resin.12 Solubilized adenylate cyclase from rat brain and heart, but not pigeon erythrocytes, binds to this resin and can be eluted from the resin when forskolin is included in the buffer. Preparations of adenylate cyclase devoid of Ns activity are recovered from this column consistent with the proposai that forskolin binds to the catalytic subunit(s). Adenylate cyclase preactivated with guanosine-5',3,y-imidodiphosphate (GppNHp) also binds to the forskolin-Sepharose column and elutes from the column as a complex of the catalytic subunit(s) and the activated Ns protein.31
-
Potentiation of Forskolin Stimulation by the Ns Protein Forskolin stimulation of adenylate cyclase can be potentiated by guanine nucleotides and the Ns protein. Forskolin stimulation of adenylate cyclase in wild type S49 lymphoma cell membranes exhibits a time lag and biphasic kinetics indicative of low (Ka=22.OllM) and high (Ka=0.35uM) affinity sites of a ~ t i o n . ~ Forskolin ~,~~ stimulation of adenylate cyclase in S49 cyc- membranes, which do not contain Ns protein, has kinetics consistent with one site of action with a Ka of 244pM. The high affinity component for activation of the adenylate cyclase in S49 membranes by forskolin requires the Ns protein. The lag time in activation of the wild type S49 adenylate cyclase by forskolin is also associated with the presence of the Ns protein and is not observed in the presence of isoproterenol. Preparations of the catalytic subunit which do not have functional Ns protein can be stimulated by the addition of Ns and GppNHp and forskolin can potentiate this stimulation.ll This potentiation requires that forskolin, Ns, GppNHp, and the catalytic subunit be present at the same time. Preactivated Ns (Ns-GppNHp) and forskolin are only additive in their stimulation of the catalytic subunit when they are present in the assay at the same time. Forskolin can potentiate GppNHp stimulation of adenylate cyclase in membranes from some tissues. This potentiation can be observed when the effect of the Ni protein is suppressed by treatment with pertussis toxin,33 or by including manganese chloride in the assay medium.34 Activation of Cyclic AMP Generation in Intact Cells - Forskolin activates adenylate cyclase in intact cells and tissues with similar characteristics as those observed for activation of the enzyme in membranes and solubilized preparations. These include preparations of rat35 and human36 adipocytes, human latelets,37 tissue slices from brain and other peripheral tissues,315 and various endocrine and secretory tissues.39 Forskolin stimulates adenylate cyclase in S49 lymphoma cells,25p32 rat astrocytomas 40341 rat pheochromoc toma cells,42 cultured pituitary cells ,43-28 cardiomyocytes,49 9 51; cultured leydig cells,51 and cultured kidney cells. 52 * 53 Forskolin increases intracellular cyclic AMP rapidly with an EC50 of about 10uM, and results in elevations of cyclic AMP over basal levels which range between two and fifty-fold, depending on cell type and tissue. Increases in intracellular*cyclic AMP by forskolin occur rapidly and are reversible. Increased levels of cyclic AMP are maintained in tissue culture over long periods of time when forskolin is kept in the culture media. Cyclic AMP phosphodiesterase inhibitors augment the increases in intracellular cyclic AMP elicited by forskolin.
29 6
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Allen, Ed.
Paralleling membrane studies, forskolin elicits increases in intracellular cyclic AMP in the cyc- mutants of the S49 lymphoma cell line which are about ten-fold less than the increase elicited in wild type S49 lymphoma cells.25 Similar results are seen in the H21a mutant which contains a lesion in Ns impairing its coupling to the catalytic subunit. A maximal stimulation of cyclic AMP synthesis by forskolin in S49 cells requires the presence of a functional Ns protein. Forskolin stimulation of cyclic AMP synthesis in C6-2B rat astrocytoma cells is decreased after the cells are grown in the presence of cycloheximide, a protein synthesis inhibitor.4O This occurs without the loss of isoproterenol or cholera toxin stimulated adenylate cyclase. It was suggested that the cycloheximide blocks the synthesis of a protein which has a high turnover rate and is required for forskolin stimulation of adenylate cy~lase.~o
-
Potentiation of Hormone Action Low concentrations of forskolin (50uM) inhibits basal, hormone-stimulated GppNHp stimulated, and forskolin stimulated adenylate c y ~ l a s e . ~ ~ This inhibition is not competitive with forskolin and has been suggested to be due to the interaction of the metal ion at a cation binding site on the catalytic subunit. Forskolin activation of adenylate cyclase can be inhibited b high concentrations of detergents and low molecular weight alcohols.74 ~~5 Inhibition of forskolin stimulated adenylate cyclase by ethanol can be observed at 0.2M and results in 50% inhibition at 1.OM. Butanol and propanol are more effective at inhibiting forskolin stimulated adenylate cyclase than ethanol. Dimethyl sulfoxide inhibits the stimulation of adenylate cyclase by forskolin much less than ethanol .75 Physiological Effects of Forskolin - Forskolin elicits physiological effects consonant with its ability to stimulate adenylate cyclase and increase intracellular cyclic AMP-. This has been studied in many different tissues and cells and a complete description of all systems is beyond the scope of this review. Secretory responses which are elicited by forskolin include: chloride-secretion across rat colon descendens,76 acid and pepsinogen secretion from astric gland ,77 ~ 7 8prolactin secretion from pituitary cells y43 *4f luteinizing hormone secretion from pituitary,79 renin secretion from perfused kidney,80 insulin release from pancreatic cells,81 ACTH release from pituitary cell^,^^,^^ and amylase secretion from pancreatic acinor cells .82 Other responses which are elicited by forskolin and have been associated with increased intracellular cyclic AMP include li 01 sis in adipocytes,35 ~ 3 6 ~ 5 4 inhibition of platelet a g g r e g a t i ~ npT2 ,~~ ,83 relaxation of smooth muscle,2,3,84 and stimulation of steroidogenesis.57 ,85 Forskolin increases cyclic AMP content in oocytes and inhibits progesterone induced meiosis. 86-92 Forskolin increases sodium transport in cultured epitheliag3 and bicarbonate transport in choroid plexus.94 Reduction of intraocular pressure b forskolin has been observed in rabbit, human, and monkey eye.95,9g Forskolin stimulates transcription of the prolactin gene in pituitaryg7 and increases the biosynthesis of enkephalin in bovine chromafin cells .98 Forskolin elicited physiological responses are usually potentiated by the natural hormone consistent with the hormonal potentiation of forskolin elicited increases in cyclic AMP. Forskolin has been shown to affect enzymes other than adenylate cyclase indirectly. Treatment of rabbit cardiac slices with forskolin results in the inhibition of the membrane bound Na+ K+ ~ ~ p ~ ~ ~ . 9 9
29 8
Sect. VI
- Topics in Chemistry and Drug Design
Allen, Ed.
The inhibition of the ATPase is probably due to a cyclic AMP mediated phosphorylation. Tyrosine hydroxylase is activated in striatal slices treated with forskolin,lOO also due to a cyclic AMP mediated phosphorylation. Structure Activity Relationships for Forskolin Analogs - Forskolin makes up 1% of the dry weight of the roots of Coleus forskohlii and is the major diterpene that is isolated from the plant. Other structurally similar diterpenes that occur naturally in the plant include 6-acetyl-7-desacetylforskolin (k), 7-desacetylforskolin (5), 1,9-dideoxyforskolin (7), and 9-deoxyforskolin @). A numEer of semisynthetic derivatiTes of forskolin have been prepared.101,102 These and the naturally occurring analogs of forskolin have been tested for their ability to activate adenylate cyclase.103
2 3
4
OH OH OH
5 OH OH Z H OH
OH OH OH OH OH
OH
OCOEt OC0,Et OSOZ-&Me-Ph OH OH OCOMe OCOMe
OH
OCOMe
OH
OH 'H
OH OH
OCOMe OCOMe
OH OH
0 0
OH OH OH OH
OCOMe OH
CH-=H, CWH, CHXH, cn=H, CH=CH2 CH=CH, CMH, CH=CH,
H H
9 o\
The 1-and 9-hydroxyl groups define an important area for forskolin's action. The 1,9-dideoxyderivative (L) is totally inactive and the 1,9,6,7-dicarbonate (9), and 1,9 sulfonate are very weak at activating adenylate cyclase. OtFer structurally related compounds which are unable to activate adenylate cyclase are 1,6-diketoforskolin, the 14,15-oxide (12), virescenol-B, abietic acid, podocarpic acid, retene, gibberellin, and other diterpenes(l3-15) - - 023983 None of the inactive diterpenes antagonize forskolin stimulation of adenylate cyclase. Derivatives of
Chap. 29
Forskolin and Adenylate Cyclase
Seamon 299
forskolin which are partially active include a number of 7-acyl 3) derivatives. The most active of these include the 7-ethylcarbonate ( and the 7-propionate (2) which are almost equipotent with forskolin. The 7-formyl, 7-desacetyl and 7-hemicsuccinate are less potent than forskolin with EC50's that range from 50pM to 150pM. The 7-tosyl derivative (4) is much less potent than the other 7-acyl derivatives. The 14,15-diKydroforskolin (11) and llphydroxy-forskolin (10) are less potent than forskolin w z h EC50's of about 50 pM. The activity of forskolin analogs does not correlate with lipophilicity.
(z),
12-t3H] forskolin (sp. act. 27 Ci/mmol) has been s nthesized and used to determine forskolin binding sites in membranes 12-[3H] forskolin binds to rat brain membranes with high affinity, Kd = 27nM, Bmax = 275 fmol/mg protein. The ability of forskolin analogs to compete for these binding sites correlates with their ability to activate adenylate cyclase.
.Io4
Therapeutic Potential - Forskolin is a potent hypotensive agent due to its peripheral vasodilatory properties. Reduction in blood pressure is observed in dogs (20ug/kg i.v.), renal hypertensive rats (50uglkg i.v.), and spontaneous hypertensive rats (10 mg/kg, p.0.) .2s105 Forskolin is also a potent positive inotropic agent as observed in isolated guinea pig heart and atrial preparations, and in dog and cat hearts ~ i t r o ,Io5 . ~ Cardiac output in anesthesized dog was increased at doses ranging from 5ug/kg to 100ug/kg, i.vm2 Forskolin antagonizes histamine or acetylcholine induced bronchoconstriction making it a potent bronchodilator.lo6 ,Io7 Forskolin inhibits platelet aggre ation in vitro and -in vivo, making it a possible anti-thrombotic agent.3$,72,83Forskolin produces a rapid and long lasting reduction in intraocular pressure upon topical application to the eye, and would appear to be a valuable agent to treat glaucoma .959 96 Forskolin has been shown to inhibit pulmonary tumor colonization in mice b B16 murine melanoma cells and is a potential antimetastatic agent .log Summary - Forskolin activates hormone sensitive adenylate cyclase in a completely unique manner. Its ability to potentiate hormonally induced increases in cyclic AMP provides a new mechanism to potentiate hormonal responses -in vitro and -in vivo; The exact site of action of forskolin still remains unclear, and it is possible that forskolin acts directly at the catalytic subunit of adenylate cyclase or at another subunit which has not yet been identified. The possibility still remains that an endogenous forskolin compound might exist, which would have the ability to either stimulate adenylate cyclase directly or to put the enzyme in a potentiated state which would be exquisitely sensitive to hormonal stimulation. It also remains to be determined if -in vivo effects of forskolin are due to the direct stimulation of adenylate cyclase by forskolin or to the potentiation of the action of natural hormones which are in the circulation. Binding sites for forskolin in membranes can now be studied using the radiolabelled forskolin, providing an easy assay to search for forskolin agonists and antagonists. The radiolabelled forskolin should also provide a tool to study the metabolism and disposition of forskolin after -in vivo administration.
300
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Allen, Ed.
In over 300 articles in the past three years forskolin has been established as a powerful tool to study adenylate cyclase in vitro and in vivo. It is also anticipated that potential new uses of forskolin as -a therapeutic agent will continue to be investigated. Studies with forskolin have thus provided medicinal chemists with a new target in the design of therapeutic agents, the adenylate cyclase enzyme. Acknowledgement - I would like to acknowledge the cooperation and support of Dr. Noel J. deSouza, Dr. Jurgen Reden, and their colleagues at Hoechst Pharmaceuticals, Bombay during our investigations on forskolin. This review is dedicated to the memory of Dr. Balbir S. Ba jwa who made such significant contributions to our understanding of the medicinal chemistry of forskolin.
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302,
302
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- Topics in Chemistry and Drug Design
Allen, Ed.
85. L. Hedin and S. Rosberg, M o l . Cell. Endocrinology, 33, 69 (1983). 86. N. Dekel and I. S h e r i z l y , FEBS Lett., 151,153 (198g. 87. S.B. Oldham, C.T. Molloy. L.G. Lipson, and T.T. Boggs, Endocrinology, 114, 207 (1984). 88. P.J. O l sie wski and W.H. Beers, Dev. Bi ol ., 100. 287 (1983). 89. S. S c h o r d e r e t s l a t k i n e and E.M. Baul i eu, Endocrinology 111, 1385 (1982). 90. R.M. S c h u l t z , R.R. Montgomery, and J.R. B e la noff, Dev. Biol. , 97, 264 (1983). 91. R.M. S c h u l t z , R.R. Montgomery, P.F. Wardbailey, and J.J. Eppig, Dev. B i o l . , 294 (1983). 92. F. Urner, W.L. Herrmann, E.E. Baulieu, and S. S c h o r d e r e t s l a t k i n e , Endoc rinology, 113, 1170 (1983). 93. A.W. C u t h b e r t , and J.A. Spayne, Br. J. Pharmacol., 2, 33 (1982). 94. Y. S a i t o and E.M. Wright. J. P h y s i o l . (Lond.), 336, 635 (1983). 95. S.P. Bartels, S.R. Lee, and A.H. Neufeld, Curr. Eye Res., 2, 673 1983). 96. J. C a p r i o l i and M. S e a r s , L ancet , 1, 958 (1983). 97. G.H. Murdoch, M.G. Rosenfeld, and E.M. Evans, Sc ie nc e , 2,1315 1982). 98. L.E. Eiden, and A.J. Hot chki ss, Neuropept ide s , 4, 1 (1983). 99. F. Spah, J. Cardiovasc. Pharmacol., 6, 99 (19847. 100. I.R. Katz, D. Smith, and M.H. MakmanT Brain Res., 264, 173 (1983). 101. S.V. Bhat, A.N. Dohadwalla, B.S. B a j w a , N.K. Dadkar, H. Dornauer, and N.J. Desouza, J. Med. Chem., 2, 486 (1983). 102. S.V. Bhat, B.S. Bajwa, H. Dornauer, and N.J. de Souza, J. Chem. Soc., P e r k i n Tra ns . I, 767 (1982). 103. K.B. Seamon, J.W. Daly, H. Metzger, N . J . de Souza, and J. Reden, J. Med. Chem., 26, 436 (1983). 104. KB. Seamon, R. V a l l a n c o u r t , M. Edwards, and J.W. Daly, Proc. Nat. Acad. S c i . U.S.A., in p r e s s (1984). 105. N . J . de Souza, A.H. Dohadwalla, and J. Reden, Med. Res. Rev., 2, 201 (1983). 106. J.F. Burka, J. Pharmacol. Exp. "her., 225, 427 (1983). 107. J.F. Burka, Can. J. P h y s i o l . Pharmacol., 61, 581 (1983). 108. K.C. Agarwal and R.E. P arks, I n t . J. Cancer, 32, 801 (1983).
Chapter 30.
Recent P r o g r e s s i n t h e R a t i o n a l Design of P e p t i d e Hormones and N e u r o t r a n s m i t t e r s
V i c t o r J. Hruby, John L. Krstenansky, and Wayne L. Cody Department of Chemistry, U n i v e r s i t y of Arizona, Tucson, AZ 85721
- Any e f f o r t t o d i s c u s s p r o g r e s s i n " r a t i o n a l design" of pept i d e n e u r o t r a n s m i t t e r s and hormones ( h e r e a f t e r r e f e r r e d t o as p e p t i d e hormones) poses c e r t a i n d i f f i c u l t i e s . P e p t i d e hormones g e n e r a l l y produce t h e i r b i o l o g i c a l e f f e c t s by i n t e r a c t i o n w i t h membrane bound r e c e p t o r s , but w e know l i t t l e about t h e s p e c i f i c chemical and p h y s i c a l ( t h r e e dimens i o n a l ) p r o p e r t i e s of t h e s e binding sites. Thus any r a t i o n a l d e s i g n must u t i l i z e o u r a b i l i t y t o deduce t h e p r o p e r t i e s of t h e r e c e p t o r system ( h o s t ) by u s i n g w e l l designed p e p t i d e hormone analogs ( g u e s t s ) , and c a r e f u l chemi c a l and p h y s i c a l a n a l y s i s of t h e s t r u c t u r e , conformation and dynamic prop e r t i e s of t h e s e analogs i n r e l a t i o n t o b i o l o g i c a l a c t i v i t i e s . -I-n--t r o d u c t i o n
U l t i m a t e l y hormone-receptor i n t e r a c t i o n s are t h r e e dimensional i n n a t u r e , i n which two complementary t o p o l o g i c a l rsurfaces i n t e r a c t t o produce a b i o l o g i c a l response. These t h r e e dimensional f e a t u r e s o f t e n can be examined by conformational r e s t r i c t i o n of t h e p e p t i d e hormones. 1-8 For l a r g e r p e p t i d e hormones 0 2 0 r e s i d u e s ) , design of e x t e n s i v e secondary s t r u c t u r e s such as amphiphilic h e l i c e s can provide important i n s i g h t s i n t o membrane binding, r e c e p t o r binding, and o t h e r a s p e c t s of b i o l o g i c a l recogn i t i o n . This approach has r e c e n t l y been reviewed9-11 and w i l l n o t be d i s c u s s e d here. Rather we w i l l c o n c e n t r a t e on smaller p e p t i d e s (0*co \ TI
(CH2) 2OCH3
Hb-.
Enocitabine is an antileukemic agent closely related to cytarabine. It appears more resistant to deamination than cytarabine, thus allowing greater in vivo phosphorylation into an active cytotoxic metabolite.
EperisomeHydmchbn'de (muscle r e l a ~ a n t ) 3 ~ , 3 9 Country of Origin: Japan Originator: Eisai First Introduction: Japan
. C2Hg-*'
Introduced Trade Name: by: MYONAL E M
/-*\ 'o=o'
k . 0 HCH2
ZH3
.--.
'0-0':~~~
Eperisone hydrochloride is a centrally acting muscle relaxant useful in the management of various spastic conditions including cervical spondylosis and cerebral palsy. It is structurally related to tolperisone. Epoprostenol sodium (platelet aggregation i n h i b i t ~ r ) ~ O , ~ l Country of Origin: Originator: First Introduction: Introduced by: Trade Names:
United Kingdom Burroughs Wellcome United Kingdom Burroughs Wellcome; Upjohn FtoLAN;CYCLO-PROSTIN
8H
Epoprostenol sodium (prostacyclin) is a naturally occurring prostaglandin indicated for the preservation of platelet function during cardiopulmonary bypass, prevention of platelet aggregation during charcoal hemoperfusion of patients in hepatic failure, and as an alternative to heparin during renal dialysis.
Fenbuprol (choleretic)4&43 Country of Origin: Originator: First Introduction: Introduced by: Trade Name:
W. Germany Klinge Pharma W. Germany Rhom Pharma VALBIL
,.=. l H
,8"8/.-O-CHz
HCliflC4Hg
Fenbuprol produces an increase in the volume of bile secretion. It is useful in patients having symptomatology associated with biliary tract dysfunction. Flutamide (antineoplastic)%45 Country of Origin: Originator: First Introduction: Introduced by: Trade Name:
USA scheting Chile Schering DROGENIL
( CH 3 2
Flutamide is an orally active, non-steroidal antiandrogen indicated for the treatment of prostatic cancer in both castrates and noncastrates.
Allen
To Market, To Market
Chap. 31
Fluvoxamine Maleate (serontonergic antidepressant)46-48 Country of Origin: Netherlands Originator: First Introduction: Introduced by: Trade Name:
Duphar Switzerland Kali-Duphar FLOXYFRAL
F3C-0,
.=.
p*-*\,
/ 3i-CH2-CH2-CH2-CH2-0-CH3 N-O-CH 2-CH 2-NH 2 ‘C4H404
Fluvoxamine maleate is the most recent of the serotonin-specific antidepressants to reach the market. In vitro and in vivo animal experiments have shown fluvoxamine t o have a marked effect on 5-HT mediated processes and little effect on norepinephrine. Clinical trials suggest similar efficacy t o imipramine and clomipramine with a somewhat lower incidence of side effects, especially anticholinergic effects. Fluvoxamine, in contrast to the tricyclic antidepressants, does not appear to produce heart rate increase, postural hypotension or prolongation of the intraventricular conduction t i m e and QT interval.
Gallopamil Hydrochloride (antianginal)49 Country of Origin: Originator: First Introduction: Introduced by:
W. Germany
Knoll W. Germany Chem. Werke Minden Trade Name: PROCORUM
CH 30
Gallopamil hydrochloride is a somewhat more potent methoxy analog of calcium channel blocker verapamil with a similar profile. It is useful in the treatment of angina and auricular arrhythmia.
Gemeprost ( a b ~ r t i f a c i e n t ) ~ ~ , ~ ~ Country of Origin: Originator: First Introduction: Introduced by:
Japan On0
MalaysiaJSingapore May & Baker
Trade Name: CERVAGEM
*/*\ */’\*/
o\*-.P+*\
CO 2CH 3
/
'a-•
Ho\+’
1.4
\*/
\*/ \*/
dH
Gemeprost, a metabolically stabilized analog of PGE1,has been demonstrated t o reliably induce termination of early pregnancy when administered as a vaginal suppository. Its effect is presumably due to both uterine contraction and rapid decline of steroid hormone levels. Minimal side effects have been reported.
Guanadrel Sulfate (antihypert ensive)%53 Country of Origin: Originator: First Introduction: Introduced by: Trade Name:
USA Cutter USA Penuwalt WLOREL
Guanadrel sulfate is an antihypertensive belonging t o the class of adrenergic neuron blocking drugs. It diminishes sympathetic vasoconstriction by inhibiting norepinephrine storage and release from neuronal storage sites. It appears similar in effectiveness and side effect profile to its structural relative guanethidine.
319
320
Sect. VII
- Worldwide Market
Introductions
a
Halometasone (topical antiinflammatory)%55 Country of Origin: Originator: First Introduction: Introduced by: Trade Name:
Allen, Ed.
Switzerland Ciba-igy Switzerland Ciba-Geigy SICORTEN
Halometasone is a Dotent,. topical - steroid useful in a variety of acute and chronic eczematous dermaioses and psoriasis. It is reportedly devoid of skin toxicity and systemic effects. Hydrocortisone Butyrate Propionate (topical antiinflammat0ry)5~ GH flCOCH 2CH 3 Country of Origin: Originator: First Introduction: Introduced by: Trade Name:
Japan Taiaho Japan Taisho
PANDEL
Hydrocortisone butyrate propionate is a potent, topical steroid reported to possess minimal systemic side effects. It is indicated in the treatment of various acute and chronic contact, eczematous, and atopic dermatoses and psoriasis. Jndalpine (serotonergic a n t i d e p r e ~ s a n t ) ~ ~ - ~ g
Country of Origin: Originator: First Introduction: Introduced by: Trade Name:
France Pharmuka France Labs. Fournier Freres UPSTENE
.’! /.B c r ‘0-.
\*/
yH
Indalpine is a non-tricyclic antidepressant with a serotonin selective profile. It is 6-7 times more potent than fluoxetine and clomipramine in inhibiting serotonin reuptake in vitro in rat brain synaptosomes. Statistically significant clinical effects within one week of onset of treatment have been reported. An anxiolytic effect may accompany the antidepressant effect. Indalpine appears devoid of anticholinergic and cardiovascular side effects and does not promote weight gain or affect appetite. sox xi cam (antiinflammatory)6&62 Country of Origin: Originator: First Introduction: Introduced by: Trade Name:
USA Warner-Lambert W. Germany Warner-Lambert
PAC=
Isoxicam is a non-steroidal antiinflammatory agent useful in the treatment of various forms of rheumatoid arthritis, osteoarthritis and musculoskeletal disorders. It is about one-tenth as potent as its structural relative sudoxicam; its similar long TQ (>30hrs.) allows once-daily dosing.
Chap. 31
To Market, To Market
Allen
Loprazolam Mesylate (hypnotic/sedative)63,64 Country of Origin: United Kingdom Originator: R o w e l First Introduction: United Kingdom Introduced by: Roussel Trade Name: DORMONOCT
*-a
p