ANNUAL REPORTS IN MEDICINAL CHEMISTRY Volume 9
ANNUAL REPORTS IN MEDICINAL CHEMISTRY Volume 9
ii
CONTRIBUTORS
. ....... Amer. M . S . . . . . . . . . Archer. R . A . . . . . . . . Bindra. J . S . . . . . . . . Bloch. A . . . . . . . . . . Campbell. W . C. . . . . . . Carlson. R . G . . . . . . . . Chang. A . Y . . . . . . . . . Chignell. C . F . . . . . . . Claridge. C . A . . . . . . . Daly. J . W . . . . . . . . . Dean. R . R . . . . . . . . . DuCharme. D . W . . . . . . . Eargle. D . H., Jr . . . . . . Flanders. L . E . . . . . . . Fleming. J . S . . . . . . . . Francis. J . E . . . . . . . . Fullerton. D . S . . . . . . . Giles. R . E. . . . . . . . . Gordon. M . . . . . . . . . . Gylys. J . A . . . . . . . . . Harbert. C. A . . . . . . . . Hardy. R . A., Jr . . . . . . Adelstein. G W
67 203 253 214 139 115 270 182 280 95 290 67 50 260 162 75 57 260 85 38 27
1
11
........ Hodson. A . . . . . . . . . Holland. G . F . . . . . . . Jerina. D . M . . . . . . . . Johnson. A . G . . . . . . . Katzenellenbogen. J . A . . . Kenyon. G . L . . . . . . . . Leitner. F . . . . . . . . . MacNintch. J . .E. . . . . . McKinney. G . R . . . . . . . Metcalf. R . L . . . . . . . Mrozik. H . . . . . . . . . Mueller. R . A . . . . . . . Partyka. R . A . . . . . . . Pereira. J . N . . . . . . . Rando. R . R . . . . . . . . Rosenthale. M . E. . . . . . Schwartz. A . R . . . . . . . Shadomy. S . . . . . . . . . Tomeszewski. J . E . . . . . Vernier. V . G . . . . . . . Welch. W . M . . . . . . . . Wheelock. E . F . . . . . . . Herzig. D . J
85 151 172 290 244 222 260 95 75 203 300 115 162 27 172 234 193 128 107 290 19
1 151
ANNUAL REPORTS IN MEDICINAL CHEMISTRY Volume 9 Sponsored by the Division of Medicinal Chemistry of the American Chemical Society Editor-in- Chief: RICHARD V. HEINZELMAN THE UPJOHN COMPANY KALAMAZOO, MICHIGAN
SECTION EDITORS MAXWELL GORDON
FRANK CLARKE
WALTER MORELAND
WILLIAM WECHTER
ACADEMIC PRESS
@
GEORGE WARREN ROBERT WILEY
New York and London 1974
A Subsidiary of Harcourt Brace Jovanovich, Publishers
ACADEMIC PRESS RAPID MANUSCRIPT REPRODUCTION
COPYRIGHT 0 1974, 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 ANY INFORMATION STORAGE AND RETRIEVAL SYSTEM, WITHOUT PERMISSION I N WRITING FROM THE PUBLISHER.
ACADEMIC PRESS, INC. 111 Fifth Avenue, New
York. New York 10003
United Kingdom Edition published by ACADEMIC PRESS, INC. (LONDON) LTD. 24/28 Oval Road, London NWl
LIBRARY OF CONGRESS CATALOG CARD NUMBER:66- 26843
PRINTED IN THE UNITED STATES O F AMERICA
V
CONTENTS
............................. ...............................
Contributors
ii
Preface
xi
I. CNS AGENTS Section Editor: Maxwell Gordon, Bristol Laboratories, Syracuse, New York
..............
1
...............
11
1.
Antipsychotic and Antianxiety Agents. Charles A. Harbert and Willard M. Welch, Pfizer, Inc., Groton, Connecticut
2.
Narcotic Antagonists and Analgesics Robert A. Hardy, Jr., Lederle Laboratories, A Division of American Cyanamid Company, Pearl River, New York
3.
Antiparkinsonism Drugs. Vernon G. Vernier, E. I. Du Pont de Nemours & Co., Inc., Newark, Delaware
4.
Psychotomimetic Agents. Richard A. Partyka and Jonas A. Gylys, Bristol Laboratories Syracuse, New York
5.
Abuse of CNS Agents Maxwell Gordon, Bristol Laboratories, Syracuse, New York
.....................
19
.....................
27
.......................
38
11.
Section Editor: 6.
PHARMACODYNAMIC AGENTS
Frank H. Clarke, Ciba-Geigy Corporation, Ardsley, New York
..................
The Etiology of Hypertension. Donald W. DuCharme, The Upjohn Company, Kalamazoo, Michigan
50
vi CONTENTS 7.
.....................
Antihypertensive Agents John E. Francis, Research Department, Pharmaceuticals Division, CIBA-GEIGY Corporation, Ardsley, New York
..............
8. Antiarrhythmic and Antianginal Agents Gilbert W. Adelstein and Richard R. Dean, Searle Laboratories, Chicago, Illinois 9.
......................
Antithrombotic Agents J. Stuart Fleming and John E. MacNintch, Bristol Laboratories, Syracuse, New York
.................
10. Pulmonary and Antiallergy Drugs Ralph E. Giles and David J. Herzig, Warner-Lambert Research Institute, Morris Plains, New Jersey
57
67
75
85
111. CHEMOTHERAPEUTIC AGENTS
Section Editor: George H. Warren, Wyeth Laboratories, Inc., Philadelphia, Pennsylvania
...........................
95
........................
107
......................
115
........................
128
11. Antibiotics F. Leitner and C. A. Claridge, Bristol Laboratories, Syracuse, New York
12. Antifungal Agents Smith Shadomy, Medical College of Virginia, Richmond, Virginia 13. Antiparasitic Agents. W. C. Campbell and H. Mrozik, Merck Institute for Therapeutic Research, Rahway, New Jersey
14. Antiviral Agents.
Andrew R. Schwartz, Hahnemann Medical College and Hospital, Philadelphia, Pennsylvania
vii CONTENTS
......................
139
.....................
151
15. Antineoplastic Agents A. Bloch, Roswell Park Memorial Institute, Buffalo, New York
16. Immunotherapy of Cancer Anita Hodson and E. Frederick Wheelock, Jefferson Medical College, Thomas Jefferson University, Philadelphia, Pennsylvania
IV. METABOLIC DISEASES AND ENDOCRINE FUNCTION Section Editor: Walter T. Moreland, Pfizer Inc., Groton, Connecticut
. . . . . . . . . . . . . . 162
17. Prostaglandins and Related Compounds. Richard A. Mueller and Lloyd E. Flanders, G. D. Searle and Co., Chicago, Illinois
. . . . . . . . . . . . . . . . . . 172
18. Disorders of Lipid Metabolism Gerald F. Holland and Joseph N. Pereira, Pfizer Inc., Groton, Connecticut
........................
19. Diabetes Mellitus Albert Y. Chang, The Upjohn Company, Kalamazoo Michigan
182
. . . . . . . . . . . . . . 193
20.
Non-steroidal Antiinflamatory Agents Marvin E. Rosenthale, Wyeth Laboratories, Inc., Radnor, Pennsylvania
21.
Cyclic Nucleotides and Drug Discovery M. Samir h e r and Gordon R. McKinney, Mead Johnson Research Center, Evansville, Indiana
. . . . . . . . . . . . . . 203
viii CONTENTS
V.
TOPICS IN BIOLOGY
Section Editor: William J. Wechter, The Upjohn Company, Kalamazoo, Michigan Section Editorial
........................
213
........................
214
22.
Anti-aging Drugs. Jasjit S. Bindra, Pfizer, Inc., Groton, Connecticut
23.
Affinity Labeling of Hormone Binding Sites. John A. Katzenellenbogen, Department of Chemistry, University of Illinois, Urbana, Illinois
24.
Mechanism-Based Irreversible Enzyme Inhibitors. Robert R. Rando, Dept. of Pharmacology, Harvard Medical School, Boston, Massachusetts
25.
Adjuvants to the Immune System. Arthur G. Johnson, University of Michigan Medical School, Ann Arbor, Michigan
26.
The Cannabinoids: Therapeutic Potentials Robert A. Archer, Lilly Research Laboratories, Indianapolis, Indiana
. . . . . . . . . . . 222
. . . . . . . . . 234
. . . . . . . . . . . . . . . . . 244 . . . . . . . . . . . . 253
VI. Section Editor: 27.
TOPICS IN CHEMISTRY
Robert A. Wiley, University of Kansas, Lawrence, Kansas
. . . . . . . . . . 260
Reactions of Interest in Medicinal Chemistry. Dwight S. Fullerton, George L. Kenyon and Dolan H. Eargle, Jr., Department of Medicinal Chemistry, College of Pharmacy, University of Minnesota, Minneapolis, Minnesota and University of California, San Francisco, California
ix CONTENTS 28.
29.
The Intramolecular Diels-Alder Reaction in Organic Synthesis. Robert G. Carlson, Department of Chemistry, University of Kansas, Lawrence, Kansas
. . 270
. . . . . . . . . . . . . . . 280
Drug Binding and Drug Action. . . . Colin F. Chignell, National Heart and Lung Institute, Bethesda, Maryland
. . . . . . . . 290
30. Cytochrome P-450 Monoxygenases in Drug Metabolism J. E. Tomeszewski, D. M. Jerina and J. W. Daly, National Institute of Arthritis, Metabolism and Digestive Diseases, National Institutes of Health, Bethesda, Maryland
31. Use of Chemical Relationships in Design of DDT-Type Insecticides. 300 Robert L. Metcalf, Department of Entomology, University of Illinois, Urbana-Champaign, Illinois
COMPOUND NAME AND CODE NUMBER INDEX.
. . . . . . . . . . . . . . . . . 309
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xi
PREFACE Our editorial policy continues to be to report annually on the more active fields of interest to medicinal chemists and to cover less regularly new or maturing biomedical topics or fields which have passed their prime. Less than half of this year's topics appeared also in last year's Annual Reports. It may be well to remind the reader that it is not our intent to provide comprehensive literature coverage for the expert in the field; plenty of other publications already serve this purpose. Our aim is to report significant highlights to enable the worker in a related field to recognize developments or concepts which may broaden his approach to his own area or even cause him to delve into a new one. Just as the increasing availability of massive computer-generated bibliographies on a particular topic poses a growing problem for a researcher who wishes to read selectively in a new field, so this same capability makes it more difficult for our chapter authors to be selective in what they choose to discuss. In fact this problem constitutes the editor's most arduous task; the ten-page chapter limit is designed both to achieve desired selectivity of subject matter and to keep the volume within a reasonable price range in this era of soaring costs. For the first time section editors have been given the opportunity to include one-page editorial overviews of their sections; among the objectives of these overviews are to highlight major new developments, emphasize common features in drug actions or disease etiologies or just to permit speculation as a stimulus for new ideas. While only one section editor chose to accept the invitation this year, it is hoped that more will do so in future volumes. A s always, your constructive suggestions for improvement or for new topics are earnestly solicited.
Kalamazoo, Michigan June, 1974
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Section I
-
CNS Agents
E d i t o r : Maxwell Gordon B r i s t o l L a b o r a t o r i e s , Syracuse, New York Chapter 1. A n t i p s y c h o t i c and A n t i a n x i e t y Agents Charles A. Harbert and W i l l a r d M. Welch P f i z e r I n c . , Groton, Conn. 06340 I n t r o d u c t i o n - Although s e v e r a l promising a g e n t s have p r o g r e s s e d t o advanced c l i n i c a l s t a g e s , no fundamentally new a n t i p s y c h o t i c o r a n t i a n x i e t y a g e n t s w e r e i n t r o d u c e d i n t h e U.S. i n 1973. Clozapine has emerged as t h e focus of c l i n i c a l and t h e o r e t i c a l i n t e r e s t i n t h e a n t i p s y c h o t i c area based on i t s extremely low i n c i d e n c e of e x t r a p y r a m i d a l s i d e r e a c t i o n s . This d i s c o v e r y h a s given new impetus t o r e s e a r c h d i r e c t e d toward an und e r s t a n d i n g of t h e biochemical and n e u r o p h y s i o l o g i c a l c o r r e l a t e s of a n t i p s y c h o t i c a c t i o n and e x t r a p y r a m i d a l m a n i f e s t a t i o n s . A c t 2 v i t y i n t h e a n t i a n x i e t y area a g a i n l a r g e l y focused on a g e n t s s t r u c t u r a l l y and mechani s t i c a l l y r e l a t e d t o t h e benzodiazepines. Extensive treatises have covered t h e c h e m i s t r y , b i o c h e m i s t r y , pharmacology and c l i n i c a l u t i l i t y of t h e b e n z o d i a z e p i n e s l and t h e n e u r o l e p t i c s . 2 A n t i p s y c h o t i c Agents - T r i c y c l i c s - S t r u c t u r e - a c t i v i t y r e l a t i o n s h i p s (SAR) of t h e t r i c y c l i c p s y c h o t h e r a p e u t i c s 3 and c l i n i c a l e x p e r i e n c e w i t h t h e l o n g - a c t i n g n e u r o l e p t i c s 4 9 5 have been reviewed. Emphasis continued t o s h i f t from s t r u c t u r e s w i t h 6 , 6 , 6 r i n g systems t o t h e 6 , 7 , 6 - t r i c y c l i c s . Among t h e l a t t e r , c l o z a p i n e (A) h a s a t t r a c t e d t h e most i n t e r e s t . A r e c e n t review6 h i g h l i g h t s t h e unique p r o f i l e of t h i s agent and t h e a u t h o r p o i n t s out some troublesome s i d e e f f e c t s (hypotension, t a c h y c a r d i a ) t h a t may l i m i t i t s c l i n i c a l use. Regardless, t h e emergence of c l o z a p i n e has reemphasized t h e f e a s i b i l i t y of d i s a s s o c i a t i n g a n t i p s y c h o t i c a c t i v i t y from t h e c a t a l e p t i c p r o p e r t i e s t h a t have been c l a s s i c a l l y regarded as a prerequisite f o r c l i n i c a l therapeutic activity. 7 a)XmH ;ZICH b ) X r C l ; Z n C H c ) X = H ; Z = N d) X I C1 ; Z = N e ) X = O E t ; Z = CH f ) X I OCH3;Z = CH _.
H
6
4
Continuing SAR s t u d i e s i n t h e d i b e n z o [ b , f ] t h i e p i n and r e l a t e d 6 , 7 , 6 systems have shown t h a t s u b s t i t u t i o n by c h l o r i n e i n t h e 9 - p o s i t i o n of p e r a t h i e p i n (&) r e s u l t s i n diminished a c t i v i t y .8 A l l e i g h t p o s s i b l e aryl-monochloro isomers of p e r a t h i e p i n have now been s y n t h e s i z e d , and of
2 -
Sect. I
-
CNS Agents
Gordon, Ed.
t h e s e only t h e 8 - C 1 isomer ( o c t o c l o t h e p i n , &) i s more p o t e n t t h a n t h e p a r e n t compound. Octoclothepin enantiomers and t h e racemate were found t o d i f f e r l i t t l e from each o t h e r i n CNS d e p r e s s a n t e f f e c t s o r i n t o x i c i t y . ' The 4-aza a n a l o g s & and 2d showed markedly reduced depress a n t and c a t a l e p t i c a c t i v i t y compared w i t h 3 and 2, r e s p e c t i v e l y . l O , l l Among s e v e r a l 8-alkoxy analogs prepared, o n l y t h e 8 - E t 0 isomer (&) e x h i b i t e d a c t i v i t y (1OX chlorpromazine [CPZ] i n t h e r o t a t i n g rod t e s t ) e q u i v a l e n t t o t h e known 8-Me0 d e r i v a t i v e ( 2 f , octometothepin) .I2 Oxid a t i o n of t h e S atom and/or 4'-N atom i n a number of d i b e n z o [ b , f ] t h i e p i n s r e l a t e d t o 1. r e s u l t e d i n a uniform l o s s of CNS d e p r e s s a n t a c t i v i t y . 1 3 EX 11-582A (3) p o s s e s s e s p o t e n t a c t i v i t y i n t h e f i g h t i n g mouse (ED50 0.9 mg/kg) and antiamphetamine t o x i c i t y (ED50 0.28 mg/kg) t e s t s . 1 4 Like c l o z a p i n e , t h i s compound was i n a c t i v e a g a i n s t apomorphine-induced emesis.
q y
a) R CF3
c1
CHCHzCHzN(CH3
3 -
)2
-
a;Q CH,;
Z
I
N
I
R
b) R
-4
I
L c1
S
C H 2 9 ; Z
=
N (CH, ) 2
S
-5
A r e c e n t paper of t h e o r e t i c a l i n t e r e s t r e p o r t e d p o t e n t n e u r o l e p t i c a c t i v i t y f o r a number of p i p e r i d y l i d e n e t h i o x a n t h e n e s (4,Z=S) and r e l a t e d s t r u c t u r e s (4, Z=O o r NCH3); f o r example, 4a and 4b were r e s p e c t i v e l y 7 and 1 8 X CPZ i n t h e r a t p t o s i s t e s t . 1 5 S i g n i f i c a n t l y , many of t h e s e compounds were more p o t e n t t h a n t h e i r corresponding t r i c y c l i c congeners w i t h nonr i g i d s i d e c h a i n s . Since k cannot assume t h e s o l i d s t a t e c o n f i g u r a t i o n t h e a u t h o r s q u e s t i o n e d earlier s p e c u l a t i o n s t h a t t h e a c t i v e of CPZ c o n f i g u r a t i o n of a n t i p s y c h o t i c s resembles t h a t of c r y s t a l l i n e CPZ. Rather, t h e i r f i n d i n g s are c o n s i s t e n t w i t h t h e S - c o n f i g u r a t i o n of a n t i p s y c h o t i c d r u g s f r e q u e n t l y d e p i c t e d by Janssen.7 I n a r e l a t e d paper, NMR s h i f t r e a g e n t s were used t o determine t h e s t e r e o c h e m i s t r y of and t r a n s aminoalkylidene xanthenes and thioxanthenes . I 6 As expected, n e u r o l e p t i c a c t i v i t y predominated i n t h e cis series.
(z),
An i m p o r t a n t pharmacokinetic s t u d y c o r r e l a t e d CPZ plasma l e v e l s w i t h t h e r a p e u t i c response and found t h a t plasma l e v e l s i n t h e range of 150-300 ng/ml u s u a l l y corresponded t o c l i n i c a l improvement . I 7 S i g n i f i c a n t l y , concomitant a d m i n i s t r a t i o n of an a n t i p a r k i n s o n agent ( t r i h e x y p h e n i d y l ) tended t o lower plasma l e v e l s of CPZ, an e f f e c t a s c r i b e d t o reduced g a s t r i c mot i l i t y and d e c r e a s e d a b s o r p t i o n of CPZ. Butyrophenones, P h e n y l p i p e r a z i n e s , and R e l a t e d Compounds - Bromoperidol (&) i s e s s e n t i a l l y e q u a l t o h a l o p e r i d o l i n potency and d u r a t i o n of a c t i o n . 1 8 The trans-4-aryl-4-methoxycyclohexylaminobutyrophenone 6b exceeded h a l o p e r i d o l on s e v e r a l b e h a v i o r a l e n d p o i n t s and exhibitedh i g h l y s e l e c t i v e (vs. 5-HT) NE uptake blockade.19 Compound &, t h e most p o t e n t (15X CPZ i n c o n d i t i o n e d avoidance response [CAR]) i n a series of s u b s t i t u t e d pyrazino[2',1':6,l]pyrido[3,4-b]indoles, was r e p o r t e d t o have a prolonged d u r a t i o n of a c t i o n and few c a r d i o v a s c u l a r
Antipsychotic and Antianxiety Agents Harbert, Welch 2
Chap. 1
effects .20 Centpyraquin (6d, compound 69-183) combined neuroleptic activity (ca. CPZ in CAR) with significant hypotensive effects.21 AHR-2277 (lenperone, 6 e ) exerted a rapid and favorable effect on psychotic symptomatology, and was claimed to produce a very low incidence of neurological side reactions.22 A double-blind study of AL-1021 (6f) in hospitalized schizophrenics failed to confirm earlier encouraging results obtained in pilot study.23 Milipertine (&) produced troublesome EPS side effects in schizophrenics at doses well below those required to produce antipsychotic activity.24 Compound j’&was one of a series reported to exhibit CNS depressant activity in animals roughly in the CPZ range.25 Oxazolidinone 7c blocked isolation-induced aggression in mice (ca. 0 . 5 X CPZ);26 a similar level of activity in this test was seen with the related compounds 8a and &.27
-6
H
7 m
7b) X
I
OCH3; Y
= H; R =
-
R
-8
COCH3 CH2CHOHCH206
8b)
R
(CH,),OCONHCH,
Miscellaneous Compounds - A benzodiazep ne, SCH- 2,041 (2, halazepam), showed evidence of efficacy against acute psychotic symptoms in an uncontrolled tria1.28 CI-686 (10)was predicted to have major tranquilizer activity of short duration on the basis of quantitative pharmaco-EEG studies.29 Butaclamol (11, - AY-23,028) possessed pharmacological actions resembling fluphenazine, with activity residing solely in the (+)-enantiomer.30,31 The SAR of several cannabinoids nas been reexamined and
Sect. I
4 -
-
CNS Agents
Gordon, Ed.
found to differ significantly from earlier reports.32 A comparison study in the same paper showed 12 to be 3-15X CPZ in decreasing motor activity and in blocking fighting behavior in mice.
10 -
-9
11 -
OH
12 -
Antianxiety Agents-Benzodiazepines and Related Compounds - The drug therapy of anxiety33~34 and the effect of benzodiazepines in controlling aggressive behavior35 were reviewed. It was argued that conformational similarities between diphenylhydantoin and diazepam36 do not constitute an appropriate basis for comparing their biological activities.37 A series of benzodiazepines with a,a-dihydroperfluoroalkyl groups at N-1 were found to successfully block metabolism at N-1 as predicted. One of these, nalazepam (Sch 12,041, 2 ) , appeared to be more specific than diazepam in anticonvulsant (pentylenetetrazole, PTZ) and traction endpoints, with lower side effect potential (therapeutic index 4X diazepam). 38 The ,39 and ID-540 (13b)hO related compound, fletazepam (Sch 15,698, were claimed to be skeletal muscle relaxants. Clonazepam (Ro 5-4023, 13c) was more active against seizures induced by i.v. lidocaine than Ro 8-4192 (E) or diazepam.41 Compound from a novel series =her of 7-azidobenzodiazepines, demonstrated sedative and anticonvulsant properties equivalent to diazepam.42
e)
e,
1 ‘
2
7 5
13 2
K 3 -N
c1
14a) 2 -NHOCH2CH2=CH2 14b) 2-NHCONHCOOC2 Hcj 14c) 2 - N 7 - H
\’. 14 -
0
Chap. 1
Antipsychotic and Antianxiety Agents
Harbert, Welch
2
Pharmacological studies indicated uldazepam (U-31,920, 3) to be an effective antianxiety drug with weak sedative and muscle relaxant prowas nearly equivalent to diazepam in perties.43 The urea derivative several behavioral and convulsant (thiosemicarbazide) end points ,44 wherewere less active in the same tests. as the related 2-imidazolidones (&) A number of 1,3-dialkylpyrazolo[4,3-e]diazepinones exhibited potent CNS activity and one of these, pyrazapon (CI-683, 2) showed anxiolytic activity equivalent to diazepam in man.45 SAR studies showed that hydrogen is preferred at N-4 and that 1,3-dialkyl substitution is superior to the 2,3-pattern of the isomeric series (16).
=
15 -
16 -
18 -
Several series of triazolobenzodiazepines (17) with CNS depressant the most potent of the 3activity have been reported. D-40TA (E), unsubstituted triazoles, was highly active as a tranquilizer, sedativehypnotic and muscle relaxant in animal^.^^-^^ Triazolam (U-33,030, =) exhibited hypnotic activity in man at 0.25-2.0 mg. 49 The anticonvulsant and antiaggressive potencies of U-31,957 (G) and U-35,005 (G) exceeded those of the corresponding diazepam analogs, but they were only weakly active in end points measuring overt depression (traction, EtOH) .50 Alprazolam (U-31,889, 17e)51 demonstrated a marked and rapid anxiolytic effect in man as had been predicted from its EEG profile.52 Chlordiazepoxide isosteres in the triazolobenzodiazepine series (e.g. 17f) were consistently inactive.46,53 The triazinodione 18was equal to diazepam on several behavioral end points and was 0.3X diazepam against PTZ convulsions and EtOH narcosis.44 The 1,5-benzodiazepine triflubazepam (ORF-8063, =)possessed antianxiety properties in man at 40-160 mg/day,54,55 with a profile similar to diazepam.56 Like most lY4-benzodiazepines it was primarily metabolized through N-dealkylation, but was otherwise hydroxylated in the N-phenyl ring rather than at C-3.57 Pharmacokinetic studies with
6 -
Sect. I
-
CNS Agents
Gordon, Ed.
t r i f l u b a z e p a m showed c l i n i c a l improvement t o b e c o r r e l a t e d w i t h blood l e v e l s of 0.5 t o 1 . 0 ng/m1.58 Clobazam w a s claimed t o be b e t t e r t o l e r a t e d t h a n c h l o r d i a z e p o x i d e i n pharmacological and t o x i c o l o g i c a l s t u d i e s . 59 The 1,5-benzodiazepine r e l a t i v e s of c h l o r d i a z e p o x i d e , 20a and E , p r o t e c t e d mice a g a i n s t c o n v u l s i o n s produced by PTZ, s t r y c h n i n e (ST) and t r e m o r i n e , and were less d i s r u p t i v e t h a n c h l o r d i a z e p o x i d e on tests of motor c o o r d i n a t i o n . 6 0
(w)
2
CH3
7
Q;Jo I 19
C6H5
OZN
19a) 7-CF3 19b) 7-C1
l o C6H5
20 -
20a) 2-M12 20b) 2-N(CH3 )2
Other S t r u c t u r e s w i t h A n t i a n x i e t y A c t i v i t y - The o x y p e r t i n e a n a l o g s 21a and 21b induced a t a x i a , d e c r e a s e d locomotor a c t i v i t y and antagonizede l e c t r o s h o c k (ES) and ST c o n v u l s i o n s i n mice 0.25 diazepam).61 I n d o l e d e r i v a t i v e s U-5092 and U-13,625 (G) p r o t e c t e d mice a g a i n s t ES-induced c o n v u l s i o n s and produced a CPZ-like t r a n q u i l i z i n g e f f e c t . 62 I n a double-blind s t u d y a g a i n s t a n x i e t y , 200 mg opipramol (22) w a s e q u i v a l e n t t o 30 mg c h l o r d i a z e p o x i d e .63 The i s o q u i n o l i n e d e r i v a t i v e 23 and t h e b e n z o t h i a z e p i n e d e r i v a t i v e 2 4 possessed marked CNS a c t i v i t y i n mice c h a r a c t e r i z e d by g e n e r a l d e p r e s s i o n and a n t i c o n v u l s a n t (ES, PTZ, ST) a c t i v i t y . 6 4 , 6 5 The R enantiomers of a series of 1 - a m i n o t e t r a l i n s (e.g. 25) s u p p r e s s e d c o n d i t i o n e d emotional response (CER) i n rats a t 10 I n t e r e s t i n g l y , t h e S isomers were devoid of CER a c t i v i t y , b u t mg/kg. possessed marked a n t i c a t a l e p t i c ( t e t r a b e n a z i n e ) a c t i v i t y . 66 Although 26a and 26b resemble known m e t a b o l i t e s of 1,4-benzodiazepine t r a n q u i l i z e r s , t h e y e x h i b i t e d o n l y v e r y weak anti-PTZ and ES a c t i v i t y , and showed no taming e f f e c t i n i s o l a t e d mice.67
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2l a
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Chap. 1
A n t i p s y c h o t i c and A n t i a n x i e t y Agents
H a r b e r t , Welch
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OCH3 25
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B i o l o g i c a l Observations and Hypotheses - The r o l e of dopamine r e c e p t o r b l o c k i n g a g e n t s i n t h e t r e a t m e n t of schizophrenia68 and t h e m e c h a n i s t i c r e l a t i o n s h i p of t h e a n t i p s y c h o t i c e f f e c t s of n e u r o l e p t i c s t o t h e i r e x t r a pyramidal (EPS) e f f e c t s @ have been reviewed. S e v e r a l hypotheses have been advanced t o account f o r c l o z a p i n e ' s low i n c i d e n c e of EPS r e a c t i o n s , 6 most of which invoke i t s a t r o p i n e - l i k e a n t i c h o l i n e r g i c a c t i v i t y . For example, c l o z a p i n e causes a g r e a t e r i n c r e a s e i n homovanillic a c i d l e v e l s i n t h e l i m b i c system than i n t h e s t r i a t u m , w h i l e h a l o p e r i d o l shows no s e l e c t i v i t y i n t h e s e p o r t i o n s of t h e b r a i n . However, when an a n t i c h o l i n e r g i c agent is coadministered w i t h h a l o p e r i d o l an e f f e c t resembling t h a t of c l o z a p i n e i s o b t a i n e d , 70 c o n s i s t e n t with r e c e n t evidence t h a t c h o l i n e r g i c pathways modulate dopaminergic a c t i v i t y i n t h e s t r i a t u m , but n o t i n t h e l i m b i c system. 7 l T h e r e f o r e , c l o z a p i n e ' s a n t i c h o l i n e r g i c properties may f o r t u i t o u s l y balance i t s EPS e f f e c t i n t h e s t r i a t u m w i t h o u t a t t e n u a t i n g i t s a n t i p s y c h o t i c e f f e c t . The r e c e n t observation72 t h a t CPZ enhances a c e t y l c h o l i n e output i n t h e s t r i a t u m of cannulated c a t s provides t h e f i r s t d i r e c t evidence t h a t n e u r o l e p t i c drugs a c t i v a t e t h e c h o l i n e r g i c s y s t e m and s u p p o r t s t h e p o s t u l a t e d e x i s t e n c e of an interdependent and a n t a g o n i s t i c c h o l i n e r g i c-d op aminer g i c neuronal network .73 F u r t h e r evidence f o r t h e e x i s t e n c e of a neuronal feedback mechanism c o n t r o l l i n g DA t u r n o v e r i n b r a i n came from a r e p o r t t h a t a n t i p s y c h o t i c drugs i n c r e a s e t h e r a t e of f i r i n g of dopaminergic neurons i n t h e s u b s t a n t i a n i g r a and t h e v e n t r a l tegmental area. 74 Recent papers a l s o suggested t h a t y-hydroxybutyric a c i d (GHB) 75,76 and y-aminobutyric a c i d (GABA) 77 may r e g u l a t e t h i s feedback mechanism. I f t h e s e f i n d i n g s are s u b s t a n t i a t e d GHB and GABA w i l l provide v a l u a b l e t o o l s t o s t u d y t h i s feedback mechanism and i t s r e l a t i o n s h i p t o n e u r o l o g i c a l m a n i f e s t a t i o n s such as c a t a l e p s y i n animals, and EPS s i d e e f f e c t s and t a r d i v e d y s k i n e s i a i n man. The import a n c e of i d e n t i f y i n g e x i s t i n g o r new b i o l o g i c a l tests t h a t r e l a t e more s p e c i f i c a l l y t o a n t i p s y c h o t i c a c t i v i t y i n man was emphasized i n two r e c e n t papers which claimed t h a t b i o l o g i c a l endpoints showing t o l e r a n c e (e.g. s t e r e o t y p e d behavior) do n o t assess p o t e n t i a l a n t i p s y c h o t i c e f f e c t s i n man, b u t r a t h e r only measure p r o p e n s i t y t o cause EPS e f f e c t s . 7 8 9 7 9 A new technique t o assess changes i n NE s y n t h e s i s i n rat b r a i n measures t h e accumulation of 3-methoxy-4-hydroxyphenylethyleneglycol (MOPEG), a major NE m e t a b o l i t e . 8 0 This method w a s claimed t o be more convenient than c o n v e n t i o n a l methods u t i l i z i n g exogeneous a-methylp-tyrosine o r l a b e l e d t y r o s i n e , and t o avoid e r r o r s t h a t might b e
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introduced by i n t e r a c t i o n of t h e s e agents w i t h t h e t e s t compound. The s e d a t i v e a c t i v i t y of n e u r o l e p t i c s appears t o c o r r e l a t e w e l l w i t h t h e i r a b i l i t y t o i n c r e a s e MOPEG levels i n b r a i n . 8 1 S e v e r a l r e c e n t r e p o r t s have i m p l i c a t e d enzyme def i c i e n c e s o r a b n o r m a l i t i e s i n t h e pathophysiology of s c h i z o p h r e n i a . Post-mortem b r a i n specimens of s c h i z o p h r e n i c s showed a s i g n i f i c a n t r e d u c t i o n i n dopamine-8-hydroxylase a c t i v i t y vs. normals i n a l l b r a i n r e g i o n s examined.82 Monoamine oxidase (MAO) a c t i v i t y w a s found t o be s i g n i f i c a n t l y lower i n t h e p l a t e l e t s of s c h i z o p h r e n i c and non-schizophrenic monozygotic twin p a i r s when compared t o normal c o n t r o l s , s u g g e s t i n g t h a t decreased MA0 a c t i v i t y might be a g e n e t i c marker f o r v u l n e r a b i l i t y t o s c h i z o p h r e n i a . 83 An N-methyltransferase capable of forming dimethyltryptamine (DMT) , a known h a l l u c i n o g e n , was found i n human red blood c e l l s , plasma, and p l a t e l e t s . 8 4 S i g n i f i c a n t l y t h e a c t i v i t y of t h i s enzyme i n p l a t e l e t s w a s h i g h e r i n p s y c h o t i c s u b j e c t s than i n nonpsychotics, a f i n d i n g a p p a r e n t l y r e l a t e d t o an u n i d e n t i f i e d d i a l y z a b l e i n h i b i t o r pres e n t i n normal s u b j e c t s , b u t n o t i n p s y c h o t i c s . A non-specific N-methylt r a n s f e r a s e i s o l a t e d from rat b r a i n w a s found t o be s i m i l a r l y c o n t r o l l e d by a d i a l y z a b l e i n h i b i t o r . 8 5 DMT and b u f o t e n i n have been i d e n t i f i e d i n t h e u r i n e of a u t i s t i c c h i l d r e n 8 6 and c h r o n i c s c h i z o p h r e n i c s , 87 b u t no d i f f e r e n c e between plasma DMT l e v e l s i n c o n t r o l s and s c h i z o p h r e n i c s w a s e v i d e n t i n a r e c e n t study.88 Although S-adenosylmethionine h a s been viewed f o r many y e a r s as t h e methyl donor r e s p o n s i b l e f o r t h e 0- and N-methylation of b i o g e n i c amines, t h e involvement of 5-methyltetrahydrof o l i c a c i d i n t h e s e t r a n s f o r m a t i o n s h a s now been f i r m l y e s t a b l i s h e d . 8 9 , g 0 Accordingly, i t h a s been p o s t u l a t e d r e c e n t l y t h a t t h e cause of schizop h r e n i a might b e an overabundance of f o l a t e coenzymes, r e s u l t i n g i n e x c e s s i v e N-methylation of b i o g e n i c amines i n b r a i n . 8 9 Diazepam appears t o e x e r t a d i r e c t a c t i o n a t t h e s p i n a l level by i n c r e a s i n g segmental p r e s y n a p t i c i n h i b i t i o n and by d e c r e a s i n g o r s u p p r e s s i n g p r e s y n a p t i c c o n t r o l s of s u p r a s p i n a l o r i g i n . 91 Benzod i a z e p i n e s appear t o d e c r e a s e NE turnover by reducing impulse a c t i v i t y i n t h e l o c u s coeruleus r a t h e r t h a n by a d i r e c t i n t e r f e r e n c e w i t h release of NE from t h e nerve terminals.92 R e p e t i t i v e dosing may, however, r e v e r s e t h i s i n h i b i t o r y e f f e c t i n t h e "reward s t r u c t u r e s " w h i l e m a i n t a i n i n g t h e i n h i b i t o r y e f f e c t upon t h e "aversive s t r u c t u r e s .'Ig3 Benzodiazepines may f o r t i f y t h e mechanisms of p h y s i o l o g i c a l s t a b i l i z a t i o n of n e u r o n a l nerve t e r m i n a l membranes by f a c i l i t a t i n g t h e formation, release o r a c t i o n of p r o s t a g l a n d i n s .94 A combination of i n h i b i t i o n of 2-M phosphodiesterase a c t i v i t y and i n h i b i t i o n of 5-HT turnover i n t h e midbrain raph6 region i n rats may be r e s p o n s i b l e f o r c h l o r d i a z e p o x i d e ' s a b i l i t y t o s u p p r e s s a g g r e s s i v e behavior .95 REFERENCES
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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.
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CNS Agents
Gordon, Ed.
Y. Kuwada, Chem. Pharm. B u l l . (Tokyo), 2382 (1973). L). M. G a l l a n t , W. C. Swanson, D. H. Mielke and R. Guerrero-Figueroa. Curr. Therap. Res.. Clin. Exp., 14, 664 (1972). S . Gershon, Psychopharm. B u l l . 9, 92 (1973). R. D. Heilman, E. W. Bauer and J. P. DaVanzo, Pharmacologist, 15. 254 (1973). R. M. Grimes, K. B. A l t o n and C. Shaw, Is, 254 (1973). G. S a k a l i s , E. Pearson. E. Kermani and S . Gerahon. Curr. Ther. Us.. C l i n . Erp., Is, 268 (1973). F. Barzaghi, R. Fournex and P. Mantegazza. Armelm.-Forsch., 23. 683 (1973). A. Bauer, P. Danneberg, K.-H. Weber and K. Minck, J. Med. Chem., l6, 1 0 1 1 (1973). G. R. A l l e n , J r . , V. G. De Vries, E. N. G r e e n b l a t t , R. L i t t e l l . F. J. HcEvoy and D. 8 . %ran, ibii., 1 6 , 949 (1973). K . P. S z g h and 0. S. Bhandari, Arzneim.-Forach., 23, 973 (1973). K . Jepson and G. Beaumont, J. I n t . Mad. Rea., 1 .145 (1973). J. L. Neumeyer and K . K . Weinhardt, J. Med. Chem., 2,808 (1973). V . Nacci, G. F i l a c c h i o n i , G. C. P o r r e t t a . G. S t e f a n c i c h and A. G u a i t a n i , Farmaco, Ed. Sci.. 28, 949 (1973). R. S a r g e s , J. R. T r e t t e r , S. S . Tenen and A. Weissman, J. Med. Chem., l6. 1003 (1973). H . OelschlYger, W. A. Behrendt and H. Hoffmann, Arzneim.-Forsch., 23, 802 (1973). J . R. Stevens, Arch. Gen. P s y c h i a t r y , 29. 177 (1973). S . Matthysse, Fed. P r o c . , Fed. Amr. SOC. Exp. B i o l . , 32, 200 (1973). N.-E. Anden and G. S t o c k , J. Pharm. Pharmacol., 25, 346 (1973). G. B a r t h o l i n i , K . G. Lloyd and H. S t a d l e r , L i f e Sci., l3. No. 4 , v i (1973). H . S t a d l e r , K. G. Lloyd, 14. Gadea-Ciria and G. B a r t h o l i n i . B r a i n R e 8 . . 55, 476 (1973). H. Corrodi, K . Fuxe and P. Lindbrink, 43, 397 (1972). B. S . Bunney. J . R. Walters. R. H . Roth and .K.G Aghajanian. J. Pharmacol. Exp. Ther.. 560 (1973). G. S t o c k , T. Magnusson and N.-E. Anden, Naunyn-Schmiedeberg'a Arch. F'harmakol. Exp. P a t h o l . 278, 347 (1973). R. Roth, J. R. Waltars and G. K . Aghajanian, L i f e S c i . , 12, No. 8. CxXXiX (1973). R. A. L a h t i and E. G. Losey, Res. Cormrmn. Chem. P a t h . Pharmacol.. 1. 31 (1974). H . Asper, M. B a g g i o l i n i . H. R. Burki, H. Lauener, W. Ruch and G. S t i l l e , 287 (1973). I. M. N i e l s e n , B. F j a l l a n d , V. Pedersen and M. Nymark, Psychopharmecologia. 2,195 (1974). J. L. Meek and N . H. Neff, J. Pharmacol. Exp. Ther.. 570 (1973). H. H. Keller, G. B a r t h o l i n i and A. P l e t s c h e r , Eur. J. Pharmacol.. 23, 183 (1973). C. D. Wise and L. S t e i n , S c i e n c e , 181,344 (1973). R. J . Wyatt, D. L. Murphy, R. Belmaker, S . Cohen, C. H. Donnelly and W. P o l l i n , 1 7 9 , 916 (1973). R. J. Wyatt, J. M. Saavedra and J. Axelrod, Am. J. P s y c h i a t r y , 130, 754 (1973). J. M. Saavedra, J. T. Coyle and J. Axelrod, J. Neurochem., 0, 743 (1973). N . Narasimhachari and H. E. Himrich, L i f e S c i . XI, g, 475 (1973). H . E . Himwich, N . Naraaimhachari, B. Heller, J. S. Spaide, L. Haakovec, M. Fujimori and K. Tabushi, "Chem1C.d l i o d i f i c a t i o n of Brain Function", Ed. H. C. S a b e l l i , R a w n P r e s s , New York, 1973. p. 297. R. J. Wyatt, L. R. Mandell, H. S. Ahn, R. W. Walker and W. J. A. Vanden Heuvel, Peychopharmacologia, 31, 265 (1973). P. Ladiiron, L i f e S c i . , 12,No. 6 , x c i i i (1973). W. C. Korevaar, M. A. Geyer, S. Knapp, L. L. Hsu and A. J. Mandell, Nature, Nev B i o l . , 245, 244 (1973). D. hlen6trey. J. Decaud-Gasarabwe and J. N. Besson, Eur. J. Pharmacol.. 2 4 , 158 (1973). P. Lidbrink and L.-0. Farnebo, Neuropharmacology, l2, 1087 (1973). R . Guerrero-Figueroa. E . Guerreo-Figueroa, G. A. Sneed and M. J. Kennedy, Curr. Ther. Res., C l i n . Exp., Ifr. 137 (1974). M. Doteuchi and E . Costa, N e u r o p h a m c o l o g y . 12,1059 (1973). L. F. Quenzer, R. S. Feldman and J. W. Moore, Psychopharmacologia. 3, 8 1 (1974). K. Meguro, H . Tawada. H. Miyano. Y. S a t 0 and
u.,
u.,
185,
w...g,
184.
-
u..
Chapter 2.
Narcotic Antagonists and Analgesics
Robert A. Hardy, Jr., Lederle Laboratories, A Division of American Cyanamid Company, Pearl River, New York
-
m i n g 1973, c l i n i c a l t r i a l reports on new agents remained Introduction few. Further d e t a i l s on agonists and antagonists described i n 1972 abs t r a c t s and preliminary reports appeared, indicating continuation of these investigations. !Be variety of new s t r u c t u r e types with analgesic or antagonist a c t i v i t y appears t o have declined i n 1973. Ebth analgesics and antagonists are t r e a t e d together i n s t r u c t u r e - a c t i v i t y discussion as d i f ferences between them remain very subtle indeed (e.g. diastereomers and enantiomers w i t h opposite actions ). Several reviews are noteworthy: IIhe well known analgesic publicat i o n s of the l a t e Dr. &than B. Eddy were brought t o a close w i t h an overview of 75 years of research toward solutions f o r morphine-type drug dependence .1 m e proceedings of an International Symposium on Agonists and Antagonists (1971) were published.2 Reviews on pentazocine3,4 and strong analgesics5 appeared.
-
New Clinical Studies Current reports do not provide d e f i n i t i v e informat i o n on any new compounds. Previous investigations a r e continuing and it remains premature t o assess t h e i r c l i n i c a l importance. However, the outlook is optimistic that significant c l i n i c a l advances a r e i n progress considering the variety of agonist and antagonist agents being studied. A 1O:l thadone:naloxone combination evaluated i n man provoked few side effects? advancing t o c l i n i c a l trials the previously described concept of using a parenterally e f f e c t i v e antagonist co bined with an o r a l l y a c t i v e narcotic t o prevent abuse of o r a l medication.
7
b, -c%j
H Q---
I
I1
An e a r l y c l i n i c a l t r i a l of oxilorphan [(- )-Bc-2605, Ia] as an antagonist f o r potential narcotic ad i c t i o n treatment indicated a d i u r e t i c e f f e c t a t low doses (2-4 mg sc).' In laboratory animals Ia was a naloxone-like antagonist, w a s almost equally e f f e c t i v e o r a l l y as parenterally, and had a prolonged d ~ r a t i o n . ~Preliminary c l i n i c a l r e s u l t s a r e cons i s t e n t with this p r o f i l e . Ihe N-cyclobutylmethyl analog (-)-I3 was a highly potent, dependence-free agonist i n animals, and preliminary c l i n i c a l t ials have been i n i t i a t e d with r e s u l t s comparable t o the animal model5 (See a l s o m p t e r 5 1.
12
Sect. I
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CNS Agents
Gordon, Ed.
Structure-Activity; Agents under Investigation iates and Wrphinans - Details of the total synthesis of the BC-2605 zries (1)were described.9 A new synthetic approach was utilized representing a &jor advance in synthetic methods for Grphinans and related compounds, including isomorphinans and hasubanan analogs. It provides naloxone analogs which are not derived from the opium alkaloid thebaine, but which have comparable activities. (- )-Ib shared antagonist signs in monkeys, and was a potent analgesic equivalent to morphine in mice (hotplate and Nilsen tests).1° Ib racemate was morphine-like in monkeys (suppression of abstinence) and was similar to (-)-Ib in mice.1° The (+)isomer was morphine-like in monkeys, less potent but somewhat longer acting, and was weak to inactive in mice. From a series of morphine derivatives recently synthesized in Hungary, azidomorphine (IIa)and azidocodeine (IIb) proved to be about 300 times and about 1 times more potent than morphine, respectively (rat hotplate).113’ -ever, physical dependence capacity (pDc) in mice, rats and monkeys was low at equianalgesic doses, unusual for a potent N-methyl opiate structure. A levallorphan metabolite isolated from rat urine was established by X-ray analysis as the @-hydroxy derivative 111.13 It was then synthesized and, like the parent drug, was an antagonist without analgesic activity. An analogous 68-h oxy metabolite of naltrexone, formed by mese examples represent the first reports reduction, was found in of C-6 isomorphine configuration metabolites.
-.IF
The bicyclic 5-;-hydroxyphenylmorphan series was extended to include the N-propyl, N-cyclopropylmethyl (CPM and N-ally1 derivatives Na,b,c, both as racemates and the (+)-is0mers.~5 They were pentazocine-like analgesics with weak or no antagonist actions, and showed no PDC in monkeys. Thus, potent antagonists cannot be obtained in this series by replacing the N-methyl group of a potent agonist with claseical antagonist substituents (IVd-3 x morphine; high PM:). 6,7-%nzomorphans - Very little new structure-activity information concerning benzomorphans appeared during 1973. The s=-amine @-hydroxy6,7-benzomorphan had analgesic activity about 4 that of codeine (mouse hot-plate) while the corresponding N-methyl derivative was inactive, the reverse of the usual relative potency.16 Rntazocine underwent a chemical change in aqueous acid conditions used to hydrolize glucuronides.17 The mass spectral fragmentation of 93- and gg-alkyl-6,7-benzomorphans, including pentazocine, indicated that the configuration at C-9 could be assigned even when only one isomer of a diastereomeric pair was
Chap. 2
Narcotic Antagonists, Analgesics
Hardy
13
18
available.
Aminotetralins - Additional details of the synthesis of 49 1,3-bridged-2aminotetralins (V) were pub1ished;lg several were equal to or more potent than morphine (rat tail-flick). V was the longest acting a tagonist tested in the Univ. of Michigan morphine-dependent monkeys." It displayed twice the duration of cyclazocine and was about 1/5 as potent as nalorphine as an antagonist. In mice it was equivalent to morphine (hotplate ).
C,
-OH
VI
V
Three diastereomeric pairs of tetralins VI were synthesized as methadone analogs.20 VIa were analgesics comparable to meperidine (mouse hotplate) with little differences-in the potency of the hiastereonkrs; substitution of hydroxyl for hydrogen increased potency somewhat. Among a series of potential antiparkinson agents, 2-methylamino-5-methoxytetralin shared analgesic activity in the codeine-propoxyphene range (mouse tailpinch), but was more toxic.21 Piperidines and Pyrrolidines - New antagonist-analgesics were reported in the N-alkyl-norketobemidone series.22 The N-awl, hexyl and heptyl compounds VIIa,b,c were strong agonists with weak antagonist properties, and no PIX! in monkeys. VIIa was an analgesic nearly 3 times more potent than the parent VIId (mouse hot-plate). This contrasts with the more traditional N-propyl, CPM and allyl analogs which were not antegonists but weak to medium strength agonists with high mX:.
c, -Heptyl d, - M VII
VIII, (- )-isomer
In the pyrrolidine series, additional details of the selective antagonist actions of N-CPM and N-ally1 analogs of profadol appeared,23,24 and CI-747 (VIII) was selected for detailed evaluation. Its analgesic potency was 40-50 times pentazocine (mouse antiwrithing ) with antagonist properties 5-6 times pentazocine. Ihe absolute stereochemistry and analgesic properties of the diastereomeric 3-allyl-prodine analogs who deduced similar conclusions. M was 13-15 times more potent than morphine (mouse hot-plate), and its (+)-enantiomer was 40 times morphine and 260 times its (-)-isomer. It was suggested that the equatorial allyl group enhanced receptor association.
14
Sect. I
-
CNS Agents
Gordon, Ed.
Similar a solute stereochemical studies of other prodines were also described.2 r29
ll
CON(C5
l2
(c6'5 a, R
-
L
R X p-C1; b, R = 3-CF -4-Cl 3
A series of 2,2-diphenyl-4-piperidinobutyramides (X) were studied in which the goal was separation of analgesic and antidiarrheal (constipating) activities.3° Ioperamide (Xa) and fluperamide ( X b ) were selected for further investigation each being more potent than diphenoxylate and codeine in protecting against castor-oil-induced diarrhea and very weak analgesics (rat,warm water tail-withdrawal). Closely related analogs combined strong analgesic properties with strong antidiarrheal activity. In this study, potent analgesic activity was an unwanted side effect. Several additional piperidines were active analgesics. The 3-methyl derivative of the potent narcotic fentanyl was 10 times more potent than the parent drug .3l N-[2- (2-Oxazolidinone-5-yl)ethyl]-4-phenyl-4-propionoxypiperidine shared morphine-like analgesic$ t e n ~ y , 3and ~ an N-(2indanyl )-analog had meperidine-like potency.
Diphenylpropylamines and Mscellaneous Compoundg - Among a series of thiophthalanes studied for analgesic activity,j4 Iu 6-062 (XI) was the most active. It was approximately equal to morphine (mice, hot-plate, tail-pinch and Nilsen tests ), produced a Straub-tail reaction and was antagonized by nalorphine. Similar thiophthalanes with a propylamino side chain had been previously studied as antidepressants, but were not analgesics and did not produce a Straub-tail.
XI
XI1
Another approach to the separation of antidiarrheal from narcotic analgesic activity was the comparison of Straub-tai V8. CoMtipating effects.35 CI-750 (XII), selected for evaluation,3 did not produce a Straub-tail, showed only weak analgesic activity and had a constipating effect about equal to diphenoxylate. Piperidinoethyl and pyrrolidinoethyl analogs of XI1 with a 1-dimethylamino group had potent narcotic actions.
2
Agonist and antagonist properties of vimi 1 stereoisomers (XIII, previously known as diviminol) were described.j? This compound has 3 asymmetric centers and 6 stereoisomers. Analgesic activity resided in a
Chap. 2
Narcotic Antagonists, Analgesics
Hardy
15
(-)-isomer (€$; several times morphine, some PDC) and antagonist activity in a (+)-isomer (S,; nalorphine-like)which were not enantiomers of each other. No other isomers had analgesic or antagonist activity. In the
racemate, the agonistic activity of % is apparently partially opposed by the antagonistic E$ isomer. The result is a racemate with codeine-range analgesic activity and a low order of pzx;'. Analgesic activity was found in a group of N-substituted cyclohexylmethylbenzamides, and AH 791 (XIV) was equipotent to morphine (mice, antiwrithing and hot-plate) and showed high PDC in monkeys. !Bis represents a structurg distinctly different from previous high PDC narcotic-like compounds . 3 The dissociative anesthetic etoxadrol ( X V ) produced analgesia at low subanesthetic doses (0.5-1 mg), measured by elevation of the electrical stimlation threshold in monkeys.39 It was comparable to morphine,but shorter in duration. XV was not active in mice (antiwrithing), perhaps since dissociative anesthetics produce different effects in rodents vg. higher species. Further studies as a parenteral analgesic were suggested. 0
(c21f,
xv
XVI A study of 24 monoaryl- and diarylphosphorus analogs of methadone revealed moderate analgesic activity in several compounds; XV was 1/4-1/5 the potency of morphine (mice, hot-plate and antiwrithing Unfortunately, the toxicity was highest in compounds with the best analgesic activity. Pharmacological implicat of the conformations of methadone and isomethadone were analyzed. Aniinflanmttory Analgesics - !&is class of compounds is discussed in detail in Chapter 20. Studies of arylalkanoic acids and derivatives continue unabated. Details of analgesic testing of these agents are frequently incomplete PharmacolORy, Biochemistry and bktabolism - !be variety of studies covering methods, mechanism of action, biochemistry, etc. continues to increase. Detailed discussion of these is outside the scope of this review; however, the following selected examples are illustrative of current trends. A full paper on biodegradable poly-lactic acid long-acting delivery
.
16
Sect. I
-
CNS Agents
Gordon, Ed.
systems f o r narcotic antagonists a ~ p e a r e d , ~following 3 up the i n i t i a l a b s t r a c t s . The use of chronic spinal dogs dependent on morphine w a s described f o r quantitative assays of antagonists .44 &tho@ f o r producing antibodies t o morphine f o r radioinnnunoassay were reviewed. F’urther studies of specific opiate rece or s i t e s based on stereos p e c i f i c binding i n nervous t i s s u e a eared.a The i n i t i a l r e s u l t s were confirmed by other investigators. 471@ m e r o l e of sodium i n enhancing antagonist binding but decreasing agonist binding i n v i t r o w a s found as an important q u a l i t a t i v e difference t h a t may be relevant t o the divergent pharmacological properties of antagonists and agonists .46 Several additional metabolic findings a r e of i n t e r e s t . The formation of codeine from morphine (or heroin) w a s observed i n a significant increase of 0-methyl transferase a c t i v i t y leading t o increased codeine i n addict urine could be a specific marker f o r the addiction s t a t e . The long sought primary amine metabolite from levomethadyl acetate was i d e n t i f i e d by combined gas chromatography-mass spectrometry (gc-ms) of r a t t i s s u e and plasma samples, and was found t o be a highly potent analgesic (1.5 mg/kg sc, mouse antiwrithing).5O Thus, the general tendency of mono- and d i demethyl congeners t o have a low order of a c t i v i t y does not apply i n t h i s s e r i e s . Both mono- and di-demethyl metabolites of g-propoxyphene were observed i n man, using combined gc-ms and deuterium-labelled propoxyphenes .51 a-(+)-Norpropoxyphene was the major metabolite, and was about 1/10as active as the parent drug (mouse antiwrithing, and rat t a i l f l i c k ) .52 The r o l e of biogenic amines a8 r e l a t e d t o narcotic e f f e c t s continues t o be an active f i e l d . Further studies appeared concerning enhanced cerebral serotonin turnover ,53 balance w i t h the catecholamine s y ~ t e m , 5 ~ dopamine turnover i n p e striatum,55 and homovanillic acid content i n t h e caudate nucleus .5 Additional d e t a i l s of the cyclic-AMP antagonism of mo phine analgesia,57 and i t s e f f e c t on tolerance and physical dependence5 were a l s o published.
fi
Referenees 1. N. B. Eddy and E. L. b y , Science, 181, 407 (1973). 2. H. W. Kosterlitz, H. 0. J. Collier and J. E. Villarreal (Eds), Agonist and Antagonist Actions of Narcotic Analgesic Ih-ugs, Univ. F’ark Press, Ehltimore (1973). 3. J. P. h p e , h g s , b 1 (1973). 4. R. N. Brogden, T. M. Speight and G. S. Avery, Rrugs, 6 (1973). 5. W. B. b a n and J. D. Wrrison, Drugs, b 108 (1973). 6. S. D. Aarwatikar and R. R. Knowles, Clin. Fharmacol. ?her., 14, 941 (1973) l 230 (1973). 7. I. J. Pachter, Science, & 8. J. G. M t t and D. R. Jasinsky, Rmmacologist, lI_, 240 (1973). 9. I. Wnkovi6, T. T. Conway, H. Wong, Y. G. Fkrron, I. J. Pschter and 7910 (1973); cf a l s o Qem. Abstr., B. Belleau, J. Am. Chem. SOC., 79, 18931~(1973).
Chap. 2
Narcotic Antagonists, Analgesics
17
Hardy
H. H. Swain, J. E. V i l l a r r e a l and M. H. Seevers, Addendum, Report 35th Ann. M t g . Corn. on Problems o f Rrug Dependence, Chapel H i l l , N. c., MY 21-23, 1973. 11. J. Knoll, S. Mrst and K. Kelemen, J. Pharm. pharmacol., 22, 929 (1973 1 12. J. Knoll, pharmacol. Res. Commun., 175 (1973). 13 J. F. Blaunt, E. Mhacsi, F. M. Vane and G. J. Wnnering, J. E d . Chem., 16, 352 (1973). 14. E. J. Cone, Tetrahedron I e t t e r s , 2607 (1973). 1.5 * H. H. Chg, T. a - i s h i and E. L. My, J. E d . Chem., 17 133 (1974). 16. J. J. Fauley and J. B. IaPidus, l6, 181 (1973f 17 * D. P. Vaughan and A. H. Beckett, J. Pharm. Pharmacol., 2_2 993 (1973 1. 18. D. I?. Vaughan and A. H. E c k e t t , i b i d . , 22, 895 (1973). 19. M. E. Freed, J. R. Potoski, E. H. Freed, G. L. Conklin and J. L. &Us, J. E d . Chem., 16, 595 (1973). 20. E. M. Kandeel and A. R. W r t i n , LbL., 16, 947 (1973). 10.
z.,
21. 22. 23
Y. C. W t i n , C. H. Jarboe, R. A. Krause, K. R. I p n , D. k n n i g a n and J. B. W.land, 6, 147 (1973). T. a - i s h i and E. L. Wy, 16,1376 (1973). R. E. B3wman, H. 0. J. C o l l i e r , P. J. Hattersley, I. M. Lockhart, D. J. Peters; C. Schneider, N.-E. Webb and M. Wright, g., 16,
z. , s.,
~
1177 (1973). 24. 25 26. 27. 28. 29.
30
31
33 34.
36 35
38
u.,
u., u., w., u.,
u.,
(19731
W. J. Welstead, Jr., G. C. Helsley, C. R. Taylor, L. B. Turnbull, J. P. DaVanzo, W. H. Funderburk, and R. S. Alphin, J. &d. Chem., 16,
32
37
R. E. EOwman, H. 0. J. Collier, I. M. Lockhart, C. Schneider, N. E. Webb and M. Wright, l6, 1181 (1973). K. H. kll and P. S. Fbrtoghese, 16 203 (1973). K. H. k11 and P. S. Portoghese, 589 (1973). M. A. B r i o , G. Camia and A. F. Casy, 16, 592 (1973). D. L. k s o n and P. S. Portoghese, l6, 1%(1973). P. S. Portoghese, Z. S. D. Gomaa, D. L. k s o n and E. Shefter, ibid., 16, 199 (1973 1. R. A. Stokbroekx, J. Vandenberk, A. H. M. T. Van Heertum, G. M. L. W. van Iaar, M. J. M. C. Van der Aa, W. F. M. Van Ever and P. A. J. Janssen, 16, 782 (1973). T. N. Riley, D. B. Hale and M. C. Wilson, J. Pharm. Sci., 62, 983
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m9 (1973).
z.,
E. Solomons and J. Sam, l6, 1330 (1973). B. Fjalland, V. Pedersen, N. lassen, P. V. Petersen and I. M l l e r Nielsen, Acta pharmacol. Toxicol., 32, 278 (1973). D. E. J h t l e r , R. F. Myer, S. M. Alexander, P. €kiss and J. A. Kennedy, J. Md. Chem., 16, 49 (1973). P. Ittss, J. A. Kennedy, J. N. Wiley, J. V i l l a r r e a l and D. E. J h t l e r , J. pharmacol. Exp. 'lher., 186, 183 (1973 ). D. Della Bella, V. Ferrari, V. Frigeni and P. Iualdi, Nature New Biol., 241, 282 (1973 ). R. T. Brittain, D. N. Kellett, M. L. Neat and R. Stables, Brit. J. Pharmacol., 158P (1973).
9,
18 39 40. 41. 42. 43
9
Sect. I
- CNS Agents
Gordon, Ed.
A. H. Tang and J. D. Kirch, Anesth. Analg. Curr. Res., 52, 577 (1973). W. H. Shelver, M. Schreibman, N. S. Tanner and V. Subba Rao, J. Md.
Chem., 17, I20 (1974 ). H. B. m g i , J. D. l b n i t z and E. Shefter, Nature New Blol., 244, 186 (1973 ) * J. G. Henkel, K. H. Bell and P. S. Brtoghese, J. Wd. Chem., 17 -, 124
(1974 1
J. H. R. Woodland, S. Yolles, D. A. Blake, M. Eelrich and F. J. Myer, 16, 897 (1973 ) * W. R. Mutin, C. G. Eades and J. A. ?hapson, Fed. Proc. 32, 687 j
44. 45
46. 47. 48. 49 50
(1973 1-
S. Spector, B. Brkowitz, E. J. Flynn and B. Fkskar, pharmacol. Rev. 25, 281 (1973 1. C. B. Fkrt, G. Pasternak and S. H. Snyder, Science, 182, 1359 (1973). E. J. Simon, J. M. Hiller and I. Edelman, €?roc. Natl. Acad. Sci., 70,
1947 (1973 1.
54.
L. Terenius, Acts pharmacol. Toxicol., 377 (1973). U. Edrner and S. Abbott, Experientia, 24, 180 (1973). R. E. BLllings, R. Booher, S. b i t s , A. Pohland and R. E. kbkhon, J. Wd. Chem., l6, 305 (1973). R. E. McMhon, H. R. Sullivan, S. L. lXle and F. J. &shall, Life Sci, 12 Pt 11, 463 (1973). R. Nickander and S. Smits, Pharmacologist, 1L 203 (1973). G. G. Yarbrough, D. M. Bxbaum and E. Sanders-&sh, J. Eharmacol. %P. !her., 14, 328 (1973). D. M. Wbaum, G. G. Yarbrough and M. E. Carter, G., %1 317
55 *
E. Costa, A. Carenzi, A. Guidotti and A. Revuelta, Life Sci.,
52
53
56 57 58.
(1973 1
m i (1973).
12(4),
L. Ahtee and I. Kd&idinen, Amopean J. pharmacol., 22, 206 (1973). I. K. Ho, H. H. Loh and E. L. Way, J. Fharmacol, Ekp. !her., l@j,
336 (1.9731.
I. K. Ho, H. H. Loh and E. L. Way,
u.,%1
347 (3.973).
Chapter 3.
Antiparkinsonism Drugs
Vernon G. V e r n i e r , E. I . du Pont de Nemours
?C
Co., I n c . , Newark, De.
-
General Knowledge of t h e n a t u r e o f t h e biochemical l e s i o n of P a r k i n s o n ' s d i s e a s e and t h e a v a i l a b i l i t y of r a t i o n a l c o r r e c t i v e t h e r a p y c o n t i n u e s t o s p u r a c t i v i t y t o f i n d improved and novel e n t i t i e s w i t h e x i s t i n g and d i f f e r e n t nodes of a c t i o n . Many u s e f u l reviews are a v a i l a b l e i n t h i s f i e l d . T h i s c h a p t e r updates t h e compilation by Vernier1. Levodopa i s reviewed by McDowell' and it and o t h e r developments are covered by Calne3. The l a r g e and growing amantadine l i t e r a t u r e i s w e l l analyzed by P a r k e s 4 . T h i s review w i l l cover t h e r a p e u t i c a g e n t s and leads under f o u r mechanisms of a c t i o n : 1. dopamine replacement, 2. dopamine r e l e a s e , 3. dopamine r e c e p t o r s t i m u l a n t s and 4. a n t i c h o l i n e r g i c s . Each agent w i l l be d i s c u s s e d under i t s presumed major mode of a c t i o n even though t h e evidence f o r it may not be d e f i n i t i v e o r t h e agent may a c t by s e v e r a l mechanisms.
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Dopamine replacement This most e f f e c t i v e t r e a t m e n t f o r P a r k i n s o n ' s disease i s s u p p l i e d by t h e - n a t u r a l l y - o c c u r r i n g amino a c i d levodopa ( I, L3,4-dihydro~yphenylalanine~'~), I n t h e u s u a l o r a l doses o f 2 . 5 t o 6 g d a i l y it e n t e r s t h e b r a i n and i s decarboxylated t o form dopamine (11)
I
I1
which presumably r e p l e n i s h e s t h e d e f i c i e n t n e u r o t r a n s m i t t e r s t o r e s i n t h e basal g a n g l i a r e s u l t i n g from midbrain s u b s t a n t i a n i g r a l e s i o n s . I n 60 t o 80% o f c a s e s a k i n e s i a , r i g i d i t y and t o a lesser e x t e n t tremor a r e moderately t o markedly improved, b u t management i s d i f f i c u l t . T h i s i s due t o numerous s i d e e f f e c t s , t h e n e c e s s i t y o f g i v i n g many doses d a i l y , and t h e slowly i n c r e a s i n g dose t i t r a t i o n , l e a d i n g t o a long induct i o n time f o r o p t i m a l e f f e c t s . Some p a t i e n t s have a s h o r t and/or v a r i a b l e t i m e of symptomatic r e l i e f f o l l o w i n g each dose. Sudden f r e e z i n g , t h e "ono f f e f f e c t , " i s o f t e n noted0. I t s causes are under study and s e v e r a l approaches t o i t s a l l e v i a t i o n are i n development ( d e t e r m i n a t i o n s o f levodopa plasma l e v e l s f o r optimal t i m e and dose t i t r a t i o n , s u s t a i n e d r e l e a s e formulations, e tc )
..
P r i n c i p a l levodopa s i d e e f f e c t s are i n d e c r e a s i n g o r d e r of incidence: 1. Nausea and vomiting, 2. p o s t u r a l hypotension, 3. c a r d i a c a r r h y t h m i a s , 4. abnormal i n v o l u n t a r y movements and 5. p s y c h i a t r i c d i s t u r b a n c e s . Considerable r e s e a r c h e f f o r t i s d i r e c t e d toward t h e d e t e r m i n a t i o n of how much o f t h e o r i g i n a l t r e a t m e n t r a t i o n a l e can b e s u b s t a n t i a t e d . Although g e n e r a l l y a g r e a t e r t h a n o r i g i n a l l y expected complexity i s being
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found, most r e s u l t s a r e consistent with o r i g i n a l expectations. Carbidopa and benserazide, two agents which a r e potent aromatic amino acid decarboxylase i n h i b i t o r s in v i t r o and in V
.
Early r e p o r t s of t h e biochemistry and pharmacology of carbidopa (111, MK 486, t h e L form of MK 485, a l s o r e f e r r e d t o as HMD) indicated i t s
I11
IV
e x t r a c e r e b r a l d i s t r i b u t i o n and potent in v i t r o and in v i v o i n h i b i t i o n of aromatic amino a c i d decarboxylasell. I t s i n t e r a c t i o n s with levodopa i n Carbidopa enhanced t h e animals were characterized by L o t t i and Porter’’. a b i l i t y of levodopa t o increase motor a c t i v i t y of mice and rats and t o reverse reserpine-induced hypothermia and suppression of locomotion and p t o s i s . Brain dopamine l e v e l s were concomitantly enhanced. I n c o n t r a s t levodopa emesis i n dogs and pigeons was attenuated. C l i n i c a l e y e r i e n c e with carbidopa and levodopa has been extensive
and f a ~ o r a b l e l ~, , ~Carbidopa alone had no influence on Parkinsonian p a t i e n t s and i s reported t o be q u i t e non-toxic. It r a i s e d plasma l e v e l s of levodopa about five-fold, thus i n d i c a t i n g about an 80% reduction of t h e required levodopa alone dose. Carbidopa doses of 100-300 mg/day combined with levodopa i n c o n t r o l l e d t r i a l s r e s u l t e d i n a rapid onset of therap e u t i c e f f e c t ( a few days o r weeks compared with months with levodopa alone) , and s t r i k i n g l y reduced t h e incidence of nausea and vomiting. P a t i e n t s on long term combined therapy achieved superior r e l i e f . Combined treatment m a y reduce cardiac arrhythmias , but t h e incidence of involuntary movements i s increased. Several groups found a 1:lO combination most usef u l with a d a i l y dose of carbidopa 100 mg: levodopa 1000 mg. Marsden14 suggests t h a t it may become t h e treatment of choice. The pharmacology of and c l i n i c a l experience with benserazide (IV,
Ro 4-4602) i s generally similar t o t h a t of carbidopalO. It i s somewhat l e s s potent in v i t r o as a decarboxylase i n h i b i t o r but in V i V O and i n t h e ~ ,d~i f~f e.r s i n c l i n i c i t s potency i s similar t o t h a t of c a r b i d ~ p a ~ , ~It penetrating t h e b r a i n i n animals at high doses. Benserazide administrat i o n , l i k e carbidopa, increases absorption of levodopa from t h e g u t , increasing plasma l e v e l s , and blocks p e r i p h e r a l conversion t o dopamine. I n t h e b r a i n t h e i n h i b i t o r s increase levodopa penetration and catecholamine ( dopamine and norepinephrine) l e v e l s .
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C l i n i c a l l y benserazide+levodopa treatment improved t h e t h e r a p e u t i c r e s u l t s , decreased t h e required levodopa dose ( t o about 1 / 5 ) and decreased t h e incidence of nausea and vomiting. It generally did not decrease t h e involuntary movements but increased them. Benserazide seems t o be well t o l e r a t e d on a long term administration a t t h e 100-400 mg/day l e v e l s , with few t r a n s i e n t changes i n l i v e r biopsies and enzyme levels15.
-
Dopamine r e l e a s e r s Schwab noted i n 1968 t h e antiparkinsonism e f f e c t of amantadine ( V ) , an a n t i v i r a l agent which has s i n c e been extensively
V
VI
t e s t e d 1 7 and approved f o r t h a t a d d i t i o n a l i n d i c a t i o n . Amantadine a f f e c t s catecholamines, primarily dopamine, and had no a n t i c h o l i n e r g i c e f f e c t s 1 8 . I t caused pressor responses i n t h e dopamine-primed dog with much l e s s e f f e c t upon norepinephrine , suggesting predominately dopamine r e l e a s e l a 3 19. Amantadine antagonized e f f e c t s of major t r a n q u i l i z e r s on s e v e r a l kinds of behavior, including t h e conditioned avoidance response22 , a f f e c t e d t h e r o t a t i o n a l a c t i v i t y of s t r i a t a l - l e s i o n e d rats and mice23 and i n t e r a c t e d wit*h a p ~ m o r p h i n,25. e ~ ~ This and considerable other biochemical and pharmacological evidence i s generally consistent with t h e r e l e a s e of s t o r e d dopamine as a m a o r element i n i t s action together with f a c i l i t a t i o n of neural release2g-28. Amantadine a l s o increased synthesis of levodopa and presumably dopamine i n rat striatum2’. Although many recent r e p o r t s d e a l with t h e s e aspects of t h e animal pharmacology, t h e exact mechanism i s not y e t f u l l y established. I n p a t i e n t s t h e onset of a c t i o n i s prompt, faster than levodopa but of l e s s i n t e n s i t y , with fewer s i d e e f f e c t s than levodopa, p a r t i c u l a r l y nausea and vomiting and dyskinesias. A t doses of 200-300 mg/day maximum b e n e f i t i s achieved soon a f t e r beginning drug and t h i s e f f e c t may be sust a i n e d over long periods, although some r e p o r t s i n d i c a t e a decline i n effect. Although no c l i n i c a l r e p o r t s on D 145 (VI, DMAA) have been found, it i s a c l o s e congener of amantadine which shows catecholamine i n t e r a c t i o n s generally similar t o it, suggesting capacity f o r dopamine r e l e a s e . I t may a l s o have some capacity f o r d i r e c t stimulation of dopamine receptors3’. Moderate t o high i . v . doses of D 145 decreased decerebrate r i g i d i t y i n cats31. Other biochemical findings have been noted32. Carmantadine (VII, Sch 15427) i s s t r u c t u r a l l y r e l a t e d t o amantadine33. It shares some of i t s pharmacological a c t i o n s , w a s e f f e c t i v e i n a
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head-turning t e s t 3 4 , and i s i n e a r l y c l i n i c a l t r i a l s . Dopamantine ( V I I I ) combined elements of both mantadine and dopamine i n i t s s t r u c t u r e , shares some pharmacological e f f e c t s of amantadine and i s i n e a r l y c l i n i c a l trials35.
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Dopmine receptor stimulants A chemical rearrangement of morphine y i e l d s apomorphine ( I X ) which i s now known t o s t i m u l a t e dopaminergic r e c e p t o r s .
Hd
X
’OH IX
S ~ h w a bdiscovered ~~ i n 1951 t h a t it r e l i e v e d tremor and r i i d i t y of parkinsonism. T h i s has been confirmed by CotziasJ7, Castaignef8 and others. It markedly diminished tremor i n monkeys39. Apomorphine i s not a p r a c t i c a l t h e r a p e u t i c agent (poor o r a l absorption requiring p a r e n t e r a l admini s t r a t i o n , s h o r t duration of a c t i o n , high incidence of nausea and emesis , i n s t a b i l i t y ) but i s an important s y n t h e t i c and b i o l o g i c a l s t a r t i n g point i n t h e search f o r new agents, s i n c e it contains common elements of t h e dopamine s t r u c t u r e . Experiments w i t h apomorphine have shed l i g h t on t h e a c t i o n of levodopa and dopamine i n t h e treatment of parkinsonism and a l s o t h e nature of drug-induced parkinsonism which accompanied t h e use of major t r a n q u i l i z e r s which antagonize apomorphine emesis and stereotypy4’. Originally tested i n cardiovascular disease , t r i v a s t a l (X, ET 495) has been proposed as a dopamine receptor stimulant w i t h b e t t e r o r a l a b s o r p I n. addition t o i t s t i o n and longer duration of a c t i o n t h a n a p ~ m o r p h i n e ~ ~ d i r e c t e f f e c t on t h e receptors t h e data suggested a weak dopamine and norepinephrine r e l e a s i n g e f f e c t . T r i v a s t a l blocked caudate EEG spindles as d i d a p ~ m o r p h i n eand ~ ~ reduced tremor i n monkeys w i t h midbrain lesions43. Others have reported i t s drug dynamics and pharmacological actions44 ,45. Reports of c l i n i c a l t r i a l s 4 6 i n 125 p a t i e n t s at doses of 20 t o 240 mg/day l e d t o p o s i t i v e conclusion of b e n e f i t i n 4 s t u d i e s (involving 83 p a t i e n t s ) and negative assessment i n 3 s t u d i e s (involving 42 p a t i e n t s ) . Side e f f e c t s were s t r i k i n g and seen i n most p a t i e n t s but could be reduced w i t h lowered doses. Dyskinesias, l i k e those w i t h levodopa, were most frequent , w i t h nausea and vomiting, daytime drowsiness ( w i t h insomnia a t
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n i g h t ) a l s o found frequently. Less common were confusion, headaches and dizziness. Most t r i v a s t a l t r i a l s continued p a t i e n t s on previous medicat i o n including levodopa s o t h a t t h e t h e r a p e u t i c r o l e of t r i v a s t a l i s not yet clear. P r e c l i n i c a l data suggest t h a t NBTI ( X I ) a t f a i r l y high doses (40 mg/ kg i . p . ) caused tremors and stereotypy i n rodents reminiscent of apomor-
XI
phine, which was blocked by chlorpromazine and other major t r a n q u i l i z e r s but not by a n t i c h o l i n e r g i c s . It r e s t o r e d locomotor a c t i v i t y i n reserpinea ~ ~ It may stimulate dopaminergic depleted rats l i k e l e v o d ~ p ,47. receptors4*. Reports of i t s c l i n i c a l s t a t u s have not appeared.
-
The peripheral a n t i c h o l i n e r g i c e f f e c t s of benapryzine Anticholinergics (XII, BRL 1288) were considerably weaker than those o f trihexyphenidyl
XI1
XI11
In but i t s c e n t r a l e f f e c t s were nearly equivalent i n animals and man4'. a p i l o t t r i a l benapryzine a t higher doses than those of trihexyphenidyl was found t o be equivalent t o i t , but w i t h possibly fewer s i d e e f f e c t s 5 0 . Two o t h e r p a r t i a l l y c o n t r o l l e d t r i a l s (13 and 28 p a t i e n t s ) indicated Benapryzine may represent a t e f f e c t i v e n e s s and s a f e t y a t 200 mg/day5'. least p a r t i a l success i n separating c e n t r a l from p e r i p h e r a l a n t i choliner i c e f f e c t s , possibly as a r e s u l t of d i f f e r e n c e i n pharmacok i n e t i c s f 3 . Direct e f f e c t s have been demonstrated by iontophoretic a p p l i c a t i o n t o c o r t i c a l neurons5". The morphanthridine e l a n t r i n e (XIII, Ex 10-029) has been t e s t e d i n man and i t s pharmacological a c t i o n appears t o be mainly, but not exclus i v e l y , r e l a t e d t o i t s a n t i c h o l i n e r g i c action55. Two c l i n i c a l t r i a l s ( 2 5 and 13 p a t i e n t s ) r e p o r t e f f i c a c y alone a t doses of 30-60 mg/day and w i t h levodopaS6,57, Side e f f e c t s were predominately of t h e a n t i c h o l i n e r g i c type. A recent s u b s t a n t i a l m u l t i c l i n i c c o n t r o l l e d study58 i n 89 p a t i e n t s found t h a t e l a n t r i n e , 40 mg d a i l y , e f f e c t i v e l y reduced tremor and possibly r i g i d i t y more than t h e same dosage of trihexyphenidyl. E l a n t r i n e caused more dry mouth, Thus e l a n t r i n e was e f f e c t i v e but not c l e a r l y more select ive
.
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Comment E a r l i e r r e s e a r c h i n t e r e s t has s h i f t e d from a n t i c h o l i n e r g i c t o catecholamine a c t i o n s . Is t h e formation o f 6-hydroxydopamine, which causes a d r e n e r g i c nerve t e r m i n a l d e s t r u c t i o n , t h e cause of t h e biochemical l e s i o n of Parkinson's d i s e a s e ? If t r u e , new p o s s i b i l i t i e s f o r c u r e o r p r e v e n t i o n through i n f l u e n c i n g t h e cause could emerge from t h e i n t e n s i v e catecholamine r e s e a r c h e f f o r t . A d d i t i o n a l dopamine releasers w i l l probably be t e s t e d s i n c e t h i s mode of a c t i o n h a s been i d e n t i f i e d . E f f e c t i v e and s e l e c t i v e dopamine reuptake b l o c k e r s should be explored as another t h e r a p e u t i c approach. D i r e c t dopamine r e c e p t o r s t i m u l a t i o n i s probably t h e most a c t i v e p r e s e n t area, I n view o f t h e complex catecholamine physiology o t h e r n e u r o l o g i c a l t a r g e t u s e s f o r t h e s e l e a d s w i l l be sought and undoubtedly found. Comb i n a t i o n s of t h e l a s t 3 approaches w i l l b e p r i m a r i l y i n i t i a t e d by c l i n i cians.
New small animal methods , supplementing oxotremorine tremor , e t c , , have made p o s s i b l e s t u d y of a d r e n e r g i c a c t i o n s based mainly upon t h e changes i n locomotor a c t i v i t y and s t e r e o t y p y e x e r t e d by amphetamine and apomorphine and by drug e f f e c t s upon t u r n i n g behavior a f t e r s t r i a t a l l e s i o n s caused by 6-hydroxydopamine, e l e c t r o l y s i s and s u c t i o n . References V. G. V e r n i e r , i n "Annual Reports i n Medicinal Chemistry, 1970,"
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C. K. Cain, ed., Academic P r e s s , N.Y. (1971) p. 42. F. H. McDowell, e d . , Symposium on Levodopa i n P a r k i n s o n ' s Disease, Neurol., 22 (5, P a r t 21, 1 (1972). D. B. Calne, e d . , "Progress i n t h e Treatment o f Parkinsonism," Advances i n Neurol., 2, 1 (1973) Raven P r e s s , N.Y. J . D. P a r k e s , Amantadine, i n Recent Advances i n Drug Research, Academic P r e s s , N.Y. (1974) i n p r e s s . G. C. C o t z i a s , M. H. Van Woert and L. M. S c h i f f e r , New Eng. J . Med.,
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L a n g r a l l and C. Joseph, Neurol. , 22 ( 5 , P a r t 2) , 3 (1972). R. B. Godwin-Austen, Advances i n Neurol., 3, 23 (1973). A. R. Damasio, A. Castro-Caldas and A. L e e , Advances i n Neurol.,
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J . D. Parkes, R. C. Baxter, A. G a l b r a i t h , C . D . Marsden and J. E. Rees, Advances i n Neurol., 3, 105 (1973). R. P. Grelak, R. C l a r k , J. M. Stump and V. G. V e r n i e r , S c i e n c e , 2,
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P. P e n t i k a i n e n , P. J. Neuvonen and H. Vapaatalo, E x p e r i e n t i a , 3, 1524 (1973). B. Cox and S. J . Tha, Europ. J. Pharmacol. , 24, 96 (1973). P. F. Von Voightlander and K. E. Moore, S c i e n c e , I& 408 , (1971). P. F. Von Voightlander and K . E. Moore, J. Pharmacol. Exp. Therap.,
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R . M. P i n d e r , Advances i n Neurol. , 3, 295 (1973). D. M. Brown, B. 0. Hughes, C . D. Marsden, J . C. Meadows and B. 476 (1973). S p i c e r , B r i t . J. Pharmacol., 0. P. W. Robinson, Advances i n Neurol., 3, 97 (1973). K. R . Hunter and 0. P. W. Robinson, C l i n i c a l Trials J o u r n a l , 2, 3
9,
(1972). 53.
D . J . Vicary, P. M. Horrocks, J . E. Rees, J , D. Parkes and C . D . Marsden, C l i n i c a l T r i a l s J o u r n a l , l0, 3 (1973). D . J . J e f f e r y , D. M. Brown and P. F. Langley, Xenobiotica, 5 , 169
54. 55.
G. Clark and J . Davies, B r i t . J . Pharmacol., 465 (1973). R . C. U r s i l l o and J. A . McCulloch, Arch. I n t . Pharmacodyn., 197,132
56. 57. 58.
E. A. S p i e g e l and H. T . Wycis, Confinia Neurol., 2,176 (1969). H. T. Wycis, Confinia Neurol. , 34, 130 (1972). E . R. Blonsky, A. D. E r i c s s o n , A . S . McKinney, A. Rix, R . I . H. Wang and A . A. Rim, C l i n i c a l Pharmacol. Therap. , 2, 46 (1974).
52.
(1971).
3,
(1972)
-
Chapter 4.
Psychotomimetic Agents
Richard A. Partyka and JOMS A. Gylys B r i s t o l Laboratories, Syracuse, New York 13201
-
'-'
Comprehensive suryeys on t h e chemistry, pharmacology2 and metabolism o f General The proceedings of conferences d e a l i n g w i t h marihvsna a n n a b i n o i d s have ap7e red. have been published. Governmental p u b l i c a t i o n s which cover t h e b i o l o g i c a l , s o c i a l An e x c e l l e n t account of e r g o t and l e g a l a s u e c t s of marihuana have a l s o appeared.'-'' has been published i n which the t a x o n o q and d i s t r i b u t i o n of the Claviceus s p e c i e s , t h e c o n s t i t u e n t s o f e r g o t and t h e i r b i o s p t h e t i c o r i g i n , and t h e production 3f e r g o t a l k a l o i d s a l l have Seen reviewed.lZ Extensive reviews on t h e ~ h e r n i s t r y ~ a:d ~ - bio~ synthesis15 o f peyote a l k a l o i d s have appeared. The biochemistr] o f psychosesL6 a s w e l l a s t h e r o l e of adrenochromel' a s a psychotomimetic agent have been t h e s u b j e c t s of reviews. -4 review o f che chemistry and pharmacology o f W m e t h v s t i c u m , t h e 18 pepper p l a n t from which t h e n a r c o t i c d r i n k kava-kava i s prepared, has been pubiished.
-
I. Chemistry A. Cinnabinoids A d e f i n i t i v e paper appeared on t h e i s o l a t i o n and s t r u c t u r a l e l u c i d a t i o n o f 1'-retrahydrocannabinol (A1-THC). (2, cannabigerol cannabichromene (3, and cannabicyclol ( 4 A d e s c r i p t i o n o f the a b s o l u f t c o n f i g u r a t i o n 3111 o f both c h i r a l c e n t e r s (C-3 and C-4) i n 1 -THC a s & was also presented. $&fls on t h e t o t 3 1 syntheses o f ( 5 ) cannabidiol (+J d 5 - T H C , (-) d l - T H C , (-) -THC, as well a s t h e racemic and ( + ) l n o d i f i c a t i o n s of t h e l a t t e r two THC'S, were
-
4.
(z),
-
(z),
-3
p
4
-5
-6
published. 20-1 Other cannabinoid r e p a r a t i o n s i n v o l v i n g t h e condensation of terpenoid moieties were a l s o described. 2 2 - 3 ~'32 P opyl and methyl analogs o f cannabinoids 2 and 6 were i s o l a t e d and characterizedz4-' fro111 n a t u r a l sources. A propyl a n a l o g o f 2 was a l s o reported.2i S y n t h e t i c cannabinoids with n i t r o g e n c o n t a i n i n g s i d e chains such a s s t r u c t u r e s i n analogous f a s h i o n according t o p r e v i o u s l y d e s c r i b e d s y n t h e t i c A unique approach t o t h e trans-aza s t r u c t u r e 4 was a l s o reported. 30 routes
7a-c w5gegprepared
L
-8
Unfortunately, b i o l o g i c a l d a t a were n o t reported f o r t h e s e i n t e r e s t i n g s t r u c t u r e s . The ready a v a i l a b i l i t y o f v a r i o u s cannabinoids by i s o l a t i o n and s y n t h e s i s l e d t o a n i n t e n s i f i e d study o f t h e i r metabolism i n v a r i o u s animal specie^.^^'^ I n general, hydroxyIation occurred i n any a v a i l a b l e a l l y l i c o r benzyl o s i t i o n of t h e THC framework with t h e p o t e n t i a l f o r f u r t h e r metabolic conversion."-' I n man, a n important
28
Sect. I
-
CNS Agents
Gordon, Ed.
m e t a b o l i t e o f 4l-TIK was t h e corresponding 7-OH d e r i v a t i v e which a l s o e l i c i t e d p o t e n t cannabinoid-like e f f e c t s . ' 9 - 4 2 S y n t h e t i c developments k e p t pace v i t h t h e i s o l a t i o n and i d e n t i f i c a t i o n the v r i us metaboli p r o ucgs. Thus, procedures f o r t h e s y n t h e s i s of 7-OE-L-f6) -TBCt3-' and 7-GIi4'-THCt6-' were described. 5 8 0 e o g routes. !i9,55,w,
a ed fl-8
m e t a b o l i t e s such a s
9
and l o w e r e a l s o d e r i v e d by s y n t h e t i c
a)
€$-OH;
b)
€$a; R2-OH;
c)
5, R2-=arbanyl;
d)
%dz=H; 1,Za-epoxy;
e)
I(l-Rz=H;
RZM; R fl
3
R34i R3fl
5"
R3.0H
2 a)
5 9 ; R2R3=carbonyl
b)
%<€I3;
c)
\G2H;
OH
R2L,+,
R2-R3=H
10 -
a)
R=OCT(CH3)C5H11
b)
RIC(CH3) <(CH3)C5HI1
c)
RIC(CH3) zC6H13
d)
RG(C5)CH(CH3)C5Hl1
11
3 A v a r i e t y of new 4 -THC d e r i v a t i v e s l l a - c condensation products i n t h e 4' p o s i t i o n . 56-
was prepared i n c l u d i n g Mannich
-
B. Lvsernic Acid S t r u c t u r e s . Sev a 1 u n s u c c e s s f u l a t t e m p t s a t a new s y n t h e s i s of l y s e r g i c a c i d (12a) were sumarized.'f A review on analogs & of. I most of which a r e p a r t i a l s t r u c t u r e s , appeared. 52 An e f f i c i e n t p r o c e s s f o r t h e p r e p a r a t i o n of v a r i o u s secondary and t e r t i a 8 ly5ergamides and t h e corresponding d i - and t e t r a h y d r o d e r i v a Nu-Demethylation o f l y s e r g i c a c i d diethylamide (LSD) was t i v e s was d e s ribed. accomplishedJz 2 s well a s t h e d i F e r i z a t i o n of a wide v a r i e t y o f e r g o t a l k a l o i d d e r i v a t i v e s t o s t r u c t u r e s of type 13.-'5 A convenient p r e p a r a t i o n o f t h e t 7 t g l y s e r g i c a c i d e s t e r 15 from t h e corresponding N-oxide 1L;was d i s c l o s e d . 56 Cyclic o r t h o t h i o esters were used i n a novel F r i e d e l - C r a f t s type approach t o t h e p r e p a r a t i o n o f v a r i o u s 2 - s u b s t i t u t e d 9,lO-dihydrolysergic a c i d d e r i v a t i v e s 12b i n c l u d i n g (R1"CH3). 5 7 A t o t a l s y n t h e s i s of (3- i s o s e t o c l a v i n e (l:c)was described.
RICH2C1,
14
Ls.
C 0 2 9 , cBzcN
13 -
S t u d i e s involving t h e unraveling of t h e complex b i o s y n t h e t i c sequence of t h e review on t h e biosyne r g o t a l h l o i d s continue 20 be r e p ~ r t e d . ~ ~A- ~comprehensive l t h e s i s of e r g o t a l k a l o i d s was publishcd.62 Of p a r t i c u l a r i n t e r e s t w a s t h e d i s c l o s u r e t h a t t h e e n o l a c e t a t e 12d was a n e f f i c i e n t l y s e r g i c a c i d precursor.63 A new a l k a l o l d 1 6 (R=(+) -1ysergyl) from a Claviceps purpurea s t r a i n was i s o l a t e d and c h a r a c t e r i z e d .
-
''
Partyka, Gylys
Psychotomimetic Agents
Chap. 4
2
z
?&.
-
PhenylalXvlaiine T,;?(?s. Anolozs o f Go?I 18a (R=C1 3r,I,X02,NH,)65 as w e l l a s t h e were prepdred. A series o f b-ajor ? - t a b o l i t a s oi ;.C:.l i n t‘.e r a t (18, R.C:12CH, C0,H)’6 bromonethoxyphe=y;isoproplrlamrnes 19 ( n i l , 2) was a l s 6 described, w i t h b r i e f r e f e r e n c e s t o phar2co:oCical a c t i v i t y . 67-i3 A h i s h l y u s e f u l method f o r the a s p e t r i c s y n t h e s i s o f p h e n y l i s o p r o p y l a ~ i n c s a s well as t h e nethodoloFy f o r t h e determination o f t h e i r Numerous analogs o f the psychotonimetic e n a n t i o n e r i c p u r i ? ” Tiere published, pheny1isopropyLznines Irere preoared \:herein the s t r u c t u r a l f e a t u r e s were i n c o r p o r a t e d i n t h e nore c o c s t r a i n e d frans::ork o f a 2-amino-1, 2 3 4-tetrahydronaphthalene ( a n i n o t e t r a l i n ) s y s t e z 21 (R=CH3, OH, @Ke, H, -0CX20-).71-g The aminoindan and aminoC.
20
19 (COH)
a)
R
bj
R
C)
a=ar
a) b)
n-1 D-2
N(W3)
(WET)
(DOJ)
21 -
t e t r a l i n 2(ib analogs o f N M were a l s o prepared.
74
The p r e p a r a t i o n o f t h e s u l f u r analog 22b o f p s i l o c i n (22a) was described. Weak s e d a t i v e and vasopressor a c t i v i t y w a s found.
--
75 -6
I n v i t r o experiments provided evidence f o r t h e bioconversion o f n y r i s t i c i n (23) t o t h e corresponding phenyliso?ropylanine d e r i v a t i v e , PNDA 77 Other workers, u s i n g t h e i n t a c t animal, demonstrated t h e presence o f s t r u c t u r e s m a s major m e t a b o l i t e s
z.
o f t h e r a t and guinea pig, r e s p e c t i v e l y . 7 8 The f u l l i m p l i c a t i o n o f t h e s e bioconvers i o n s with respect t o b e h a v i o r a l o b s e r v a t i o n s i n animals79 a s w e l l a s i n man remains t o be determined. X review on nutmeg was published.80
D.
O t h e r - S t u a i e s continued on t h e measurement o f p h y s i c a l chemical parameters o e l f i d a t e s t r u c t u r e a c t i v i r y p a t t e r n s i n psychotomimetic compounds. Thus X-ray, in ordlif-’ nu& and t o t a l o r b i t a l e n e r g i e s a 4 were used t o study molecular conformation and i t s r e l a t i o n s h i p t o psychotonimetic a c t i v i t y i n t h e l y s e r g i c a c i d series. The conformations o f p s i l o c i n and mescalice we85 examined by molecular o r b i t a l and c l a s s i c a l p o t e n t i a l function calculations C o r r e l a t i o n s o f uv s p e c t r a , ‘6 n a t i v e fluorescence87 and a s s o c i a t i o n constants*8 with t h e psychotomimetic a c t i v i t y of v a r i o u s methoxylated phenylisopropylanines were made. Quantum mechanical s t u d i e s Of
30
Sect. I
- CNS Agents
Gordon, Ed.
t h e r e l a t i o n s h i p between p s y c h o t o n i n e t i a t i v i t y and t h e s t r u c t u r e s o f v a r i o u s a n t i c h o l i n e r g i c a g e n t s were a l s o published. g9-Fo
-
11. Pharnacolow. A. Cannnbinoids. Acute and Fhronic t o x i c i t y , t e r a t o l o g y , and reproduction c, t u d i e s on crude m a r i h u n a e x t r a c t , lL-TMC and o t h e r cannabinoids were p e r f o m e d i n animals. Concrolled human s t u d i e s f a i l e d to r e v e a l any drug r e l a t e d p e m n e n t e f f e c t s 2 * 11, 95althuugh chronic narihuana smokers had impaired 96 c e l l u l a r mediated i m l i n i ty. I n animals, b L - T H C showed s i o n s o f a n a l g e t i c , a n t i p y r e t i c , a n t i i n f l a m a t o 97-100 +f
r,,
and anticonvi?lsant p r o p e r t i e s , lo'-' but no a n t i d e p r e s s a n t e f f e c t s . lo3 I n man, 5 THC was i n a c t i v e a s a n a n a l g e g i c i n experimental ? a i n . l o 4 A'-THC caused hypothermia i n s e v e r a l a n i r a l s p e c i s ? i''-i v h i l e camplex and b i p h a s i c e f f e c t s were seen on rodent a g g r e s s i v e behavior. *L*h-lll ll-THC p o t e n t i a t e d t h e e f f e c t s o f d e p r e s s a n t compounds, 'I2 b u t e x h i b i t e a no c l e a r cut antzgonism towards amphetamine. S i m i l a r f i n d i n g s were a l s o r e p o r t e d f o r s y n c n e t i c cannabinoids. Cannabidiol and cannabinol, which were considered t o b e b i o l o g i c a l l y i n a c t i v e , e x h i b i t e d a n t i c o n w l s a n t a c t i v i 114 and i n t e r a c t e d with J l - T H C both p h a m c o l o g i c a l l y and m e t a b o l i c a l l y i n animals Furthermore, intravenously, cannabinol was found t o be weakly a c t i v e i n man."7 The phannac o l o g i c a l m o p e r t i e s of s e v e r a l s y n t h e t i c 13-THC d e r i v a t i v e s have been d e s c r i b e d i n animals. 7 , f O
'
I n animal o p e r a n t procedures, ?'-THC impaired performince mainly a f f e c t i n g t y p o r a l d i s c r i m i n a t i o n and s h o r t term mecory. 2,5 However, t h e e w re r e p o r t s t h a t A -THC n i g h t f a c i l i t a t e a c q u i s i t i o n of avoidance perfonrance. l l L 1 2 % Tolerance t o t h e e f f e c t s o f LI-THC developed r a p i d l y , was long l a s t i n g , and d i d rigi in.^ Hovev r, t h e r e were s e v e r a l b e h a v i o r a l parameters which f a i l e d t o e x h i b i t tolerance.121'e Thus f a r , only t h e fhesus monkey I n man, e x h i b i t e d s i g n s resen'nling p h y s i c a l dependence upon withdrawal of 1 t o l e r a n c e t o cannabis p r e p a r a t i o n s was less apparent and no t r u e p h y s i c a l dependence The s o - c a l l e d "reverse" t o l e r a n c e p o s t u l a t e d i n Fan could n o t b e was seen.8, lo, r e c e n t l y confirmed.125-6 ll-THC decreased s l e e p d u c t i o n time i n man, however, a Extensive humen s t u d i e s on "hangover" phenomenon was observed the nexc day. cannabinoids d e a l i n g with s u b j e c t i v e e f f e c t s mental function, memory, w t o r and d r i v i n g performance were published. 5 , 11, 128-31
not appear t o b e metabolic i n
'"
The s i m i l a r i t y o f cannabinoids t o o t h e r known psychotomimetic compounds was appeared t o be d i f f e r e n t from LSD i n terms of p h y s i o l o g i c a l i n v e s t i g a t e d . \'-THC and psychotomimetic p r o f i l e s i n man ( r e f . 4 , p. 196). Howe er, c e r t a i n s i m i l a r i t i e s and d i f f e r e n c e s wereaceen between marihuana and e t h a n 0 1 ~ ~w ~ i t h- ~an i n d i c a t i o n o f No c r o s s - t o l e r a n c e could b e demonstrated between ll-THC and c r o s s - t o l e r a n c e . li, IJ5 morphine, mescalice o r LSD. '7 136 I n animals,
;.I -THC produced hypotensive responses, bradycardia
reduced venous
r turn137 and a n t i h y p e r t e n s i v e e f f e c t s i n c e r t a i n l a b o r a t o r y models. 138-9 Xn nan, Ap-THC caused tachycardia, c o n j u n c t i v a l reddening, p r e s s o r e f f e c t s , impairment of Some r e c e n t evidence i n d i c a t e d t h a t ll-THC v a s o r e f l e x e s and ECG changes. reduce i n t r a o c u l a r p r e s s u r e and p u l m n a r y a i r r e s i s t a n c e i n man.
might
S t u d i e s an a b s o r p t i o n , p l a s m l e v e l s orwan d i s t r i b u t i o n , 143-7 u r i n rv and fecal excretio.., and n e t a b o l i c conversion^'^, 3g7 14'-' o f 'll-THC a n d / o r hi$6)-THC i n animals and man were published.3i,33 The c u e s t i o n o f whether A1-THC o r 7-OHdI-THC was m i n l v t e s p n s i b l e f o r t h e e f f e c t s observed w i t h A1-THC ha n o t been resolved.106,1J8 However, i t has been e s t a b l i s h e d t h a t 7-OH-A -THC was p r e s e n t in t h e m u s e b r a i n a f t e r A1-TXC admLnistration, and was found t o be more p o t e n t than L1-THC based on b r a i n concentrations.151-2. On t h e b a s i s of lasma l e v e l s , e x c r e t o r y p a t t e r n s , and concomitant drug e f f e c t s i t appeared t h a t 7+)H-l7-THC most l i k e l y w a s r e s p o n s i b l e hydroxylated m e t a b o l i t e s , such a s s t r u c t u r e s f o r t h a c t i v i t y i n m n . 39, 41, 153 ga.b15&and fi proved t o be inactit:fi31 The metabolism o f DlClP was s t u d i e d i n several animal s p e c i e s . 155
H
The s t r u c t u r e - a c t i v i t y r e l a t i o n s h i p s and r e l a t i v e p o t e n c i e s of v a r i o u s
Chap. 4
Psychotomimetic Agents
31
Partyka, Gylys
cannabinoid; were ?resented. 339156 i t should be noted t h a t d t f f e r e n c e s i n t h e potency of h1-~f1cand .?,1(6)-.IHC, a s w e l l as v a r i o u s 33-T%C d e r i v a t i v e s and analogs, e x i s t e d depending on t h e test animal and procedure ~ s e d . ~ ' , ' ~ ' Despite i n c r e a s e d knowledge, t h e pharmacological c l a s s i f i c a t i o n o f cannabinoids remains e l u s i v e . Any 1.abeling such a s "depressant" o r "hallucinogenic" should be avoided because o f t h e complex and unique p r o p e r t i e s of t h i s c l a s s o f compounds.
-
B. Lyseroic Acid and Tr;ot.-iie Tvxs. I r p o r t a n t c o n t r i b u t i o n s towards t h e unders t a n d i n g of t h e a c t i o n s of psychotomimetic substances, e.g. t h e r o l e o f s e r o t o n e r p i c neurones, s i t e s o f c e n r r a l a c t i o n , and development o f t o l e r a n c e and c r o s s - t o l e r a n c e were revie:ied. 16, 15s-62 The i n f l u e n c e of e q o t a l k a l o i d s on p i c u i c a r y p r o l a c t i n and prolactin-dependent processes :.:as a l s o reviewed. '63 The metabolism o f n i c e r g o l i n e (17) was s t u d i e d i n v a r i o u s a i i m l s p e c i e s , including nan. LSD appeared t o e x e r t s e l e c t i v e e f f e c t s on s e r o t o n e r g i c neurones i n t h e b r a i n stem raphe n u c l e i , however, it i s u?.dctermined whether i t a c t s by blocking o r mi.micking 5-hydroxytryptamine ( 5 - h i ) . 165-r Sufotenin and N, N-dimethyltryptamine (DHT) appeared t o a c t s i m i l a r l y t o LSD on t h e raphe n u c l e i , w h i l e mescaline and DOY appeared t o be d i f f e r e n t . Atropine, 2-3r-LSD, 2nd c h l o r o p r o r a z i n e v e r e i n a c t i v e on raphe The p o s s i b l e involvement o f a c e t y l c h o l i n e , y-aminobutyric a c i d I;A3A) neurones. 1'L-Z -and tryptaminc i n t h e c e n t r a l a c t i o n o f Fsychotomirnetic a g e n t s was imp1icated.l
5'
The phenomena o f t o l e r a n c e and c r o s s - t o l e r a n c e among psychotomimetic a e e n t s were i n t e n s i v tud'ed. A c r o s s - t o l e r a n c e was observed between LSD and tryptamine o r mes a i n e , n4z, Q 7 6 - f b u t n o t Setween LSD o r mescaline and amphetamine o r LSD and DKT. 17'-' I n t e r o c e p t i v e e f f e c t s induced by mescaline, p s i l o c y b i n and LSD were poorly d i f f e r e n t i a t e d by t h e rat. ls'-l D e s p i t e s t r u c t u r a l h e t e r o g e n e i t y , t h e psychoto2imetic a g e n t s i n
e n e r a l caused
an i n c r e a s e i n b r a i n 5-;iT l e v e l s and a decrease i n i t s turnover rate.1'9,184-5 The e f f e c t s on b r a i n norepinephrine (NE) and dopamine were l e s s c l e a r l y defined, b u t a l o v e r i n g o f b r a i n l e v e l s and h i g h e r turnover r a t e s o f NE have been reported. i82-5 S p e c i f i c and s e n s i t i v e r a d i o i m u n e a s s a y s were developed f o r a n a l y s i s o f LSD and mescaline i n b i o l o g i c z i f l u i d s . 186-8 Turthermore, a n t i b o d i e s were o b t a i n e d which e x h i b i t e d high s p e c i f i c i t y t o LSD and c r o s s - r e a c t i v i t y t o o t h e r psychotomimetics r e l a t e d t o tryptarnine and phenethylamine. ls9
The d i s t r i b u t i o n o f LSD i n t h e r a t b r a i n e x h i b i t e d p a t t e r n s mimicking r e g i o n a l l o c a l i z a t i o n o f g-kT.l'o The c e n t r a l e f f e c t s of LSD were antagonized by s e v e r a l s t e r o i d a l agentsAg1 and 3, N-diethyl-l-methyl-l,Z, 5 , 6 - t e t r a h y d r o n i c o t i n a n i d e . 192 Furthermore, an a c t i v e i m u n i z a t i o n by p r o t e i n c o n j u g a t e s coPtaining l y s e r g i c a c i d o r tryptophan prevented c e r t a i n c e n t r a l e f f e c t s of LSD i n mice. L53 LSD f a c i l i t a t e d a c q u i s i t i o n of l e a cd behavior and improved l i g h t d i s c r i m i n a t i o n and r e c e n t memory i n rats and monkeys. 1'e-7 There TJBS l i t t l e w e l l &ocumented evidence t o i m p l i c a t e LSD f o r causing chromsomal damage and t e r a t o l o g i c e f f e c t s b o t h i n r a n and animals. The d e t e r m i n a t i o n of a N-ethyltransferase i n h m n blood1" and brainzo0 t h a t was capable o f converting t r y p t a n i n e i n t o DMT vas d e s c r i b e d . The presence o f a n endogenous substance capable of E e t a b o l i z i n q C:T i n normal s u b j e c t s was p o s t u l a t e d . lg9 Chronic schizophrenics excreted EXC and bufotenin i n t h e i r urine, v h i l e normal s u b j e c t s d i d not. 201-2
-
C. Phenvlnlkvlamine Types. The human p h a m c o l o g y of mescaline, i t s congeners and phenylisopropylamine a n a l o s s was reviewed. '03 X comprehensive study on t h e a c u t e t o x i c i t i e s 0 5 mescaline and i t s d e r i v a t i v e s revealed t h a t t h e do was n o r e s e n s i t i v e than t h e rhesus monkev t o t h e t o x i c e f f e c t s o f t h e s e compounds. 264 Rats f a i l e d t o d i f f e r e n t i a t e 2,3,4-trimetho?cyphen lethylamine, a non-psychotomimetic a n a l o g of mescaline, from mescaline i t s e l f . 25'
A e e o s e l e c t i v i t y o f LSD, DO31& tl DOB !8c. and DOET 18b f o r c e n t r a l ::ns demonstrated i n t h a t the g - c o n f i g u r a t i o n e l i c i t e d t h e n o t e p o t e n t effects2"-' e f f e c t , w h i l e p e r i p h e r a l l y such a d i f f e r e n t i a t i o n w a s not shown. 209-10 110 such clear
32
Sect. I
_.
-
Gordon, Ed.
CNS Agents
d i f f e r e n t i a t i o n with r e s p e c t t o psychotomimetic e f f e c t s could be shown w i t h g-(-) o r 5-(+)-am hetamine, although t h e 2 - i s o s e r was more p o t e n t a s a s t i m u l a n t than t h e 11isomer. 2P1
-
S t r u c t u r e - a c t i v i t y requirements i n t h e phenylisopropjllamine s e r l e s were presented. 203 R e l a t i v e potency r e l a t i o n s h i p s i n v a r i o u s experimental procedures f o r phenylisopropylsmine and D>fT analogs were discussed. 212-3 A t r a n s o i d conformation f o r mescaline a t t h e r e c e p t o r l e v e l was supported by t h e g r e a t e r b e h a v i o r a l e f f e c t s o f 28a over 28b i n t h e
'''
W M caused a b e r r a n t g r o s s b e h a v i o r a l p a t t e r n s w i t h a concomitant h persynchronous i t a l s o produced a l e t h a l hyperthermia i n r a b b i t s . The c a r d i o v a s c u l a r a c t i v i t y of X?.lwas c h a r a c t e r i z e d by z p r e s s o r response, tachyphylsxis and p e r i p h e r a l v a s o d i l a t i o 2 folloved by v z s o c o n s t r i c t i o n . 217 The e n t r a l and p e r i p h e r a l e f f e c t s induced by 3 O Y 57ere s u s c e p t i b l e t o 5-iiT a n t a g o n i s t s . 21s-7 Or%3n d i s t r i b u t i o n , , metabolic conversions and t h e e x c r e t o r y p a t t e r n o f 20014 were s t u d i e d i n l a b o r a t o r y ~ d t o b e responsible f o r the a c t i v i t y animals.218-22G The unchanoed C O ~ ? D O U E appear observed. 219 I n man, 30XT,r23E0B221 and M..DA2'2 caused a d l d euphoria a l o n ? w i t h a f e e l i n g of enhanced awareness a t r e l a t i v e l y lot.! doses. Various a m i n o t e t r a l i n s produced smooth muscle c o n t r a c t i o n s i n v i t r o probably through 5-HT r e c e p t o r s . 223 2 h i n o - 7 - h y d r o x y t e t r a l i n caused c e n t r a l e f f e c t s s i m i l a r t o those seen with mescaline and LSD i n rats. 224
EEG i n t h e cat.215
-
A new thebaine d e r i v a t i v e 2 6 p r o v e d t o be a n extremely p o t e n t psychotoD. Other mimetic agent, q u a l i t a t i v e l y mimickinz cyclazocine i n man 225 A novel c h o l i n e s t e r a s e inhibitor caused s e v e r e sensory d i s t u r b a n c e s i n man. 2i6 Low doses of N, N-dipropyltryptamine were evaluated a s a p o t e n t i a l a d j u n c t i n psychothera v 227 The biochemical e f f e c t s o f psychotomimetic a n t i c h o l i n e r g i c drugs were reviewed. 52i I n man, sensory d i s r u p t i v e e f f e c t s produced by a t r o p i n e , scopolamine and D i t r a n were similar and could
a
27 -
20 -
b e c l a s s i f i e d a s simple d e l i r i u m r a t h e r than a s psychotomimetic syndromes. 229
In rats,
c y c l a ~ o c i n e 2 3and ~ l e ~ a l l o r p h a nproduced ~~~ similar e f f e c t s t o t h o s e seen w i t h LSD.
Chap. 4
Psychotomimetic Agents
Partyka, Gylys
33
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Psychotomimetic Agents
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Chapter 5.
Abuse of CNS Agents
Maxwell Gordon, Bristol Laboratories, Syracuse, New York Introduction. The p a s t few years have seen a marked increase i n t h e abuse of CNS agents around the world. It has a l s o seen the passing of Nathan Browne Eddy, who f o r t h i r t y years had done so much t o put a s c i e n t i f i c pharmacological b a s i s 54,55,233(see a l s o 4,8,17,45,58,87,88,131,140,172, 185 under much of the heroin abuse research being done today. Abuse of drugs, both l i c i t and i l l i c i t has been a worldwide e idemic and has involved agents as d i v e r s e as o i a t e s 2,21,31,73,78,83,8~1105,1i6, B7,1 8 8 9 190, 191,215 am hetarnine 5,117,178,307, LSD 16,189,201,202, barbitur a t e s 59,60,61,9bs288, o t h e r hallucino ens 5O,52,93,95,182,183,184,193,2O4 marihuana 20,399 68,85,93, 95 109 110,11f, 141 142,198,211, cocaine 7 1, e t c . , i n a d d i t i o n t o a spate of glue s n i f f i n g , abuse of nutmeg, snuff, oregano and, of course, the ancient scourge alcohol 6. The hallucinogens a r e covered i n chapter 4 of t h i s volume. The l i t e r a t u r e of alcoholism is so v a s t t h a t no attempt can be made i n t h i s limited space t o cover i t . Furthermore t h e r e is a r e a l question i n our s o c i e t y whether w e consider i t t o be a drug s i n c e , f o r example, i t s use is not regulated by the Food and Drug Administration. Sedative abuse (hypnotics, t r a n q u i l i z e r s ) is a serious, much studied problem. However, not even suggestions of a s o l u t i o n a r e a t hand,so t h i s c l a s s of drugs too w i l l not be reviewed.
=ate Abuse. The o p i a t e area of abuse q u a l i f i e s f o r review i n these pages on two counts. F i r s t , i t is one of t h e most s e r i o u s medicallsociological1 legal/economic problems of our times, and secondly, approaches t o the treatment o r prevention of o p i a t e abuse have yielded c o n t r i b u t i o n s t o medicinal chemistry of the c l a s s i c a l kind, and thus a review of t h e s e approaches is appropriate t o t h e readership of t h i s volume. Economic Aspects of Opiate Abuse. One of the driving forces behind t h e rapid expansion of heroin abuse has been the enormous p r o f i t 92,128,132 t o be made from heroin processing, smuggling 9s99, and s a l e . This p r o f i t motive, when added t o t h e ease of contagion of the heroin h a b i t among s u s c e p t i b l e populations (e.g. peer groups), has made the drug abuse battle a d i f f i c u l t one f o r a l l concerned. Only now, a f t e r extensive e f f o r t s t o i n t e r d i c t heroin t r a f f i c , t o a r r e s t and convict t r a f f i c k e r s , and t o e n r o l l as many a d d i c t s as possible i n t o treatment programs, has the explosive growth of heroin abuse shown signs of leveling o f f . Regrettably t h e t o t a l number of a d d i c t s is s t i l l r i s i n g , a l b e i t more slowly, and estimates of t h e number of heroin a d d i c t s i n the U.S. range as high a s 800,000 38, a l though estimates of 300,000 t o 400,000 a d d i c t s are more coarmonly c i t e d . Opium harvesting i s q u i t e labor i n t e n s i v e (about 200 man-hours per kg opium 92 a d hence opium tends t o be grown commercially i n a r e a s where lowc o s t , short-term labor is a v a i l a b l e . Climatic requirements f o r the opium poppy a r e not g r e a t , and Papaver somniferum grows i n areas as d i v e r s e as Bulgaria, China, Greece, India, I r a n , Laos, Mexico, North Africa, Pakistan, Thailand, Turkey, and t h e U.S.S.R.
Chap. 5
Abuse of CNS Agents
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Most i l l e g a l opium products smuggled i n t o t h e U.S. i n recent y e a r s have from Turkey v i a France, where morphine was converted t o heroin. However, Turkey has only accounted f o r about 5% of t h e world production, so c l o s i n g out opium c u l t i v a t i o n i n Turkey (subsidized by t h e U.S.) had little o r no e f f e c t on i l l i c i t heroin supplies i n t h e U.S., although it d i d cont r i b u t e t o a shortage of medicinal opium exacerbated by crop f a i l u r e s i n India. Furthermore, l e g a l c u l t i v a t i o n of opium i n Turkey i s about t o be r e i n s t i t u t e d f o r economic reasons. copne
Recent p r i c e s paid t o Turkish farmers f o r l e g a l opium have been about $7.00 per kilogram, but black market p r i c e s have been as high a s $250,000 per kilogram. A t t h i s p r i c e of crude opium, the c o s t of t h e heroin i n a $5.00 "bag" i n t h e U.S. i s less than one cent. Hence, because of t h e p r o f i t s made by farmers, i t was u n l i k e l y t h a t a u t h o r i t i e s would succeed i n completely choking o f f Turkish black market supplies, and even i f they did, t h e world black market has access t o even less expensive Southeast Asian opium. The l a t t e r source developed t o a high degree during t h e U.S. involvement i n t h e V i e t Nam war, and enormous amounts of heroin were smuggled i n t o t h e U.S. using m i l i t a r y p o s t a l systems. With t h e phasing out of U.S. m i l i t a r y a c t i v i t i e s i n Southeast Asia, a l t e r n a t i v e delivery systems w i l l doubtless be developed. An a d d i t i o n a l problem i n dealing with o p i a t e abuse i s t h a t i f opium prices should rise p r o h i b i t i v e l y , s y n t h e t i c a l t e r n a t i v e s l i k e methadone would be made a v a i l a b l e to s o p h i s t i c a t e d black market organizations. Methadone has been found by a d d i c t s t o be i n d i s t i n i s h a b l e from heroin on i n j e c t i o n i n double-blind experiments 103,216, 21r218,219. The e x t r a c t i o n of morphine from crude opium and i t s conversion t o heroin a r e r e l a t i v e l y simple processes t h a t r e q u i r e only rudimentary equipment o r t r a i n i n g . The synthesis of o p i a t e s de novo r e q u i r e s more equipmerc and t r a i n i n g , but should be q u i t e a v a i l a b l e t o the black market c o n s i d e r i m t h e economic rewards. Bearing i n mind t h e $25.00/kg p r i c e paid f o r Turki s h crude opium (which re r e s e n t s a c o s t of about $2.50/kg of morphine), it has been reported 128*p32 t h a t morphine base sells f o r up t o $2000/kg, t h a t 407. pure heroin sells f o r $20,00O/kg, and t h a t 1-37, pure heroin sells f o r $400,00O/kg on the 'street'. The l a r g e p r o f i t s from heroin production and d i s t r i b u t i o n underline the d i f f i c u l t y i n stopping smuggling of heroin. A look a t t h e l o g i s t i c s of i n t e r n a t i o n a l movement of f r e i g h t provides f u r t h e r discouragement s i n c e the e n t i r e U.S. heroin requirements are less than f i v e tons a year, which can e a s i l y be l o s t amid t h e more than 100,000,000 t o n s of cargo t h a t e n t e r the U.S. by sea each year 221 o r aboard t h e thousands of f l i g h t s t h a t e n t e r t h e U.S. from abroad each year carrying about 200,000,000 persons through customs. Since t h e d a i l y heroin requirement i s only about 1 2 kg, and considering t h a t a s i n g l e person can r e a d i l y c a r r y $500,000 worth of heroin on one t r i p , the problem of i n t e r d i c t i n g heroin smuggling can be appreciated. Government E f f o r t s . The epidemic of heroin abuse i n t h e U.S. has led t o considerable a c t i v i t y by Federal, S t a t e and Local Governments 144 t o d e a l with t h e problem. I n the White House a Special Action Office f o r Drug Abuse Prevention was established. The FDA has exerted considerable e f f o r t i n the area of policing drug treatment program~56,57,~04. The National
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Sect. I
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CNS Agents
Gordon, Ed.
I n s t i t u t e s of Health mounted and funded considerable research e f f o r t i n t h e a r e a of 0 i a t e abuse 24,~5,~6,~6,49,76,134,135,136,138,143,160,166,167, 170*177918192p2 he Con r e s has contributed m n e and focussed attention on t h e problem 28,~3,8~,9~,1~1,118,129,144,157,159,~74,210, the kpartment of J u s t i c e has increased i t s e f f o r t s 97, and the c o u r t s have made use of devices l i k e c i v i l commitment t o encourage treatment 29,108. On the i n t e r n a t i o n a l scene, the UN has likewise addressed t h e problem of o p i a t e abuse 98,150,203.
Geographic
D i s t r i b u t i o n of Heroin Abuse. Although o p i a t e abuse o r i g i n a t e d i n China and Southeast Asia L14 i t has f i r s t of a l l spread t o the major population c e n t e r s of the U.S. 1,12,37,38,40,41,54,55,69,L21,180, p a r t i c u l a r l y New York 165, Puerto Rico 148 and California, where the HaightAshbury scene became infamous 195*140s 191 From the U. S. t h e heroin epidemic i n the past decade spread t o En land i 3 , t h e Continent 5 3 , 1 1 3 ~ 1 ~ O , e s p e c i a l l y Sweden 79, and t o Japan Ifo6, and t h e rest of the more a f f l u e n t p a r t s of t h e world l i k e Australia, South Africa, e t c . Opiate Antagonists. Martin e t a1 133 have shown t h a t an i n j e c t i o n of a n a r c o t i c antagonist i n t o a non-dependent s u b j e c t w i l l block t h e s u b j e c t i v e o p i a t e - l i k e e f f e c t s of a subsequent i n j e c t i o n of heroin o r o t h e r o p i a t e . Thus, he postulated t h a t a long-acting antagonist could "immunize" an addict from the subjective e f f e c t s of o p i a t e abuse. The e x i s t i n g antagon i s t s l i k e naloxone, cyclazocine and naltrexone have c e r t a i n drawbacks l i m i t t h e i r usefulness a s blocking agents i n t r e a t i n g heroin
Naloxone
Cyclazocine
Naltrexe.e_
Naloxone, although a "pure" antagonist, has a short duration and poor o r a l a c t i v i t y . Hence, a 24-hour o r a l blocking dose must be measured i n grams. Thus, considerations of cost and p a l a t a b i l i t y would tend t o r u l e out t h i s substance. Cyclazocine has a d e s i r a b l e duration of a c t i o n (about 24 hours) and good o r a l a c t i v i t y . However, i t s hallucinogenic l i a b i l i t y may prevent i t s a c c e p t a b i l i t y and widespread use. For example, an "induction" period of two weeks o r longer i s required t o build up t o a 24-hour o r a l blocking dose of 4 mg. Naltrexone has a duration of a c t i o n i n t e r mediate between naloxone and cyclazocine and i s r e l a t i v e l y devoid of "agonist" s i d e e f f e c t s . However, although i t has the most promising prof i l e of the t h r e e agents, i t s r e l a t i v e l y s h o r t duration of e f f e c t and i t s r a t h e r high o r a l dose requirement (approximately 60-100 mg f o r 24-hour blocking a c t i v i t y ) a r e disadvantages, e s p e c i a l l y i n considering i t for incorporation i n t o sustained r e l e a s e implants. Both naloxone and naltrexone s u f f e r frola the drawback t h a t they provide no "reinforcement" t o the heroin addict being t r e a t e d with them. Cyclazocine s u f f e r s from the serious disadvantage of dysphoric a c t i v i t y a t e f f e c t i v e dose levels. Thus, new agents which a r e devoid of these problems a r e d e s i r a b l e . Antagonists
Chap. 5
Abuse of CNS Agents
Gordon
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Oxilorphan, a new t o t a l l y s y n t h e t i c antagonist synthesized by Monkovic e t a1 146,147, has many of the p r o p e r t i e s of t h e i d e a l antagonist agent. The s t u d i e s of J a s i n s k i e t a1 comparing cyclazocine t o oxilorphan 234 showed t h a t oxilorphan had a n o r a l potency about equal t o t h a t of cyclazoc i n e and a d u r a t i o n of e f f e c t a t least equal t o t h a t of cyclazocine, thus p r o j e c t i n g 24-hour heroin blocking a c t i v i t y f o r a 4 mg dose. A r e l a t i v e lack of hallucinogenic s i d e e f f e c t s a t 4 mg doses were seen i n former a d d i c t s 230. Thus, a projected 24-hour blocking dose of 4 mg can be given o r a l l y t o the post a d d i c t without any induction period and without any hallucinogenic e f f e c t . Additionally,oxilorphan produces some "euphoric" component which may assist i n keeping post a d d i c t s i n a n antag o n i s t program 230.
It should be mentioned t h a t oxilorphan d i f f e r s from cyclorphan only i n t h e presence of t h e 14-hydroxy group. Cyclorphan i s a potent analgesic as w e l l a s a potent hallucinogenic agent i n man, resembling cyclazocine. So it w i l l be seen t h a t introduction of a 14-hydroxy group i n t o cyclorphan 46,147 reduces i t s agonist and hallucinogenic e f f e c t s , much as is seen with naloxone i n comparison with, f o r example. nalorphine.
a6 Nalorphine
Cyclorphrn
Oxilorphan
lcvo-BC-2910
Oxilorphan has been subjected t o p a r e n t e r a l chronic t o x i c i t y and Phase I o r a l and p a r e n t e r a l t e s t i n g . I n a double-blind comparison of oxilorphan with cyclazocine 234 conducted a t t h e Addiction Research Center a t Lexington, Kentucky, oxilorphan was found t o have an hallucinogenic p o t e n t i a l one-fourth t h a t o f cyclazocine. Levo-BC-2910 an analog of cyclazocine, has been evaluated i n sub-acute t o x i c o l o g i c a l t e s t i n g by the NIMH i n preparation f o r Phase I c l i n i c a l t e s t ing. The findings comparing t h e antagonist a c t i v i t y of oxilorphan, LeVO-BC2910 and s e v e r a l reference agents are summarized i n Table I below 230. The most u s e f u l modality f o r dealing with t h e s o c i e t a l a s p e c t s of o p i a t e abuse (as w e l l as some of t h e medical and personal a s p e c t s ) has been methadone maintenance. I n consideration of t h e pharmacology o f o p i a t e dependence 4,17,45,58,87,88,131,140,172,185, Dole and Nyswander 51 proposed t h a t maintenance o f o p i a t e a d d i c t s on r e l a t i v e l y l a r g e doses of o r a l methadone would i n h i b i t drug seeking behavior, "block" t h e subjective e f f e c t s of p a r e n t e r a l heroin i n j e c t i o n because of t h e tolerance induced, break the needle h a b i t because of lack of reinforcement by t h i s route, and make the a d d i c t more a v a i l a b l e f o r p s y c h i a t r i c treatment and r e h a b i l i t a t i o n , breaking t h e cycle of crime and punishment. Methadone maintenance has been
Sect. I
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Gordon, Ed.
CNS Agents
Table I Narcotic An:agonint and Agonist (Analgesic) A c t i v i t y o f OxilorDhan. . . levo-BC-2910 and Reference /.Pence ED5o rnglkg 8 . c .
Compound Oxilorphan levo-BC-2910 Nal trexone Cyclazocine Cyclorphan Levallorphan Nalorphine Pentazocine Naloxone HC1 Horphfne s u l f a t e
Narcotic Antagonist A c t i v i t y Oxymorphone -orphone Morphine Straub T a i l Narcosis Antag.-RTI 0.19 0.074 0.027 0.81 0.32 0.29 1.14 12.0 0.09
-
.
0.03 0.013 0.006 0.12 0.10 0.32 0.58 10.1 0.02
-
0.012 0.008 0.003
0.040 0.032 0.086 0.38 12.2 0.010
-
Agoaist (Analgesic) Activity Bat nouse Writhing Writhing 12.8
44 9 0
0.046 0.031 26.3 0.77 3.7 XI0
0.70
8.0 30 35.6 0.029 0.028
11.9 0.70 0.95 XI0 0.16
3, 43 947948,s 1,62,66,70, widely em lo ed in the u S 15 s 1 8 ~ 2 2 ~ 2 27~32,36342 72 73 5 78 8 2 91 100 101, i02,107,119, ip1423, 127,145,149,151,152,154,162, 163, l d , 168: 186,192,136 and elsewhere Addicts entering a methadone maintenance program commonly go through a detoxification program 34 and preinduction physical examination. Treatment is commonly started at low daily doses building up to a maintenance level of 40-120 mgfday, tailored to each individual. Most methadone is dispensed and ingested in clinic, since diversion of take-home supplies presents a problem discussed later in this review. Dose adjustment is important since too low a dose will permit heroin abuse, whereas too high a dose can lead to dangers while driving 112 or operating machiner , and can interfere with the rehabilitative process as well 62,65,196,191;. Most methadone treatment centers belong to city or state agencies and are federally funded, althou h private methadone maintenance clinics have been operated as well 15,f56,213 A number of drug free treatment programs also exist 46,161,162,163,164,176,192,196 under names like Synanon, Phoenix,etc. Complications arise due to multiple drug abuse 26,35,67,123, and these are countered by multi-modality treatment and comnunit partici ation and preventive 62 and education programs 1, 10,30,86,126,189*200,20f. The drug abuse of addicts while in treatment programs can usefully be monitored by urinalysis 8*158,176,186 at frequent but irregular intervals, and by following deaths or serious in uries that are reported on addicts by offiMonitoring of crimes, cial sources 7,89,90,115,116,114,130,137,155,179. especially burglaries, shoplifting and robberies, also provides a good quantitative index of heroin addict activity, and large scale treatment in centers like New York or Washington, D.C. has resulted in marked drops in arrest incidence, both in the case of individuals in treatment and for the community as a whole 44,153. Inevitably the epidemic of drug abuse has iven rise to unorthodox a ptoaches ranging from transcendental meditation to heroin maintenance 69991,173 which we deplore. Research on Less Abusable Forms of Methadone and Other Oral Opiates. As mentioned earlier, the diversion of methadone to street use from maintenance programs is a serious problem since Jasinski et a1 lo3 have shown in double blind trials in post addicts that methadone and heroin are indis-
Abuse of CNS Agents
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tinguishable on injection. It has been found 32,103, 230, 224 that a 20:l mixture of methadone-naloxone is indistinguishable from methadone alone when taken orally for maintenance (or analgesia), but taken parenterally by the opium dependent subject it will precipitate abstinence and taken by the non-dependent subject the opiate effect will be eliminated, or postponed for several hours only to appear in an attenuated form. This combination relies on the fact that naloxone given orally is only about 1/100th as effective as it is parenterally. The methadone-naloxone dosage form has been further refined by the development of a gum-formulated tablet that resists dissolution and injection (thus preventing precipitation of severe abstinence in highly dependent subjects like methadone maintenance patients), and by the development of a color coding system for '"take home" methadone-naloxone tablets which permit s tracking diverted street" methadone to its diversion source. The gum formulation mentioned above, although giving identical methadone blood levels to those obtained from oral ingestion of a solution, 24 hours after dosing of each, produces less sedation and fewer "highs" than are seen with solutions. The addition of 1 mg of naloxone to 100 ml of paregoric 230 produces a product which is indistinguishable from paregoric alone when taken orally as intended. When boiled down, filtered, and injected, however, the combination will produce abstinence in the dependent subject, as does the methadone-naloxone. Other agonists are also being combined with naloxone t o deter oral abuse (e.g. oxycodone). Development of Totally Synthetic Analgesics and Antitussives. There has been a long standing interest in developing non-abusable analgetics and antitussives which do not depend on opium supplies s4955. As a result of limitations in cultivation of opium in Turkey, and crop failures in India, the U . S . has had to dip into opium stockpiles, and this has accentuated the interest in synthetic analgetics and antitussives. Two agents of current interest are butorphanol 1469147, 230 and nalbuphine 17. Butorpha-
Bu torphiu~: (levo-DC-2627)
Nalbuphine
no1 is approximately equivalent in analgesic potency to cyclazocine and approximately equal in antagonist potency to nalorphine. In animals, the analgesic activity of butorphanol is 4-10 times that of morphine, 25-70 times that of pentazocine and 1-2.5 times that of nalbuphine, which is chemically related to butorphanol. Butorphanol has antagonist activity which is approximately 30 times that of pentazocine and 4-6 times that of na lbuphine
.
Butorphanol demonstrated less physical dependence liability than pentazocine a determined in the mouse jumping test 131,172. In the Rhesus monkey 219 butorphanol did not suppress abstinence in the withdrawn mor-
44
Sect. I
- CNS Agents
Gordon, Ed.
phine dependent animal. I n a d d i t i o n , exacerbation of abstinence was observed. Physical dependence l i a b i l i t y t e s t i n g i s now i n progress by Dr.D. R. J a s i n s k i a t t h e Addiction Research Center, Lexington, Kentucky. Nalbuphine showed mixed agonist/antagonist e f f e c t s i n dependence t e s t i n g , with p r e c i p i t a t i o n of abstinence i n morphine dependent s u b j e c t s , but a l s o drugseeking behavior i n persons subjected t o d i r e c t a d d i c t i o n experiments. C l i n i c a l l y , butorphanol has been evaluated i n normal volunteers f o r s a f e t y and tolerance both o r a l l y and p a r e n t e r a l l y . Single intramuscular doses ranging from 0.1 t o 20 mg were studied. There was no i n d i c a t i o n of hallucinogenic a c t i v i t y even i n one volunteer who received the 20 mg dose 230. Butorphanol 230 and nalbuphine 231 have been t e s t e d c l i n i c a l l y f o r analgesia p a r e n t e r a l l y and found t o be approximately 10 times morphine and equipotent t o morphine respectively. I n summary, then, w e see t h a t progress has been made i n the a r e a of o p i a t e abuse by a p p l i c a t i o n of c l a s s i c a l medicinal chemical approaches of modifying various components of b i o l o g i c a l a c t i v i t y by s t r u c t u r a l modificat i o n , finding s y n t h e t i c s u b s t i t u t e s f o r n a t u r a l products which improve on nature, optimizing agonist and antagonist properties, etc. It is hoped t h a t these r e s u l t s w i l l encourage f u t u r e researchers t o make s i m i l a r prog r e s s i n d i f f i c u l t a r e a s l i k e b a r b i t u r a t e abuse, alcoholism, e t c . References 1. 2.
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n.
s.
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103. 104. 105.
136. 107. 108. 109. 110. 111. 112.
113.
114. lls. 116. 117. 118. 119.
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J. 8. Prykmdn, i n Drug Abuse, Proc. I n t e r n . C o d . , C.J.D. Z a r a f o n e t i r , Ed., Lea and P e b i g e r , 1972, p. 527. C. R. Gay, Contemporary Drug Problems, P a l l 1972. p. 735. 1 . P. G a r f i e l d , I=, p. 953. F. R. Gearing, i!):~j, Sprinq 1972, p. 191. B. J. George, J r T n Drug Abuse, Proc. I n t e r n . Conf.. C.J.D. Z a r a f o n e t i a , Ed., Lea and F e b i g e r , 1962, p. 459. F. B. G l a s e r , I n t l . J. of Addictions, 6, 615 (1971). M. M. G l a t t , Brit. J . A d d i c t i o n s , 63, 111 (1968). L. Goldberg, i n Drug Abuse, Proc. I n t e r n . Conf., C.J.D. Z a r a f o n e t i r , Ed., Lea and P e b i g e r , 1972, p . 27. P. 8. Goldberg, 3eaiing w i t h Drug Abuse. A Report t o t h e Ford Foundation, Ed. by P. M. Wald and P. B. R u t t , Praeger P u b l i s h e r s , New York, 1972, p. 300. I. Goldenberg and E . Keatinae, Contemporary Drug Problems, F a l l 1973, p. 363. B. M . Goldsmith, W . C . Capel, K. J. Waddel and G. T. S t e w a r t , C l i n . Toxicol. 2 - 7 3 (1972). A. G o l d s t e i n , The Journal, Addiction Research Foundation, Toronto, 2, 7 (1974). C. W. Corodetsky. Drug Dependence (NMH)(51) Oct. 1970, p. 18. L. Grinspoon, Contemporary Crug Problems, F n l l 1972, p. 811. P. G. Hansnond, i>-L< , Summer 1973, p. 247. L. S. H a r r i s and A. K. P i e r s o n , J. Pharm. and E x p t l . Therap., 1 4). 141 (1964). L. S. H a r r i s , A. K. P i e r s o n , J. R. Dembinski and W. L. Dtwey. Arch. I n t . Pharmcodyn., 165, 165 (1967). T H e l p e r n , Buman Pathol., 2. 13 (1972). M. Helpern. N. Y. Lnw J o u r n a l , Mar. 28. 1972. R. M. Hochstedler, Contemporary !Jrug Problems, S m e r 1972, p. 517. J. F. Holahan, Dealing v r t h Drug Abuse. A Report t o t h e Ford Foundation, Ed. by P. @I. Walt and P. B. H u t t , Praeger P u b l i s h e r s , New York, 1972, p. 255. L. E. H o l l i s t e r , i n !kug Abuse, Proc. I n t e r n . Conf., C.J.D. Z a r a f o n e t i e , Ed., Lea and F e b i g e r , 1972, p. 321. L. E. Hollfs:er, i-, p. 421. 8. Holmscedt and A . Linnarson. p. 291. C. M. IdestrClol. i n 2 u g Abuse, Proc. I n t e r n . Conf., C.J.D. Z a r a f o n e t i r , Ed., Lea and FebtgGr, 1972. p. 217. J. E . I n g e r s o l l , Committee on Problems o f Drug Dependence Mtg., May 22, 1972, Ann Arbor, Mich., p . 14. J. E . I n g e r s o l l , in Drug Abuse, Proc. I n t e r n . Conf., C.J.D. Z a r a f o n e t i s , Ed., Lea and F e b i q e r , 1972, p. 449. J. E. I n g e r s o l l , 14. Y. Law J o u r n a l , Mar. 2 7 , 1972, p . 25. C. E. I n t u r i s s i and K. Verebely, C l i n . Pharmacol. Therap. 2,633 (1972). J. H. J a f f e , i n Crug Abuse, Proc. I n t e r n . Conf.. C.J.D. Z a r a f o n e t i s , Ed.. Lea and F e b i g e r , 1972, p. 161.. J. H. J a f f e , in Yearbook of Drug Abuse, L. B r i l l and E. H a m , Eds., B e h a v i o r a l P u b l i c a t i o n s , N . Y. 1373, p . 103. D. R. J a s i n s k i e t a l . , Co!mfttce on Problems o f Drug Dependence, 442, (1972). D. R. J a s i n s k i , i n Yearbook of Drug Abuse, L. Brill and E. Harms, Eds., B e h a v i o r a l P u b l i c a t i o n s , N. Y. 1973, p. 37. Z a r a f o n e t i s . Ed., Lea and P e b i g e r , A. Koplan, i n Drug Abuse, Proc. I n t e r n . Conf., C.J.D. 1972, p. 191. M. Kato, , p. 67. E. Kaufrnan. Contemporary Drug Problems, Spring 1972, p. 207. D. C. Kay, i n Yearbook of Drug Abuse, L. B r i l l and E. Harms, Eds., Behavioral P u b l i c a t i o n s , H. Y . 1973, p . 17. M. H. K e e l e r , Am. J . P s y c h i a t r y , 125, 386 (1968). K. H. Keeler, C. B. R e i f l e r and M. 8. L i p r i n , Am. J. P s y c h i a t . 384 (1968). E. M. Kennedy, N. Y. Law J o u r n a l , Mar. 28, 1972. P. K i e l h o l z , i n Drug Abuse, Proc. I n t e m . Conf., C.J.D. Z a r a f o n e t i r , Ed., Lea a n d P e b i g e r , 1972. p. 361. P. X i e l h e l z , Sb,p. 363. E. I. Koch, N. 1. Law J o u r n a l , Mar. 28, 1972. A. J . R. Koumans, i n Drug Abuse, Proc. I n t e r n . Conf., C.J.D. Z a r a f o n o t i s , Bd., Lea and P c b i g e r , 1972. p. 331. A. J. R. Koumans, i 3 g , p. 385. J. C . Iramer, ibbi, p. 253. J. C. Kraner, i n Yearbook of Drug Abuse, L. B r i l l and E. Harm, Eds., B e h a v i o r a l Publicat i o n s , N. Y.. 1973, p. 265. M. J. Kreek, L. %dcs, 5 . Kane, J. Knoblcr and R. M a r t i n , 4nn. I n t e r n . Mod. 77,598 (1972).
!b-e,,
N,
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120* T. b n d , che J o u r n a i , Addaction Research Foundation, Toronto, 3. 8 (1976). 121. J . 8 . b n g e r , Contemporary Drug Problemr, F a l l 1972. p. 8U. 122. J. Langrod, L. B r i l l . J. Lowinson and H. Joseph, in Major Modalities in t h e Treatment o f Drug Abuse, L. B r i l l and L. L i e b e m a n , Eds.. Behavioral P u b l i c a t i o n r , N.Y., 1972. p. 107. 123. L. Langrod , H. Joseph and K. Colgan. ibid, p. 167. Contemporary Drug Probtcmr, F a l l 1972. 124. D. L a r r i e r , M. C l i n e , P. Brophy and A. >=hen, p. 707. 125. D. Laskowitr, L. B r i l l and J. B. J a f f e , in Major M o d d i t i e s in t h e Treatment o f Dmg Abuse, L. B r i l l and L. Lieberman. Edr., Behavioral P u b l i c a t i o n s , N. Y . , 1972, p. 65. 126. L. b n s f o d . i n Yearbook of Drug Abure, L. B r i l l and E. Hams, Eds., B e h a v i o r a l P u b i i c a t i o n s , N. Y . 1973, p . 303. 127. H. L. b n n a r d , L. J. E p s t e i n and M. S. Rosenthal, Science, 176,881 (1972). 128* A. C. L e s l i e , H. Y. Law J o u t n a i , Uar. 28, 1972, p. 35. 129. L. Lieberman and L . B r i l l , in Yajor M o d a l i t i e s in t h e Treatment of Dmg Abuae. L. B r i l l and L. Lieberman, Eds., Behavioral P u b l i c a t i o n s , N. Y., 1972, p. 67. 130. D. 8. Louria, i n Drug Abuse, Proc. I n t e r n . Conf., C.J.D. Z a r a f o n e t i r . Ed., Lea and P e b i g e r , 1972. p. 505. 131. C. Maggiolo and F. Huidobro, Acta Phvsiol. Latinamer., y. 70 (1961). 132. S. T. Maidlow and A. Berman. Am. J . Pub. Health, 62, 1357 (1972). 133. W. R. M a r t i n & . W. Corodetsky and T. R. McClane, C l i n . Pharmacol.Ther.,L, 455 (1966). 134. W. R. M a r t i n , Am. J . Hosp. Pharm., 22, 133 (1965). 135. W. R. U a r t i n , in Dmg Abuse, R o c . I n t e r n . Conf., C.J.D. Z a r a f o n e t i s , Ed.. Lea and P e b i p e r , 1972, p. 153. 136. W. R. M a r t i n , in Yearbook of Drug Abuse, L. B r i l l and E. b m r , Edr., B e h a v i o r a l P u b l l c a t r o n . N. Y . , 1973, p . 1. 137. H. M. Mason, W. J. R. T a y l o r and M. V. Deming, C l i n . T o x i c o l . 2. 70 (1972). 138. D. W . Hatheson. S. J. Lynch and R. W. E a r l , Clin. T o x i c o l . , 2, 67 (1962). 139. E. May, a a l i n g w i t h W g Abuse. A Report t o t h e Ford Foundation, Ed. by P. M. Wdld and P. 8. h t t , Praeger F u b l i s h e r s , Nev York, 1972, p. 345. 140. E. W. Maynert, in Orug Abuse, Proc. I n t e r n . Conf., C.J.D. Z a r a f o n e t i r , Ed., Lea and P e b f g e r , 1972, p. 199. 141. W. H. McClothlin, Contemporary Drug Problems, Summer 1972, p. 467. 142. W. H. McGothlin and L. U. Werc, Am. J . P s y c h i a t . , 125, 370 (1968). 163. J . Mendelson, The J o u r n a l , Addiction Research Foundation, Toronto, 2, 7 (1973. 164. P. H. Meyers, i n Drug Abuse, Proc. I n t e r n . Conf., C.J.D. Z a r a f o n e t i s , Ed., Lea a n d P e b i g e r , 1972, p. 515. 145. A . D. M o f f e t t , I. H. Soloway and M. X . C l i c k , Clin. T o x i c o l . 5, 68 (1972). 146. I. Monkovic. A b s t r a c t s of 1 3 t h N a t i o n a l Med. Chem. Symp., ACS Div. Med. Chem., Iova C i t y , Iowa (1972). 147. I. Monkovic, T. T. Conway, H. Wang, Y. G. Perron, I. J. P a c h t e r and 8. B e l l e a u , J. Am. Cham. SOC. 95, 7910 (1973). 148. R. Morales-Boyer, in Yearbook of Drug Abuse, L. B r i l l and E. Harms. Eds., k h a v i o r a l P u b l i c a t i o n s , N. Y . 1973, p. 179. 149. J . C. Hunns, G. Geis and B. H. J u l l i n g t o n , i n Major M o d a l i t i e s in t h e Treatment of Dmg Abuse, L. B r i l l and L. L i e b e m a n , Eds., Behavioral R t b l i c a t i o n s , N. Y.,1972, p. 191. 150. J . Nepote. in Drug Abuse, W o e . I n t e r n . Conf., C.J.D. Z a r a f o n e t i s , Ed. Lea and F e b i g e r , 1972, p. 491. 151. R. G. Newman, Contemporary Drug Problems, Spring 1972, p. 183. 152. R. G. Newman, @, F a l l 1972, p. 605. 153. R. C. tiewman, S. Bashkow and M . C a r e s , i b f d , P a l l 1973, p. 417. 154. A. W. Nichols and P. R. Torrens, Arch. I r M e d . 127, 903 (1971). 155. W. W. Nichols, in Crug Abuse, Proc. I n t e r n . Conf.. C.J.D. Z a r a f o n e t i s , Ed., Lea and P e b i g e r , 1972, p. 93. 156. J . T. Nix, C l i n . T o x i c o l . 2. 62 (1972). ls7. J . W. O l i v e r , in Drug Abuse, Proc. I n t e r n . Conf., C.J.D. Z a r a f o n e t i a , Ed., Lea a n d F e b i g e r , 1972, p. 429, 158. K. D. P a r k e r , ;Sld , p. 129. 159. C. Pepper. N. Y. Law JOUrMl. Mar. 28. 1972. 160. C. C . - P e t e r s o n and M. R. Wilson, J r . , Mayo C l i n . Proc. 46, 468 (1971). 161. J. C. P o l l a r d , in Drug Abuse, Proc. I n t e r n . Conf., C.J.D. Z a r a f o n e t i s , Ed., Lea and P e b i g e r . 1972. D. 377. 162. T. G f a l s k y , Contemporary Drug Problems, SuPsler 1972, p. 399. 163. E. Ramiret. in Hajor k l o d a l i t i e s in t n e Treatment o f Drug Abuse, L. B r i l l and L. Lieberman, Eds., Behavioral P u b l i c a t i o n s , N. Y . 1972. p. 43. 164. E. Ramiret. i s , p. 55. 165. C. R. Rangel, N. Y. 5aw JOutM1, Mar. 20, 1972. I~
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166. 167. 168. 169.
170. 171. 172. 173. 174. 175. 176. 177. 178. 179.
180. 181. 182. 183. 184. 185. 186. 107.
186. 189. 190. 191. 192. 193. 194. 195. 196. 197. 198. 199. 200. 201. 202 * 203. 204. 205. 206. 207. 208. 209. 210. 211. 212.
213.
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CNS Agents
Gordon, Ed.
i n Major M o d a l i t i e s in t h e Treatment of Drug Abura, L. B r i l l and L. Lieberman, Edr., Behavioral P u b l i c a t i o n r , N. Y., 1972, p. 7. 0. S. Ray, C. V. Mosby Co.. S t . Louis, 1972. A. Richman. M. E. P e r k i n r , 8. B i h a r i and J. J. Pishman, Am. J. P u b l i c H e a l t h 62, 1002(1972). L. Robbine, B. S. Robhins and M. Stem, Druq IBwndmnce, (NMII) (5) O c t . 1970, p. 1. C. C. Rohrs, J . P. Murphy and J. Desen-Cerber, in D r u g Abuse, Proc. I n t e r n . Conf., C.J.D. Z a r a f o n e t i s , Ed., Lea and F e b i g e r , 1972, p. 571. M. S. Rosenthal. i n Yearbook of D r u g Abuse, L. B r i l l and E. Harrps. Eds., B e h a v i o r a l F u b l i c a t i o n s , N. Y. 1973, p. 83. J. K. S a e l e n s , P. R. Granat and W. K . Sawyer, Arch. I n t . Pharaucodya., E. 213 (1971). H. Samuels, Contemporary D r u g Problems, S m e r 1972. p. 517. J. 8 . Scheuer, i n Drug Abuse, Proc. I n t e r n . Conf., C.J.D. Z a r a f o n e t i s , Ed., Lea and P e b i g e r , 1 9 i 2 , p. 423. S. H. Schuman, p. 513. p. 233. J. W. Schweitzer and A. J. F r e i d h o f f , M. H. Seevers, , p. 9 . M. L. S e e v e r s , N. Y. Lav J o u r n a l , Mar. 28, 1972. S. 8. S e l l s , L. R. Chatham and R. L. Retka, Contemporary Drug Problems, P a l l 1972, p.665. R. P. S h a f e r , e t a l , Second Report o f N a t i o n a l C&ssion on Marihuana a n d Drug Abuse, March 1973. M. Shepherd, Lancet 1 (7636) 31 (1970). A. T. S h u l g i n , in Psychotomimetic Drugs, D. H. Efron. Ed., Raven Prarr, 1970, p. 21. A. T. S h u l g i n , T. Sargent and C. Naranjo, Nature, 221, 537 (1969). R. E. S h u l t e s . B u l l . N a r c o t i c s , 3.15 (1969). E. Siegmund, R. Cadnus and P. Lu, R o c . SOC. Exp. Biol., 9s. 729 (1957). J. M. Singh, M. D. C a a t e t and J. T. Nix, C l i n . Toxicol. 5, 72 (1972). C.J.D. Z a r a f o n e t i s , Ed., i c a a d D. - . E. Smith. i n Drug Abuse. Proc. I n t e r n . Conf., P e b i g e r , 1 9 j 2 , p. 271. D. E. Smith, i n Major M o d a l i t i e s i n t h e Treatment of Drug Abure, L. Brill and L. Lieberman, Eds., Behavioral P u b l i c a t i o m , N. Y., 1972, p. 267. D. E. Smith and A . J . Rose, i n Major M o d a l i t i e s i n t h e Treacment of h g Abuse, L. B r i l l 6nd L. Lieberman, Eds., Behavioral P u b l i c a t i o n s , N. Y., 1972, p. 257. D. E. Smith and D. Bentel, in Yearbook of Drug Abuse, L. B r i l l and E. Halmu, E d i . . Behavioral P u b l i c a t i o n s . N. Y., 1973. p. 157. R. C . Smith, in Drug Abuse, Proc. I n t e r n . Conf., C . J . 3 . Z a r a f o n e t i s , Ed., Lea and P e b i g e r , 1972, p. 455. R. C. SmFrn, =$ , p. 519. S. H. Snyder, L. A . ? a i l l a c a and H. W e i q a r t n e r , Am. J . P s y c h i a t . 125, 357 (1969). R. Stephens and E. C o r c r e l l , Brit. J . Addict. 67, 45 (1972). J. Sokol, i n Yearbook of Drug Abuse, L. B r i l l and E. Hams, Edn., Behavioral P u b l i c a t i o n s , N. Y., 1973. p. 169. W. J. R. T a y l o r , C . D. Chambers and C. E. Bowling, I n t l , J. C l i n . Pharmacol. Therap. T o x i c o l . 6, 28 (1972). D. A. T r e f i e r t , Contemporary D r u g Problems, Suxner 1973, p. 239. D. A. T r e f f e r t . l&, F a l l 1973, p. 393. J . T. U n g e r l e i d e r , in b h g Abuse, Proc. I n t e r n . Conf., C.J.D. Z a r a f o n e t i s , Ed.. Lea arid Pebiger. - . 1972. D. 339. J. T. U n s e r l e i d e r , :I=, p . 501. J. T. U n g e r l e i d e r , D. D. F i s h e r , S . R. Goldsmith. M. Fuller and E. Forgy. Am. J . P s y c h i a t . 125, 352 (1958). T T . U n g e r l e i d e r and M. P u l l e r , in Yearbook of Drug Abuse, L. B r i l l and E. Harms, Eds., Behavioral P u b l i c a t i o n s , N. Y. 1973. p. 367. B. A . Van Der Kolk, Contemporary Drug Problems, Summer 1973, p. 263. P. M. Wald and P. B. Hutt, Dealing w i t h D r u g Abuse. A Report t o t h e Ford Foundation, Ed. by P. M. Wald and P. B. Hurt, P r a e g e r P u b l i s h e r s , New York, 1972, p. 3. P. M. Wald and A. Atrams, , p. 123. A. T. Weil, , p. 329. N. Weiner, in Drug Abuse, Proc. I n t e r n . Conf., C.J.D. Z a r a f o n c t i s , Ed.. Lea and P e b i g e r , 1972, p. 243. D. R. Uesson. C. R. Gay and D. E. Smith, Contemporary Drug Roblem6, Sunmer 1972, p. 453. J. H. Willis, Contemoorary D r u g Problems, Summer 1972. p. 501. L. L. Wolff, N. Y. Law J o u r n a l , Mar. 28, 1972. B. A. Wolfson and D. B. Louria, p. 225. S. F. Yoller, i n Yearbook of Drug Abuse, L. Brill and E. Harms, Ed.., B e h a v i o r a l Publicat i o n s , N. Y. 1973, p. 239. A. Zaks and M. Peldzmn. J . Am. Ned. Ansn. 222, 1279 (1972).
B. W. Rasor,
G,
m,
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w,
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219. 220. 221. 222. 223. 224. 225. 226. 227. 228. 229. 230. 231. 232. 233. 234.
Abuse of CNS Agents
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N. E. Zi a b e rg, Contemporary Drug Problems, S p r i n g 1972, p. 263. 1. E. Z in b e rg, ib:d , P a l l 1972, p. 747. W. R. M a r t i n and H. F. F r a s e r , J. Pharm. E xptl. Therap. 133, 388 (1961). J. R. J a f f e , Arch. Gen. P sychi at . 15, 525 (1971). It. Goodall, Psvchol. Today, 6 , 1 3 6 ( 1 9 7 2 ) . I. J . P a c h t e r , S ci ence, 179, (1923). M. Moore, P o l i c y Concerning Drug Abuse i n New York S t a t e , Vol. 111, t h e Hudson I n s t i t u t e (1970). pps. 67-73. E. A. P r e b l e and J . 3. Casey, I n t . J. Addi ction, 4 , 12 (1967). P. M. Brecher. (1972) " L i c i t and I l l i c i t Drugs" L i t t l e . Brown. p. 42. R. Dougherty, unpubl i shed d a t a . S. D. P a m a t i k a r and S. R. Knowles, Clin.Pharmaco1.Therap. 1 4 ,941 (1973). A. V. P i r c i o , H. Gordon and I. J . P a c h t e r , Pharmacologist 1 2,263 (1971). A. W . P i r c i o . Htg. Ned. Chem. 3 i v . Am. Chem. SOC., Iowa C i t y , June 1972. J . F . C a l i m l i n , W. :I. W ardel l , L. Lasagna, A. 3. G i l l i e s and H. T. Davis, C l i n . Pharmacol. Therap., I n P r e s s . H. Blumberg, H. 8. Dayton, and P. S . Wolf, Pharmacologist lo, 159 (1968). H. H. Swain, J . E. Villareal and H. H. S eeve rs , C o m t e on Problems of Drug Dcpendencc, 1973, p. 695, 696. M. Gordon, A. U. P i r c i o , F. S. Caruso, and I. J. P a c h t e r , Mtg. Cmte o n Problems of Drug Dependence, Mexico C i t y. March 11. 1974. R. W. Houde i n "Diagnosis and Management of Cancer," ed. by R. L. Cla rk, Yearbook M e d i c a l P u b l i c a t i o n , Chicago, I l l . (1971) p. 489, Vol. 111. U. H. F o r r e s t , Ccste on Problems of Drug Dependence. p. 247 (1971). N. 8 . Eddy, The N a t i o n a l Research Council Involvement i n t h e O pia te Problem, 192801971, N a t i o n a l Academy o f S ci ence, Vashington, D. C., 1973. D. J a s i n s k i , Mtg. Comte on Problems cf Drug Dependence, Mexico C i t y , M a r c h 11, 1974.
VL
S e c t i o n I1 Editor:
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Pharmacodynamic Agents
Frank H. Clarke, Ciba-Geigy L t d . , A r d s l e y , New York Chapter 6 .
The E t i o l o g y of Hypertension
Donald W. DuCharme, The Upjohn Company, Kalamazoo, Michigan Hypertension i s one of t h e g r e a t e s t medical c h a l l e n g e s of o u r t i m e . I t i s e s t i m a t e d t h a t upwards of 40 m i l l i o n Americans a r e a f f l i c t e d by t h i s " s i l e n t disease",l Although g r e a t p r o g r e s s i n t h e t r e a t m e n t o f h y p e r t e n s i v e d i s e a s e u n q u e s t i o n a b l y h a s been made i n r e c e n t y e a r s , t h e u l t i m a t e t h e r a p y remains t o b e d i s c o v e r e d . The development of d r u g s which a c t s p e c i f i c a l l y upon v a r i o u s systems t o a m e l i o r a t e t h e symptom ( e l e v a t e d a r t e r i a l p r e s s u r e ) of t h i s d i s e a s e have, indeed, c o n t r i b u t e d a g r e a t d e a l t o c l i n i c a l management. They have enabled c l i n i c i a n s t o e f f e c t i v e l y lower blood p r e s s u r e i n t h e m a j o r i t y of p a t i e n t s , and minimize many of t h e a d v e r s e s i d e e f f e c t s which were a s s o c i a t e d w i t h e a r l i e r forms of therapy. There i s l i t t l e doubt t h a t s t i l l b e t t e r d r u g s can b e developed. For example, a d i u r e t i c which does n o t a l t e r potassium and u r i c a c i d e x c r e t i o n o r g l u c o s e metabolism, a c e n t r a l l y a c t i n g s u p p r e s s a n t of s y m p a t h e t i c v a s o c o n s t r i c t o r n e r v e f u n c t i o n which i s devoid of s e d a t i v e a c t i v i t y , a p e r i p h e r a l v a s o d i l a t o r which does n o t cause f l u i d r e t e n t i o n , a b e t a a d r e n e r g i c b l o c k i n g a g e n t which does n o t a f f e c t b r o n c h i a l smooth muscle f u n c t i o n , o r an a n t a g o n i s t of p e r i p h e r a l a d r e n e r g i c neurones which i s s p e c i f i c t o t h o s e neurones i n n e r v a t i n g blood v e s s e l s . The l i s t of pot e n t i a l improvements i n e x i s t i n g a g e n t s i s v i r t u a l l y e n d l e s s , and many would v e r y s i g n i f i c a n t l y improve c l i n i c a l therapy. The p o i n t remains, however, t h a t r e g a r d l e s s of how s p e c i f i c t h e s e a g e n t s become t h e y s t i l l p r o v i d e o n l y p a l l i a t i v e therapy. The r e a l c h a l l e n g e and hope i s t o d i s cover and develop a g e n t s which c o r r e c t t h e u n d e r l y i n g cause of t h e d i s ease. Before one can d e s i g n a r a t i o n a l program t o d i s c o v e r a g e n t s which w i l l "cure" h y p e r t e n s i o n , t h e c a u s e must b e d e f i n e d , While t h e c a u s e of h i g h blood p r e s s u r e can be d e f i n e d and c o r r e c t e d s u r g i c a l l y o r t h e r a p e u t i c a l l y i n a s m a l l p e r c e n t a g e of t h e h y p e r t e n s i v e p o p u l a t i o n , f o r t h e v a s t m a j o r i t y i t cannot, This l a r g e group of p a t i e n t s w i t h h y p e r t e n s i o n of unknown e t i o l o g y a r e c a t e g o r i z e d a s having " e s s e n t i a l " h y p e r t e n s i o n . I t i s t h i s group of p a t i e n t s which d i c t a t e s t h e need f o r a major r e s e a r c h e f f o r t t o uncover t h e cause o r causes of t h e d i s e a s e p r o c e s s and t o d i s cover and develop a g e n t s which w i l l e f f o r d o p t i m a l t h e r a p e u t i c c o n t r o l . I n t e r e s t i n g l y , i t h a s been p o s s i b l e t o induce h y p e r t e n s i o n i n l a b o r a t o r y animals a t w i l l by a v a r i e t y of methods f o r n e a r l y f o r t y y e a r s 2 , y e t t h e primary d i s t u r b a n c e which promotes t h e h i g h blood p r e s s u r e i n t h e s e a n i mals remains an enigma,
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Vascular R e s i s t a n c e The major hemodynarnic a b n o r m a l i t y i n h y p e r t e n s i v e p a t i e n t s and l a b o r a t o r y animals i s an e l e v a t e d 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 . Suggested e t i o l o g i c f a c t o r s which may c o n t r i b u t e t o t h e 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 a r e enhanced humoral v a s o c o n s t r i c t o r s u b s t a n c e s , d e c r e a s e d humoral v a s o d i l a t o r s u b s t a n c e s , enhanced neurogenic v a s o c o n s t r i c t o r a c t i v i t y , expanded e x t r a c e l l u l a r f l u i d volume, "auto-
Chap. 6
Etiology of Hypertension
DuCharme
51 -
regulatory" vasoconstriction and structural changes in the arterial walls. There is little question that the composition of arteriolar walls in hypertensive animals is modified. The wall is thickened in relation to the l ~ m e n 3 - 7 ~Furthermore, resistance to flow in hypertensive vessels is increased even after maximal vas~dilatation~,~, and reactivity of hypertensive vessels to vasoconstrictor stimuli is enhanceds,l0. Although most investigators believe the structural changes in the vessel walls are secondary to the increased intravascular pressure, some question remains as to whether the functional changes represent primary or secondary events'
Renin-Angiotensin-Aldosterone System - The major unresolved problem is the mechanism responsible for initiation of the abnormal vascular resistance. Humoral vasoconstrictor substances released from the kidney have been prime suspects since it was through restriction of renal blood flow that Dr. Goldblatt first induced hypertension in laboratory animals? The subsequent discovery and elucidation of the renin-angiotensin-aldosterone system, and the participation of this system in the hypertensive process has occupied the minds and efforts of many researchers and has been reviewed frequentl?l2 - I 4 . Recent studies which employed immunologic techniques to render animals insensitive to the vasoconstrictor action of angiotensin or specific angiotensin receptor blocking agents have demonstrated that the renin-angiotensin system is involved in the development of some but not all forms of experimental hypertension. Animals made hypertensive by restricting blood flow to one kidney and leaving the contralateral kidney intact generally exhibit a lowered blood pressure in response to angiotensin I1 blockade15'le. This is particularly true during the first weeks after onset of the hypertensive process. After the hypertension is well established, however, the blood pressure response to angiotensin I1 antagonism is much less consistent. On the other hand spontaneously hypertensive animals, metacorticoid hypertensive animals or animals rendered hypertensive by unilateral renal artery constriction and contralateral nephrectomy generally fail to demonstrate a decreased blood pressure in response to angiotensin I1 blo~kade~~'~~. Interestingly, immunization with a crude renal cortical extract markedly lowered the blood pressure of dogs with renal hypertension (bilateral renal artery constriction or unilateral renal artery constriction and contralateral This observation, coupled nephrectomy) of several years duratioli?o'2'. with those in which a specific angiotensin I1 antagonist was employed, suggests the possibility that the animals were immunized against a substance of renal cortical origin other than renin. This possibility is supported by more recent work which demonstrated that passive transfer of renin immunity to dogs with chronic renal hypertension did not restore blood pressure to n~rmal"~.Furthermore, active immunization of rabbits against angiotensin I1 failed to prevent the development of either perinephritic hypertension or single kidney Goldblatt hypertensionz4," 5. It is apparent that the results obtained with immunization procedures have varied considerably depending upon the animal species, the specific immunization technique, and the type of experimental hypertension employed. The causative role of the renin-angiotensin-aldosterone system in the genesis of hypertension, therefore, remains in question.
52
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Pharmacodynamic Agents
Clarke, Ed.
Other Renal Prohypertensive Factors - The lack of agreement between in-vestigators as to the etiologic significance of the renin-angiotensin system in experimental hypertension has prompted some to search for other renal factors which might be involved. Grollman has described a substance designated nephrotensin which was isolated from renal venous blood of patients with surgically remediable hypertension and from animals with renal ischemia hypertension?6'"7. Nephrotensin is presumably a polypeptide different from angiotensin I or I1 which exerts a potent vasoconstrictor action, and appears to be causally related to the hypertension associated with renal ischemia. The participation of this substance in human essential hypertension remains to be determined. Several investigators have suggested that the increase in vascular resistance associated with hypertension is a secondary, "autoregulatoryll response triggered by an increase in cardiac outpu?""l. If this is true, then the factors responsible for the increased cardiac output could become of greater etiologic importance than those which increase only vascular resistance. Cardiac output may be increased because of a change in renal function which promotes fluid retention and thereby enhances venous return to the heart?". Alternatively, venous return and cardiac output may be enhanced in response to increased venous smooth muscle tone-?-?.A vasoconstrictor material of renal origin different from angiotensin I1 which markedly contracts vascular capacity has been demonstrated ,35. ~ ~ The chemical in dogs subjected to severe hypovolemic h y p ~ t e n s i o n nature of this material and its role in the pathogenesis of hypertension remain to be determined.
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Kallidin-Kinin System Recent work has also suggested a role for the kallidin-kinin system in the pathogenesis of hypertensiod 6 . Significant increases in urinary excretion of kallikrein were found in spontaneously hypertensive and DOCA-salt hypertensive rats, while levels found in rats with renal hypertension and patients with essential hypertension were lower than those in respective controls. Additional data suggests that the kallidin-kinin system may be intimately involved in the regulation of salt and water excretion by the kidnef6, ,?',
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Renal Antihypertensive Factors In addition to a prohypertensive mechanism it has been recognized for many years that the kidney possesses an antihypertensive mechanism. Loss of the antihypertensive function may be as important to the pathogenesis of hypertension as activation of the hypertensive mechanism. Work in this area has been kindled in recent years by the demonstration that explants of renomedullary tissue will prevent the onset of renoprival hypertension or lower the blood pressure of Extracts of renal medullary tissue animals with renal hyperten~ion~"'~' have been shown to lower the blood pressure of animals with experimental hypertension by a number of investigators. Extracts possess both an acute transient hypotensive action and a slow onset, protracted effect. The The substance acute effect is due to the presence of prostaglandin^^'^^^ responsible for the delayed response is also lipid in character, however, it appears to be a neutral lipid and not in the prostaglandin fa~nily~"~~.
_ I _
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The s o u r c e of t h e r e n a l medullary a n t i h y p e r t e n s i v e s u b s t a n c e s app e a r s t o be t h e i n t e r s t i t i a l c e l l s l o c a t e d p r i m a r i l y i n t h e p a p i l l a . G r a n u l a t i o n of t h e s e c e l l s and presumably s e c r e t i o n h a s been demonstrated t o r e f l e c t changes i n blood p r e s s u r e of animals s u b j e c t e d t o e x p e r i m e n t a l hyperten~ion~~~ R e~c'e.n t l y , r a b b i t i n t e r s t i t i a l c e l l s have been grown i n t i s s u e c u l t u r e * 9 . The c e l l s were o b t a i n e d from a u t o t r a n s p l a n t s of r e n a l medulla which prevented r e n o v a s c u l a r and r e n o p r i v a l t y p e s of h y p e r t e n s i o n . E x t r a c t s of t h e c u l t u r e d c e l l s were shown t o c o n t a i n t h e t h r e e r e n a l medullary p r o s t a g l a n d i n s PGE2, PG% and PGF2 The p o s s i b l e r o l e of p r o s t a g l a n d i n s i n t h e p a t h o g e n e s i s of a r t e r i a l h y p e r t e n s i o n i s of g r e a t i n t e r e s t , s i n c e some p r o s t a g l a n d i n s n o t o n l y d i l a t e p e r i p h e r a l r e s i s t a n c e v e s s e l s but a l s o possess n a t r i u r e t i c a c t i v i t y 0 . I t i s t h e r e f o r e tempting t o s p e c u l a t e t h a t a d e f i c i e n c y of r e n a l p r o s t a g l a n d i n s might lead t o a r t e r i a l h y p e r t e n s i o n . R e l e a s e of p r o s t a g l a n d i n s i n t o t h e r e n a l venous blood has been demonstrated i n r e s p o n s e t o vasoc o n s t r i c t o r s t i m u l i o r r e n a l i ~ c h e m i a ' ~ ' ~ ~Most . s t u d i e s have suggested t h a t t h e primary v a s o d i l a t o r p r o s t a g l a n d i n r e l e a s e d from t h e kidney i s PGE2, b u t because of i t s r a p i d metabolism by t h e lungs PGE2 could n o t f u n c t i o n a s a g e n e r a l v a s o d i l a t o r hormone. A l t e r n a t i v e l y , r e n a l p r o s t a g l a n d i n s may e x e r t an a n t i h y p e r t e n s i v e a f f e c t through an a c t i o n w i t h i n t h e kidney t o m a i n t a i n normal r e n a l r e s i s t a n c e and p r e v e n t sodium and water accumulation. Lee has suggested t h a t PGAs, on t h e o t h e r hand, may be c i r c u l a t i n g a n t i h y p e r t e n s i v e hormones, s i n c e t h e y a r e much more r e s i s t a n t t o m e t a b o l i c d e g r a d a t i o n on p a s s i n g through t h e lungs55. There i s c o n t r o v e r s y , however, p e r t a i n i n g t o t h e a b i l i t y of t h e kidney t o form A prostaglandin^^^. Nonet h e l e s s , two groups have r e p o r t e d t h a t c i r c u l a t i n g l e v e l s of PGA a r e lower i n h y p e r t e n s i v e t h a n normotensive i n d i v i d u a l s , and a r e v i r t u a l l y a b s e n t i n t h e blood of a n e p h r i c 5y. While t h e h y p o t h e s i s t h a t p r o s t a g l a n d i n s f u n c t i o n a s a n t i h y p e r t e n s i v e hormones i s c e r t a i n l y a t t r a c t i v e , i t i s obvious t h a t much a d d i t i o n a l work i s n e c e s s a r y f o r v e r i f i c a t i o n .
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The autonomic nervous system a l s o i s i m p l i c a t e d Autonomic Nervous System i n t h e p a t h o g e n e s i s of h y p e r t e n s i o n . S e v e r a l i n v e s t i g a t o r s have suggested t h a t heightened s y m p a t h e t i c nerve a c t i v i t y may u n d e r l i e t h e i n i t i a l l a b i l e phase of hypertension6"' 'O. This h y p o t h e s i s i s supported by e x p e r i m e n t a l s t u d i e s which demonstrated t h a t removal of t h e sympathetic nervous system c h e m i c a l l y o r immunologically prevented t h e o n s e t of g e n e t i c o r mineraloc o r t i c o i d hypertension60, 61. On t h e o t h e r hand, t h e s e p r o c e d u r e s were i n e f f e c t i v e i n r e d u c i n g t h e blood p r e s s u r e of animals i n which t h e hypertension w a s established p r i o r t o Conversely, t h e o n s e t of r e n a l h y p e r t e n s i o n i s n o t prevented by sympathectomy, b u t maintenance of t h i s form of e x p e r i m e n t a l h y p e r t e n s i o n a p p e a r s t o be dependent upon a n i n t a c t , f u n c t i o n a l s y m p a t h e t i c nervous system64. Involvement of t h e sympat h e t i c nervous system i n h y p e r t e n s i v e d i s e a s e i s a l s o suggested by t h e a n t i h y p e r t e n s i v e e f f i c a c y of t h e many sympathetic s u p p r e s s a n t drugs. Many c l i n i c a l s t u d i e s have been conducted i n an a t t e m p t t o q u a n t i t a t e t h e d e g r e e of s y m p a t h e t i c n e u r o n a l involvement i n v a r i o u s forms of human h y p e r t e n s i o n . These s t u d i e s have shown t h a t n e u r a l l y mediated v a s o c o n s t r i c t o r t o n e i s en-
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hanced i n most h y p e r t e n s i v e s u b j e c t s , b u t i t s r o l e i n t h e maintenance of t h e h y p e r t e n s i v e s t a t e i s c o n t r o v e r s i a l . The r e a d e r i s r e f e r r e d t o two . r e c e n t a r t i c l e s which a d d r e s s t h i s q u e s t i o n i n g r e a t e r d e t a i l 6 5 Y ~ ~There would seem t o be l i t t l e doubt t h a t t h e autonomic nervous system i s i n volved i n t h e h y p e r t e n s i v e process. Whether i t p l a y s a primary o r p e r missive r o l e i n t h e p a t h o g e n e s i s of t h e d i s e a s e , however, i s i n q u e s t i o n .
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Genetic F a c t o r s The f a c t t h a t t h r e e independent groups have s u c c e s s f u l l y bred s t r a i n s of h y p e r t e n s i v e r a t s i n d i c a t e s t h a t g e n e t i c f a c t o r s a r e a l s o e x t e n s i v e l y involved i n t h e g e n e s i s of h y p e r t e n ~ i o n ~ ~ ' ~Many ". investig a t o r s b e l i e v e t h a t t h e spontaneously h y p e r t e n s i v e r a t (SHR) i s t h e model of e x p e r i m e n t a l h y p e r t e n s i o n which most c l o s e l y resembles e s s e n t i a l human h y p e r t e n ~ i o 70. n ~ ~ ~The e t i o l o g y of t h e h y p e r t e n s i o n which occurs i n t h e s e s t r a i n s , however, has n o t been d e f i n e d . The r e s u l t s of many s t u d i e s which have been conducted w i t h SH r a t s a r e h i g h l y q u e s t i o n a b l e , because app r o p r i a t e c o n t r o l animals a r e u n a v a i l a b l e . Normotensive r a t s of even t h e same s t r a i n o b v i o u s l y a r e g e n e t i c a l l y d i f f e r e n t from t h e i r h y p e r t e n s i v e r e l a t i v e s , and, t h e r e f o r e , a r e q u e s t i o n a b l e c o n t r o l s . The one e x c e p t i o n appears t o be t h e Dahl s t r a i n . These animals r e q u i r e a d m i n i s t r a t i o n of s a l t t o induce t h e hypertension. Therefore, one can have g e n e t i c a l l y s i m i l a r animals w i t h and w i t h o u t h y p e r t e n s i o n . Renal t r a n s p l a n t a t i o n experiments w i t h t h e Dahl SH r a t s have shown t h a t t h e kidney i s d e f i n i t e l y involved i n t h e p a t h o g e n e s i s of t h e h y p e r t e n ~ i o n ~ ~ - ~The ' . specific etio l o g i c f a c t o r s , however, have n o t been d e f i n e d . Although many f a c t o r s undoubtedly modify t h e p a t h o g e n e s i s of hypert e n s i o n , t h e b u l k of evidence p o i n t s toward t h e kidney a s t h e f o c a l p o i n t . F u t u r e s t u d i e s d i r e c t e d toward a c a r e f u l d e f i n i t i o n of r e n a l prohypert e n s i v e and a n t i h y p e r t e n s i v e f a c t o r s coupled w i t h r e n a l e x c r e t o r y f u n c t i o n and autonomic nerve a c t i v i t y would appear t o be most l i k e l y t o y i e l d t h e u l t i m a t e s o l u t i o n t o t h i s complex problem. References 1. 2.
3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14.
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15. A. R. C h r i s t l i e b , T.U.L. B i b e r , and R. B. H i c k l e r , J. Clin. I n v e s t . , 48, 1506, 1969. 16. 7 Bing and K. Poulsen, Acta Path, Microbiol. Scand., 7 8 , 6, 1970. 17. H. R. Brunner, J. D. Kirshman, J. E. S e a l e y and J. H. Laragh, Science, 174, 1344, 1971. 18. T T . P a l s , F. D. Masucci, G. S. Denning, F. Sipos and D. C. F e s s l e r , C i r c . Res., 2, 673, 1971. 19. F. M. Bumpus, S. Sen, R. R. Smeby, C. Sweet, C. M. F e r r a r i o and M. C. Khosla, Circ. Res., 32, Suppl. 1, 150, 1973. 20. G. E. Wakerlin, R. B. B i r d , B. B. Brennan, M. H. Frank, S. H. Kremen, I. Kuperman and J. H. Skom, J. Lab. Clin. Med., 4 l , 708, 1953. 2 1 . S. H. Kremen and G. E. Wakerlin, Proc. SOC. Exptl. B i o l . Med., 90, 9 9 , 1955. 22. E. Haas and H. G o l d b l a t t , Amer. J. P h y s i o l . , 197, 1303, 1959. 23. R. W. H i l l , J. E. Chester and P. E. Wisenbaugh, Lab. I n v e s t . , 22, 404, 1970. 24. I. Eide and H. Aars, Nature (London), 222, 571, 1969. 25. W. J. Louis, G. J. Macdonald, V. R e n z i n i , G. W. Boyd and W. S. P e a r t , Lancet, 1, 333, 1970. 26. A. Grollman, Proc. Soc. Exptl. Biol. Med., 1120, 1970. 27. A. Grollman and V. S. R. Krishnamurty, Amer. J. P h y s i o l . , 221, 1499, 197 1. 28. J. M. Ledingham and R. D. Cohen, Clin. S c i . , 22, 69, 1962. 29. J. M. Ledingham and R. D. Cohen, Can. Med. Assoc. J., 90, 292, 1964. 30. C. M. F e r r a r i o , I. H. Page and J. W. McCubbin, Circ. R e s . , 27, 799, 1970. 31. T. G. Coleman, H. J. Granger and A. C. Guyton, Circ. Res., 28, Suppl. 11, 76, 1971. 32. T. G. Coleman, J. D. Bower and H. G. Langford, C i r c . , 42, 509, 1970. 33. J. Brod, M. Cachovan, H. -U. Pixburg and P. Harmjanz, Therapiewoche, 45, 4258, 1973. 34. W. DuCharme and L. Beck, J. Pharmacol. Exp. Ther., 177, 56, 1971. 35. J. R. Powell and D. W. DuCharme, Amer. J. Physiol., 2,168, 1974. 36, H. S. Margolius, R. G. Geller, W. deJong, J. J. P i s a n o , and A. Sjoerdsma,Circ. Res., 2, Suppl. 11, 125, 1972. 37. M. Marin-Grez, P. Cottone and 0. A. C a r r e t e r o , Amer. J. P h y s i o l . , 9, 794, 1972. 38. E. E. Muirhead, J. A. Stirman, W. Lesch and F. Jones, Surg. Gynecol. O b s t e t . , 103, 673, 1956. 39. E. E. Muirhead, F. Jones and J. A. Stirman, J. Lab, Clin. Med., 56, 1 6 7 , 1960. 40. E. E. Muirhead, J. A. Stirman and F. Jones, J. Clin. I n v e s t . , 39, 266, 1960. 41. E. E. Muirhead, G. B. Brown, G. S. Germain and B. E. Leach, J. Lab. Clin. Med., 7 6 , 641, 1970. 42. R. B. H i c k l e r , D. P. L a u l e r , C. A. S a r a v i s , P. I. Vagnucci, G. S t e i n e r and G. W. Thorn, Can. Med. Ass. J., 90, 280, 1964. 43. E. G. D a n i e l s , J. W. Hinman, B. E. Leach and E. E. Muirhead, Nature (London), 215, 1298, 1967. 44. E. E. Muirhead, J. W. Hinman, E. G. D a n i e l s , M. Kosinski and B. Brooks, J. C l i n . I n v e s t . , 41, 1387, 1962.
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45. J. B. Lee, R. B. H i c k l e r , C. A. S a r a v i s and G. W. Thorn, C i r c . Res., 13, 359, 1963. 46. E. E. Muirhead, B. Brooks, M. K o s i n s k i , E. G. Daniels and J. W. Hinman, J. Lab, Clin. Med., 67, 778, 1966. 47. R. C. Muehrcke, A. K. Mandal, M. E p s t e i n and F. I. V o l i n i , J. Lab. C l i n , Med., 7 3 , 299, 1969. 48. L. Tobian, M. I s h i i and M. Duke, J. Lab. Clin. Med., 73, 309, 1969, 49. E. E. Muirhead, G. Germain, B. E. Leach, 3. A. P i t c o c k , P. Stephenson, B. Brooks, W. L. B r o s i u s , E. G. D a n i e l s and J. W. Hinman, C i r c . Res., 31, Suppl. 11, 161, 1972. 50. 7 C. McGiff and H. D. I t s k o v i t z , Circ. Res., 33, 479, 1973. 51. J. C. McGiff, K. Crowshaw, N. A. Terragno and A. J. Lonigro, Circ. Res., 27, Suppl. I , 121, 1970. 52. J. C. McGiff, K. Crowshaw, N. A. Terragno, K. U. Malik and A. J. Lonigro, Clin. S c i . , 42, 223, 1972. 53. E. W. Dunham and B. G. Zimmerman, Amer. J. P h y s i o l . , 219, 1279, 1970. 54. J. C. McGiff, K. Crowshaw, N. A. Terragno, A. J. Lonigro, J. C. S t r a n d , M. A. Williamson, J. B. Lee and K. K. F. Ng, Circ. Res., 27, 765, 1970. 55. J.' B. Lee, New Engl. J. Med. , 277, 1073, 1967, 56. R. M. Zusman, B. V. Caldwell, P. J. Mulrow and L. S p e r o f f , P r o s t a g l a n d i n s , 2 , 679, 1973. 57. J. B. Lee, A. A t t a l l a , V. K. Vance, C. Elwood and A. Prezyna, J. Clin. I n v e s t . , 52, SOa, 1973. 58. E. D. F r o h l i c h , V. J. Kozul, R. C. T a r a z i and H. P. Dustan, Circ. Res., 27, Suppl. I , 55, 1970. 59. B. Folkow, M. H a l l b i c k , Y. Lundgren, R. S i v e r t s s o n and L. Weiss, C i r c . Res., 32, Suppl. I , 2 , 1973. 60. G. H a e u s l e r , L. Finch and H. Thoenen, E x p e r i e n t i a , 28, 1200, 1 9 7 2 . 61. B. Folkow, M. Hallbxck, Y. Lundgren and L. Weiss, Acta P h y s i o l . Scand., 84, 512, 1972. 62. Ayitey-Smith and D. R. Varma, B r i t . J. Pharma., 40, 175, 1970. 63. D. W. C l a r k , C i r c . Res., 28, 330, 1971. 64. L. D. Dorr and M. J. Brody, Proc. SOC. Exptl. B i o l . Med., 123, 155, 1966. 65. R. C. T a r a z i and H. P. Dustan, Clin. S c i . , 44, 1 9 7 , 1973. 66. P. I. Korner, J. Shaw, J. B. Uther, M. J. West, R. J. McRitchie and J. G. Richards, Circ. 48, 107, 1973. 2 2 7 , 1969. 67. K. Okamoto, I n t . Rev, Exp. Path. , 7 2 7 , 1958. 68. F. H. Smirk and W. H. H a l l , Nature (London), 69. L. K. Dahl, M. Heine and L. T a s s i n a r i , Nature (London), 3,480, 1962. 70. S. Udenfriend and S. S p e c t o r , Science, 1155, 1972. 71. L. K. Dahl, K. D. Knudsen and 2. J u n e c h i , Circ. Res., 27, Suppl. 11, 277, 1970. 7 2 . L. K. Dahl, M. Heine and K. Thompson, C i r c . Res., 34, 94, 1974. 73. L. Tobian, Am. J. Med., 52, 595, 1972.
1,
182,
176,
Chapter 7.
A n t i h y p e r t e n s i v e Agents
John E . F r a n c i s , R e s e a r c h Department, P h a r m a c e u t i c a l s D i v i s i o n CIBA-GEIGY C o r p o r a t i o n , A r d s l e y , N. Y. 10502 New P r o d u c t s - I n F e b r u a r y 1973, S c h e r i n g C o r p o r a t i o n i n t r o d u c e d d i a z o x i d e ( I ) as H y p e r s t a t I . V . l , t h e f i r s t a n t i h y p e r t e n s i v e a g e n t t o r e a c h t h e U. S . market i n t e n y e a r s . I t was a l s o marketed t h i s y e a r i n S w i t z e r l a n d and Germany? S u g g e s t e d u s e i s f o r emergency i n t r a v e n o u s a d m i n i s t r a t i o n t o h o s p i t a l i z e d p a t i e n t s w i t h m a l i g n a n t h y p e r t e n s i o n , f o r which i t i s claimed t o b e a welcome and e f f e c t i v e t r e a t m e n t ? I n a r e c e n t c l i n i c a l s t u d y i t was o r a l l y e f f e c t i v e , sometimes combined w i t h a B - a d r e n e r g i c b l o c k e r , i n some p a t i e n t s w i t h s e v e r e and r e f r a c t o r y forms of h y p e r t e n s i o n : Debrisoquin ( D e c l i n a x , 11) w a s i n t r o d u c e d i n October i n S w i t z e r l a n d by HoffmannLaRoche? Long known f o r i t s u t i l i t y i n s e v e r e h y p e r t e n s i o n , t h i s d r u g gave good r e s u l t s i n a r e c e n t s t u d y o f m i l d t o m o d e r a t e l y s e v e r e hypert e n s i v e s ? P r o p r a n o l o l ( I n d e r a l , 111) t h e o n l y @ - b l o c k e r a v a i l a b l e on t h e U. S . m a r k e t 6 , i s n o t y e t i n d i c a t e d f o r t h e t r e a t m e n t of h y p e r t e n s i o n i n t h e U.S.A!
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-
111
Drups i n C l i n i c a l I n v e s t i g a t i o n Although many s i n g l e d r u g s were e v a l u a t e d , c l i n i c a l s t u d i e s i n 1973 r e f l e c t e d a growing t r e n d t o c o m b i n a t i o n t h e r a p y . C l o n i d i n e ( C a t a p r e s , ST-155 , IVa) , marketed i n Europe, w a s found t o b e e f f e c t i v e a t low d o s e s w i t h o u t p o s t u r a l h y p o t e n s i o n o r l a s t i n g s e d a t i v e s i d e e f f e c t s i n two l o n g term s t u d i e s ? y g A comparison w i t h r e s e r p i n e i l l u s t r a t e d c l o n i d i n e ’ s l a c k of d e p r e s s i v e r e a c t i o n s ” and t h e c o m b i n a t i o n w i t h c h l o r t h a l i d o n e (Combipres) w a s even more e f f e c t i v e t h a n c l o n i d i n e alone:’ The d a n g e r o f h y p e r t e n s i v e rebound and o t h e r s i d e e f f e c t s on One s t u d y d e s c r i b e d t h e sudden w i t h d r a w a l from c l o n i d i n e was n o t e d ? ,1 d r u g as s a f e and e f f e c t i v e f o r emergency t r e a t m e n t 1 4 whereas a n o t h e r r e p o r t e d s e r i o u s p r e s s o r e f f e c t s when p a t i e n t s w i t h a c c e l e r a t e d h y p e r t e n s i o n were t r e a t e d i n t r a v e n o u s l y ! New p h a r m a c o l o g i c a l s t u d i e s ’ 6 - 2 1 on t h e d r u g and i t s m a j o r m e t a b o l i t e , 4 - h y d r o x y - c l o n i d i n e Y s u p p o r t a mode of a c t i o n i n v o l v i n g s t i m u l a t i o n o f c e n t r a l a - r e c e p t o r s . These r e c e p t o r s may b e c o n n e c t e d t o a n i n h i b i t o r y n e u r o n , t h e a c t i v a t i o n of which c a u s e s a d e p r e s s i o n of p e r i p h e r a l s y m p a t h e t i c a c t i o n and a b l o o d p r e s s u r e f a l l ? ’ The r e l a t e d compound IVb, i n p h a s e I o f c l i n i c a l t r i a l s i n Europe, i s a c t i v e a t 0.2-0.4 mg./day i n man w i t h maximal h y p o t e n s i o n a t 4-5 hoursZ3
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The mechanism of a c t i o n o f indoramine ( V ) has been r e ~ i e w e d ? A~ r e c e n t c l i n i c a l t r i a l , however, was d i s a p p o i n t i n g due t o t h e s i d e e f f e c t s ~bserved?~ Guanabenz (WY 8678, BR 750, FLA 137, V I ) i n a n i m a l s shows a potency l e s s t h a n c l o n i d i n e b u t g r e a t e r t h a n g u a n e t h i d i n e w i t h a mechanism I n i t i a l c l i n i c a l t r i a l s showed showing s i m i l a r i t i e s t o b o t h d r u g s ? 6,2 t h e same potency r a n k i n g ? 8 y 2 9 The d r u g w a s e f f e c t i v e and f u r t h e r improved by h y d r o c h l o r t h i a z i d e a d d i t i o n T 8 The p r i n c i p a l s i d e e f f e c t w a s d r o w s i n e s s .
Ar=2 ,6 - d i c h l o r o p h e n y l N-CO- CGH5 Ar-3-( 2-chloro-4-methyl) V H VI th ieny 1 Guanadrel (U-28288D, V I I ) , e q u i p o t e n t t o g u a n e t h i d i n e i n t h e c l i n i c , a p p e a r s t o o f f e r more s t a b l e b l o o d p r e s s u r e c o n t r o l d u r i n g t h e day w i t h less f r e q u e n t d i a r r h e a ? ' S t u d i e s on g u a n c y d i n e ( V I I I ) c o n t i n u e d t o s u p p o r t t h e mechanism of a d i r e c t v a s c u l a r e f f e c t 3 ' w i t h p a r t i a l d e p l e t i o n o f norepinephrine s t o r e s observed only a f t e r high c h r o n i c d o s i n g under certain conditions?' I
IVa: IVb:
NH
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IX
M i n o x i d i l (U-10,858, I X ) i s a u s e f u l h y p o t e n s i v e a g e n t i n uremic p a t i e n t s w i t h s e v e r e h y p e r t e n ~ i o no~r ~ t h o s e w i t h m a l i g n a n t o r a c c e l e r a t e d h y p e r t e n s i o n r e f r a c t o r y t o c o n v e n t i o n a l d r u g s ? 4 Compared t o h y d r a l a ~ i n e ? ~ t h e d r u g was more p o t e n t , l o n g e r a c t i n g and showed no s i g n s o f t o l e r a n c e development. It c a u s e s a marked i n c r e a s e i n serum r e n i n a c t i v i t y , 3 6 ' 3 7 c a r d i a c h y p e r a ~ t i v i t y qand ~ sodium r e t e n t i o n 3 4 ' 3 5 b u t c o - a d m i n i s t r a t i o n of a @ - b l o c k e r and a d i u r e t i c c o u n t e r a c t s t h e s e s i d e effect^$'^^-^^ The pharmacological p r o f i l e i s t h a t of a p o t e n t , o r a l l y e f f e c t i v e , long a c t i n g h y p o t e n s i v e i n r a t s , dogs and monkeys w h i c h works p r i m a r i l y by a p e r i M i n o x i d i l i s n o n - t o x i c i n r a t s , monkeys p h e r a l v a s o d i l a t o r mechanism? and m i n i p i g s b u t c a u s e s a d e g e n e r a t i v e l e s i o n i n t h e r i g h t a t r i u m of dogs a f t e r p r o l o n g e d d a i l y d o s i n g . D i s c r e p a n c y between t h e p r o l o n g e d a c t i o n and r a p i d c l e a r a n c e from plasma was e x p l a i n e d by s p e c i f i c r e t e n t i o n of t h e I t may i n t e r f e r e w i t h some c e l l u l a r comd r u g i n v a s c u l a r smooth muscle. ponent ( c a l c i u m ? ) of t h e c o n t r a c t i l e p r o c e s s ? ' Another p e r i p h e r a l d i l a t o r , p r a z o s i n (CP-12299-1, X ) gave good r e s u l t s i n a r e c e n t t r i a l (1.5 t o 15 mg./day) even w i t h o u t a d i u r e t i c 4 ' i n m o d e r a t e l y s e v e r e hypert e n s i v e s . A n o t h e r member o f t h i s s t r u c t u r a l t y p e , t r i m a z o s i n (CP-19106, X I )
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showed a s t a t i s t i c a l l y s i g n i f i c a n t a n t i h y p e r t e n s i v e e f f e c t a t 50-150 mg/ day i n a group of 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 t 2 The combination drug B r i n e r d i n e ( dihydroergocr i s t i n e -c lopamide -res e r p i n e ) i s c 1i n i c a 1l y e f f e ~ t i v e and ~ ~ more , ~ ~ a c t i v e than any o f i t s s e p a r a t e i n g r e d i e n t s o r combination of two.
Recent c l i n i c a l t r i a l s of p r o p r a n o l o l (111) c o n t i n u e t o i l l u s t r a t e i t s u t i l i t y a s a h y p ~ t e n s i v ew~i t~h a c t i v i t y improved by a d d i t i o n of a v a s o d i l a t o r such as h y d r a l a z i n e i n s e v e r e h y p e r t e n ~ i o no~r ~w i t h hydroc h l o r t h i a z i d e i n mild t o moderate h y p e r t e n s i o n t 7 There i s i n c r e a s i n g evidence t h a t t h e drug reduces plasma r e n i n t 8 - 5 1 Combining p r o p r a n o l o l w i t h a d r e n e r g i c b l o c k e r s and a d i u r e t i c has a l s o been recommended?2 Int r a c e r e b r o v e n t r i c u l a r ( i c v ) s t u d i e s of p r o p r a n o l o l i n animals by a number of investigator^^^-^^ have l e d t o t h e view t h a t i t s hypotensive a c t i v i t y i s c e n t r a l l y mediated and depends on t h e i n t e g r i t y of n o r a d r e n e r g i c neurons. Blockade of p-adrenoceptors i n t h e b r a i n i s claimed t o cause hypotension and b r a d y ~ a r d i a ? ~However, i t i s noteworthy t h a t p r a c t o l o l which does n o t ( E r a l d i n , I C I 50172, X I I ) , a c a r d i o s e l e c t i v e c r o s s t h e b l o o d - b r a i n b a r r i e r 5 lowers blood p r e s s u r e v e r y w e l l i n This i s a l s o t r u e f o r a l p r e n o l o l ( A p t i n , X I I I ) 6 2 - 6 5 and I . C . I . 66082 Other p-blockers shown t o lower blood p r e s s u r e i n c l i n i c a l (XIV)$6'67 t r i a l s t h i s y e a r were p i n d o l o l (Visken, LB-46, XV) f 8 - 7 1 t i m o l o l (MK-950,
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S e c t . I1
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Pharmacodynamic Agents
C l a r k e , Ed.
X V I ) j 2 - 7 4 ~ h i w~ ahs c o n s i d e r a b l y more p o t e n t t h a n p r o p r a n o l o l i n animals 7 5 ~ 7 6 and man, and o x p r e n o l o l ( T r a s i c o r , X V I I ) j 7 - 7 9 w h i c h w a s a l s o v e r y s u c c e s s f u l when combined w i t h c y c l o p e n t h i a z i d e s O i n a l a r g e s c a l e s t u d y . Tolamolol (m 6558, XVIII) c a u s e d a pronounced b l o o d p r e s s u r e f a l l i n 3 o f 4 normot e n s i v e humans?' Many a n a l o g s have b e e n d e s c r i b e d Though c a r d i o s e l e c t i v e @ - b l o c k e r s a r e a n improvement o v e r n o n - s e l e c t i v e @ - b l o c k e r s , f o r example, i n p a t i e n t s w i t h a s t h m a , e a c h r e d u c e s c o n t r a c t i l e f o r c e a l o n g w i t h h e a r t r a t e . A " c h r o n o s e l e c t i v e " B-blocker i s s t i l l t o b e f o u n d . 8 3
N a t u r a l P r o d u c t s and R e l a t e d Compounds - A v a s o d e p r e s s o r f a c t o r (VDF) was i s o l a t e d from c a n i n e plasma and a o r t i c t i s s u e and human plasma.84 The p o l y p e p t i d e , m o l e c u l a r w e i g h t -G!OOO, c o n t a i n i n g g l u t a m i c a c i d , p r o l i n e and g l y c i n e as p r i n c i p a l components , r a p i d l y lowers b l o o d p r e s s u r e i n r a t s when g i v e n i . v . VDF l e v e l s i n c r e a s e i n dogs d u r i n g induced hemorrhagic shock and h i g h l e v e l s a r e s e e n i n p a t i e n t s w i t h d i f f e r e n t t y p e s o f shock. A t r i d e c a p e p t i d e c a l l e d n e u r o t e n s i n i s o l a t e d from b o v i n e h y p o t h a l a m i i s composed o f Lys, Arg,, Asx, Glx,, P r o 2 , I l e u , L e u 2 , T y r Z a 5 and i s a c t i v e i n r a t s a t 100 pmoles/kg. S y n t h e t i c p e p t i d e s o r i g i n a l l y i s o l a t e d from Bothrops jararaca venom, s p e c i f i c 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, c o n t i n u e t o a p p e a r p r o m i s i n g as d i a g n o s t i c t o o l s and p e r h a p s i n t r e a t m e n t o f r e n a l h y p e r t e n s i o n ? 6 - 8 8 A s t u d y of 57 r e l a t e d p e p t i d e s i n d i c a t e s t h a t t h e C-terminal sequence Pro-Gln-Ileu-Pro-Pro is r e q u i r e d f o r The significant i n h i b i t i ~ n . ~ ~ a n g i o t e n s i n I1 a n t a g o n i s t e f f e c t o f S a r 1 - a l a 8 - a n g i o t e n s i n I1 was confirmed i n dog m o d e l s g 0 ,91 and i n man.92 Hypotension w a s a c h i e v e d i n h y p e r t e n s i v e p a t i e n t s w i t h h i g h r e n i n l e v e l s b u t n o t i n t h o s e w i t h low o r normal l e v e l s . In vitro studies indicate ( I l e u 8 ) - a n g i o t e n s i n I1 t o b e t h e most p o t e n t a n t a g o n i s t y e t d e v e l o p e d , b u t s u b s t i t u t i o n o f s a r c o s i n e f o r a s p a r t i c a c i d i n t h e one p o s i t i o n i n c r e a s e s i n v i v o a ~ t i v i t y . ' The ~ p e p t i d e from t h e n a t i v e r e n i n s u b s t r a t e Asp-Arg-Val-Tyr-Ileu-His-Pro-Phe-His-Leu-Leu-Val-wr-Ser has b e e n m o d i f i e d s l i g h t l y t o produce c o m p e t i t i v e i n h i b i t o r s of r e n i n . 9 4 The s h o r t e r p e p t i d e His-Pro-Phe-His-Leu-D-Leu-Val-wr is a l s o a potent i n h i b i t o r . 95 A r a c h i d o n i c a c i d is h y p o t e n s i v e i n s p o n t a n e o u s h y p e r t e n s i v e r a t s l i k e l y due t o c o n v e r s i o n t o PGE, s i n c e a c t i v i t y i s i n h i b i t e d by a n t i i n f l a m matory a g e n t s ? 6 The a n t i h y p e r t e n s i v e e f f e c t of PGA, i n man i s t h o u g h t m a i n l y due t o i t s e f f e c t on r e n a l c i r c u l a t i o n where it c a u s e s v a s o d i l a t a t i o n and e x c r e t i o n o f water and sodium. I t a l s o i n h i b i t s a n g i ~ t e n s i n . ~ ~ The h y p o t e n s i v e a c t i v i t y o f A 9 - t e t r a h y d r o c a n n a b i n o l ( X I X ) i n a n i m a l s s u g g e s t s i t as a new l e a d . 9 8 , 9 9 Development of t o l e r a n c e t o t h i s e f f e c t may i n v o l v e a n immunogenic r e a c t i o n . ' O O Methylapogalanthamine ( X X ) , h y p o t e n s i v e i n c a t s , dogs and r a b b i t s , has a d r e n o l y t i c , s p a s m o l y t i c and s e d a t i v e p r o p e r t i e s . I t h a s b e e n approved f o r w i d e c l i n i c a l u s e i n t h e U.S.S.R?" A naphthoquinone ( X X I ) from t h e fungus Corynespora c a s s i c o l a i n h i b i t s catechol-o-methyltransferase i n v i t r o and i s a h y p o t e n s i v e o f l o n g d u r a t i o n ( 2 4 h r s . ) i n s p o n t a n e o u s h y p e r t e n s i v e r a t s a f t e r 12.5 mg/kg i . p . l o 2 F u s a r i c a c i d ( 5 - n - b u t y l p i c o l i n i c a c i d ) a n a l o g s w i t h c h l o r i n e o r bromine i n t h e 3- a n d / o r 4 - p o s i t i o n o f t h e s i d e
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c h a i n a r e more p o t e n t dopamine-@-hydroxylase i n h i b i t o r s t h a n t h e p a r e n t compound!03,104 The 4 ' - c h l o r o a n a l o g (FD-008) c a u s e s marked s u s t a i n e d h y p o t e n s i o n i n s p o n t a n e o u s h y p e r t e n s i v e r a t s a t 12.5-25 mg/kg. p . o . I o 5
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O t h e r Compounds A f f e c t i n g A d r e n e r g i c T r a n s m i s s i o n The p r e d o m i n a n t l y c e n t r a l mode of a c t i o n o f a-methyldopa h a s r e c e i v e d s t r o n g support106-110 and t h e mechanism has been more p r e c i s e l y d e f i n e d . The d r u g is m e t a b o l i z e d i n t h e CNS t o a - m e t h y l n o r e p i n e p h r i n e which may a c t on b r a i n a - a d r e n o c e p t o r s t o c a u s e h y p o t e n s i o n , as do o t h e r p o t e n t a - s t i m u l a n t s g i v e n i c v , 2 2 y 1 1 1 o r may a c t i n d i r e c t l y by r e l e a s i n g n o r e p i n e p h r i n e from a d r e n e r g i c n e u r o n s . The h y p o t e n s i o n c a u s e d by p-hydroxynorephedrine (PHN) i n man i s more l i k e l y due m a i n l y t o a p e r i p h e r a l f a l s e t r a n s m i t t e r mechanism s i n c e t h i s compound d o e s n o t p e n e t r a t e t h e b l o o d - b r a i n b a r r i e r . l 1
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The p o t e n t a - b l o c k e r 12473JL ( X X I I ) has been s e l e c t e d f o r c l i n i c a l t r i a l from a number o f o x a z o l i d o n e s w i t h a n t i h y p e r t e n s i v e , a n t i a r r h y t h m i c and a n a l g e s i c a ~ t i v i t y . ' ~ Compound ~ , ~ ~ ~ X X I I I i s reported t o be a potent a - b l o c k e r w i t h prolonged action.'15 The p i p e r a z i n e X X I V , p r o b a b l y a n
Et XXII
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a - b l o c k e r , i s a c t i v e a t 0.2 mg/kg S . C . i n m e t a c o r t i c o i d r a t s 1 1 6 b u t d i s p l a y s CNS d e p r e s s a n t p r o p e r t i e s . Methoxyl s u b s t i t u t i o n i n t h e i n d o l e nucl e i r e d u c e s a c t i v i t y . The t r i c y c l i c s t r u c t u r e XXVa, o r a l l y a c t i v e a t low d o s e s i n h y p e r t e n s i v e r a t s and n e u r o g e n i c d o g s , shows a - b l o c k i n g and n o r e p i n e p h r i n e r e l e a s i n g p r o p e r t i e s : l 7 Several r e l a t e d t r i c y c l i c compounds a r e q u i t e a c t i v e though none a s a c t i v e i n a l l models as XXVa.118y119 L i n e a r i t y of t h e t r i c y c l i c system a p p e a r s e s s e n t i a l f o r a c t i v i t y .
62 -
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R e l a t e d compounds r e p o r t e d as a c t i v e i n animal models are XXVb'20 ( a l s o a p l a t e l e t a g g r e g a t i o n i n h i b i t o r ) and XXVI?21 W-9465-2 (XXVIIa), a c t i v e i n r e n a l and DOCA h y p e r t e n s i v e rats a p p e a r s g u a n e t h i d i n e - l i k e : 2 2 b u t may a l s o r e l e a s e a n t i d i u r e t i c hormone.'23 The most a c t i v e member of a s e r i e s of 48 a n a l o g s i s XXVIIb.lZ4
XXVa
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\S-2395 ( X X V I I I ) , though a n o n - s p e c i f i c p - b l o c k e r , i s much more p o t e n t than p r o p r a n o l ~ l ~ CP-240-S ~~ ( X X I X ) i s more a c t i v e t h a n p r o p r a n s l o l a s a h y p o t e n s i v e i n spontaneous h y p e r t e n s i v e r a t s though less B-blocking.126 The D C I v a r i a n t XXX i s t h e most p o t e n t i n a s e r i e s of @ - b l o c k e r s showing p o s i t i v e i n o t r o p i c and h y p o t e n s i v e a c t i v i t y o r a l l y i n r a t s and d o g s ? 2 7 A s t u d y of t h e a-methyl a n a l o g s of p r o p r a n o l o l and p r a c t o l o l shows t h a t t h e s u b s t i t u t i o n reduces potency b u t i n c r e a s e s c a r d i o s e l e c t i v i t y . '
OH OCH~CHCH~NHCH(M~)~
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P e r i p h e r a l V a s o d i l a t o r s and Other A n t i h y p e r t e n s i v e s L-6150 ( X X X I ) , which has h y d r a l a z i n e - l i k e a c t i v i t y w i t h l e s s t o x i c i t y , shows no lupus erythemat o s u s - l i k e syndrome i n C o l l i e dog s t u d i e ~ . ~A ~group ~ , ~of ~3-amino-4~ halosydnone imines r e l a t e d t o PR-G-138 ( X X X I I ) show comparable o r a l a c t i v i t y t o t h e p a r e n t compound i n h y p e r t e n s i v e r a t s (ca. 10 mg/kg).13' Diazoxide a n a l o g s w i t h t h e benzene r i n g r e p l a c e d by t h i o p h e n e , e.g. XXXIII, a r e a c t i v e i n DOCA r a t s and i n dogs.132 The n i c o t i n i c a c i d ester H - 1 ( X X X I V ) i s a h y p o t e n s i v e and v a s o d i l a t o r i n spontaneous h y p e r t e n s i v e rats.l33
n
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Ant i h yp er t e n s i v e Ag en ts
Chap. 7
Francis
63 -
XXXV lowers b l o o d p r e s s u r e i n h y p e r t e n s i v e r a t s m a i n l y by a p e r i p h e r a l mechanism.134 Abbott 41596 ( X X X V I ) , t h e b e s t o f a s e r i e s of amino a c i d amides o f dopamine, c a u s e s r e n a l v a s o d i l a t i o n o f 3 h o u r s d u r a t i o n a t 12 mg/kg p.0. i n d o g s ? 3 5 It w i l l b e t e s t e d c l i n i c a l l y f o r p o t e n t i a l p r o p h y l a c t i c u s e i n h y p e r t e n s i o n and i n r e n a l d i s e a s e s . A s e r i e s o f phosp h o d i e s t e r a s e i n h i b i t o r s from Merck show c a r d i o s t i m u l a n t , b r o n c h o d i l a t o r and h y p o t e n s i v e p r o p e r t i e s i n a n i m a l s , I n t h i s s e r i e s , compound X X X V I I shows good h y p o t e n s i o n a t d o s e s t h a t show no c a r d i a c ~ t i m u 1 a t i o n . l ~The ~ c l o n i d i n e a n a l o g X X X V I I I i s a c t i v e a t 0.5 mg/kg p . o . i n r e n a 1 h y p e r t e n s i v e
0
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HOp , & M e
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XXXVII
XXXVIII
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rats? Sandoz 44-549 (XXXIX) c a u s e s s u s t a i n e d h y p o t e n s i o n and bradycardia i n n o r m o t e n s i v e and DOCA rats a t v e r y low d o s e s b u t s l i g h t s t r u c t u r e modif i c a t i o n s d e c r e a s e a c t i v i t y markedly? 3 8 The h y d r o x y g u a n i d i n e s XL, a c t i v e i n low d o s e s i n r e n a l h y p e r t e n s i v e r a t s and h y p e r t e n s i v e d o g s , a p p e a r t o a c t by a d i r e c t c e n t r a l e f f e c t . 1 3 ’ W-1984 ( H C 1 s a l t : W-2587) ( X L I ) i s o r a l l y a c t i v e i n h y p e r t e n s i v e r a t s , dogs and monkeys and i t , t o o , i s t h o u g h t t o a c t ~ e n t r a 1 l y . l C~e~r t a i n 2-carbamoyl-4-nitropyridine-N-oxides a r e h y p o t e n s i v e i n r a t s , dogs and humans. The b e s t of t h e s e a p p e a r s t o b e ~
~
1
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.
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XLI
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64 -
Sect.
TI
- Phanwcodynamic
Clarke, E d .
Agents
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. 39. 40. 41. 42. 43. 44. 45. 46. 47. 48. 49.
P. DeHaen, New Product Surveya, 13 (1973) P. DeHaen, ibid.. 70 (1973)
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a,
a,
lm,
Chap. 7
Antihypertensive Agents
Francis
65
50. F. R. Buhler, J.H. Laragh, E. D.Vaughan, H. R. Brunner, H.Gavrasand L.Baer,Amer. J.Cardiol.,Z, 511 (1973) 51. E. L. Bravo, R.C.Taraziand H. P. Dustan, J. Lab. Clin. Med. 83,119 (1974) 52. F. O.Simpson, Curr. Ther. 14,91 (1973) 53. C.T. Dollery, P.J. Lewis, M.G. Myersand J.L. Reid, Brit. J. Pharmacol.a,343P (1973) 54. N. Winer. Clin. Res.2,15A (1974) 55. M. D. DayandA.G. Roach, Federation Proc.a,724Abstr.,Abstr.2879(1973) 56. N. Ram and H.L. Garvey, Federation Proc. g,724Abstr., Abstr. 2880 (1973) 57. R.K. Srivastava,V. K. Kulshrestha, N. Singh and K.P. Bhargava, Eur. J. Pharmacol.a,222 (1973) 58. B.L. Bayer, P. Mentzand W. Foerster, Arch. Int. Pharmacodyn. Ther.a0,341 (1972) 59. L. Offerhausand P.A. Van Zwieten, Europ. J. Clin. Invest.,& 258 (1973) 60. M.D. Eslerand P. J. Nestel, Brit. Heart J.%,469 (1973) 61. R.A. Wood,T.M. Forrester, A.W. Johnstonand K.N.V. Palmer,Clin. Trials J. lJ,53 (1973) 62. J.A. Vedin, C.-E. Wilhelmsson and L. Werko, Brit. Heart J. 1285 (1973) 63. G. Berglund and L. Hansson, Acta Med. Scand. 193,547 (1973) 64. J.Castenfors, H. Johnsson and L. Oro, Acta. Med. Scand., 193,189 (1973) 65. C. FarsangandZ. Nagy,Orv. Hetil.,l-, 196 (1973);C.A.B, 143784e(1973) 549 (1973) 66. L. Hansson, H. Aberg, S. Jameson, B. Karlberg and R. Malmcrona, Acta. Med. Scand. 67. B.R.Graham, D.W. Littlejohns, B.N.C. Pritchard, B.Scalesand P. Southorn, Brit. J. Pharmacol.,@, 154 P (1973) 68. S. deMuckadell and F. Gyntelberg,Europ. J. Clin. Pharmacol.,S, 210 (1973) 69. I. Persson and J. Ulrich, Europ. J. Clin. Pharmacol.5,151 (1973) 70. M. Safar. Y. Weiss, A. Sobel, G. Lagrue and P. Milliez, Nouv. Presse Med. 2,2685 (1973) 71. P.E. Van Coller, J. Int. Med. Res. 1,561 (1973) 72. G. Lohmolleand E. D. Frohlich, Europ. J. Clin. Invest.& 251 (1973) 73. J.A. Franciosa, E.D. Freisand J. Conway, Circulationg, 118 (1973) 74. P.A. Poole-Wilson, J. Int. Med. Res. 1,580 (1973) 75. A.Scriabine,M.L.Torchiana, J.M.Stavorski,C.T. Ludden, D.H. MinskerandC.A.Stone,Arch. Int. Pharmacodyn.a5,76 (1973) 76. R.A.Hall, R.D. Robsonand N.N.Share, Proc. Canad. Fed. Biol.Sci.,lL3,113(1970) 77. A.J. Marshall and D.W. Barritt, Brit. J. Clin. Pract.z,337 (1973) 78. J.Tuckman, F. Messerli and J. Hodler, Clin. Sci. Molec. M e d . , g (Suppl I),159s (1973) 79. G. Muiesan, M. Motoleseand A. Colombi, Clin. Sci. Molec. M e d . 9 (Suppl I),163s(1973) 80. W.A. Forrest, Brit. J. Clin. Pract.,z, 331 (1973) 81. R.H. Briant, C.T. Dollery, T. Fenyvesi and C. F. George, Brit. J. Pharmacol. @, 106 (1973) 82. J. Augstein, D.A. Cox, A.L. Ham, P.R. Leeming and M. Snarey, J. Med. Chem 11,1245 (1973) 83. R.E. Goldstein, Circulation 4_2,443 (1973) 84. J. Rosenthal, J. Paddock and W. Hollander, Circulation Res. 32/33,Suppl. I, 1-169 (1973) 85. R. Carrawayand S.E. Leeman, J. Biol. Chem. 6854 (1973) 86. H.S. Cheung and D.W. Cushman, Biochem. Biophys. Acta, 23,451 (1973) 87. H.F. Loyke, J. Lab. Clin. Med. 82,406 (1973) 88. A. Bianchi, D.B. Evans, M. Cobb, M.T. Peschka.T. R. Schaefferand R.J. Laffan, Europ. J. Pharmacol. 23,90 (1973) 89. D.W. Cushman, J. Pluscec, N.J. Williams, E.R. Weaver, E.F.Sabo, 0.Kocy, H.S. Cheungand M.A. Ondetti, ExperientiaD, 1032 (1973) 90. D.T. Palsand F.D. Masucci, Europ. J. Pharmacol. 23,115 (1973) 91. B.G. Zimmerman, J. Pharmacol. Exp. Therap. l-,486 (1973) 92. H.R. Brunner, H. Gavras,J.H. Laragh and R. Keenan, Lancet W/11,1045 93. F.M. Bumpus, S.Sen, R.R. Smeby, C.Sweet, C.M. Ferrarioand M.C. Khosla, Circulation Res.32/33, SUPPI.I, 1-150 (1973) 94. I . Parikhand P. Cuatrecasas, Biochem. Biophys. Res. Commun.,&I, 1356 (1973) 95. E. Haber, K. Poulsen and J. Burton, Clin. Res. 716 (1973) 96. M. Cohen, J. Sztokalo and E. Hinsch, LifeSci. 13,317 (1973) 97. A. Hornych, M. Safar, N. Papanicolaou, P. Meyerand P. Milliez, Ann. Med. interne I.%, 355 (1973) 98. R.B. Williams, K.R. Lamprecht and I.J. Kopin, Psychopharmacologiaa, 269 (1973) 99. I.Cavero,T. Solomon, J.T. Buckleyand B.S. Jandhyala, Res. Commun, Chem. Pathol. Pharmacol. 6. 527 (1973)
s,
m,
w,
a,
_-66
Sect. I1 - Pharmacodynamic Agents
Clarke, Ed.
100. G. Nahas, I.Schwartz, J. Palacekand D.Zagury, C.R.Acad. Sci., Ser. D , m , 6 6 7 (1973) 101.U. B. Zakirov, Kh. U. Alievand I.M. Karnilov, Farrnakol. Toksikol.tj, 708 (1972) 102.H. Chimura, T. Sawa, Y. Kumada, F. Nakamura, M. Matsuzaki,T.Takita, T.Takeuchi and H. Umezawa, J. Antibiot.z, 618 (1973) 103. H. Hidaka,T,Asanoand N.Takernoto, Mol. Pharmacol.,Q,172 (1973) 104. H. Umezawa,T.Takeuchi, K. Miyano,T. KoshigoeandH.Harnano,J.Antibiot.,& 189(1973) 105. Y. Ishii, Y. Fujii,and H. Urnezawa,Jap. J. Pharrnacol.a, Supp1.,53,Abstr. 76 (1973) 106. M. D. Day, A.G. Roach and R.L. Whiting, Europ. J. Pharrnacol., 21,271 (1973) 107. L. Finchand G. Haeusler, Brit. J. PharrnacoI.,a,217 (1973) 108. A. Heiseand G. Kroneberg, Naunyn-Schrniedebergs Arch. Pharrn.279,285 (1973) 109. M.L.Torchiana,V.J. L0tti.C. M. Clarkand C.A.Stone,Arch. Int. Pharrnacodyn.2&5,103(1973) 110. M.J.Antonaccio, R.D. Robson and R. Burrell, Europ. J. Pharrnacol.,a, 9 (1974) lll.T.BaurnandA.T.Shropshire, NeuropharrnacologyQ,49(1973) 112. R. E. Rangno, J. S. Kaufrnan, J. H. Cavanaugh, D. Island, J. T. Watson and J. Oates, J. Clin. Invest., 52,952 (1973) 113.TMaillard, M.Langlois,V.V.Tri, P. Delaunay, R. Morin, M. Benharkate, C. Manuel and F. Motosso. Chim.Ther.8,393 (1973) 114. J. Maillard, M. Langlois, P. Delaunay, T. V. Van, J. Chenu, R. Morin, M. Benhartake, C. Manuel and F. Motosso, J. Med. Chern. 13,1123 (1972) 115. L. I.Wiebe,R.T.CouttsandA.A. Noujaim,Arzneirn.-Forsch.,a,466 (1973) 116. K. Brewster, D.M. Green, R. M. Pinderand P.B.J.Thornpson, Clin. Ther.,& 169 (1973) 117. J.P. Buyniski, R.L. Cavanaugh and M.E. Bierwagen, Res. Cornrnun. Chern. Pathol. Pharmacol.5, 647 (1973) 118.T. Jen, P. Bender, H. Van Hoeven, B. Loev, J. Med. Chern. 16,407 (1973) 119. T. Jen, B. Daniel, F. Dowalo, H. Van Hoeven, P. Bender and 8. Loev, J. Med. Chern. 12,633 (1973) 120. Bristol-Myers Co., Ger. Offen. 2,305,575 (9.22.72) 121. G.V. Kovalev,S.M. Gofrnan,S.V. Ivanovskaya, M.V. Punshina,V.I. Petrov,A.M. Sirnonovand I. N. 88 (1973) Tyurenkov. Farrnakol. Toksikol. 122. D. Deitchrnan, A. W. Gornoll and G. R. McKinney, Federation Proc. =,750Abstr., abstr. 3021 (1973) 123.D.Deitchrnan and A.W. Gornoll, Proc. SOC.Exp. Biol 144,203 (1973) 124. W.L. Matier, D.A. Owens, W.T. Comer, D. Deitchrnan, H. C. Ferguson, R.J. Seideharnel and J.R. Young, J. Med. Chern. 13,901 (1973) 125. M. Laubie, H. Schrnitt, P. Mouille, G. Cheyrnol and J.C. Gilbert, Arch. Intern. Pharrnacodyn. 201,334 (1973) 126.J. Roba, G. Larnbelin and A. F. deschaepdryver, Arch. Inter. Pharrnacodyn. 200,182 (1972) 127. E. Bellasio and F. Cristiani, Ger. Offen 2,220,337 (11.23.72) 128. B. Levy, Brit. J. Pharmaco1.@,514 (1973) 129. E. Baldoli,A.Sardi, V. Dezulian, M. Capelliniand G. Bianchi,Arzneirn.-Forsch.,a, 1591 (1973) 130. A. Sardi, A. M. Caravaggi and E. Baldoli, Naunyn-Schrniedebergs Arch. Pharrnacol. 279,301 (1973) 131.M.Gotz,K.GrozingerandJ.T.Oliver, J.Med.Chern. 13,671 (1973) 132. Schering Corporation, US. 3,641,017 (2.8.72); U S . 3,733,409 (5.15.73) 133. K.Tsururni, H. FujimuraandY. Suzuki,OyoYakuriZ, 175 (1973) 134. E. Bellasio, A. Carnpi, A. Trani, E. Baldoli, A. M. Caravaggi and G . Nathansohn, Farmaco, Ed. Sci., 164 (1973) 135. P.H. Jones, J.H. Biel, C.W. Ours, I.L. Klundt and R. L. Lenga, 165th A.C.S. Metting, Dallas (April) MEDI-9 (1973); F.N. Minard, J.C. Cain and D.S. Grant, ibid., MEDI-10; P. Sornani, R. Hollingerand F.N. Minard, ibid., MEDI-11. 136.J.H. Jones,W.J. Holtz and E.J.Cragoe, J. Med. Chern 16,537 (1973) 137.WanderAG.,Ger.Offen.2,258,318 (10.12.72) 138. L.D. Boyajy, R.E. Manning, R. Mclntosh, F. Schaefer, M. Herzig, J. Schaafand H.H.Trapold, 166th A.C.S. Meeting, Chicago (August), MEDI-22 (1973) 151 (1973) 139. D.M. Bailey,C.G.DeGrazia,H.E. Lape,R. Frering,D. FortandT.Skulan, J. Med.Chern. E, 140.J.F. SchaeferJr., R.R. Loetzer, G. B. Phillips, B. J. Kletzkin and F.M. Berger, Federation Proc. 32,750 Abstr.,abstr. 3020 (1973) 141.J.M.D. Aron-Samueland J.J. Sterne, Ger. Offen. 2,213,843 (10.12.72)
s,
a,
Chapter 8. Antiarrhythmic and Antianginal A g e n t s Gilbert W. Adelstein and Richard R. Dean, Searle Laboratories, Chicago, 111.
ANTIARRHYTHMIC AGENTS Introduction - Cardiac arrhythmias generally result from enhanced automaticity or disturbances in conduction. These disturbances cause alterations in cardiac rate, regularity, origin of cardiac impulse or sequence of activation of the atria and ventricles.1-6 8-Receptor Blocking Agents - Current synthetic efforts in @-blockers has focused mainly on compounds of generic structure 1. By alteration of the ArOCH2CH (OH)CH2NHCH (CH3)2
3,4-dihydro-1(2H)-naphthalenon-5-yl 4-ACNHCgHq4-MeOCH2CH2C6H44-MeOCH2CH20C6H42-CH2=CHCH2C6H48-thiochromanyl 1,2,3,4-tetrahydro-l,4-ethano-5-naphthyl 2-acetyl-7-benzofuranyl 7-indenyl 5-methyl-8-coumarinyl 2,3-Me2CgH34-CgHqCONH2 2,3,4,5-tetrahydro-1H-2-benzazepin-l-on-7-y1 4-indolyl 4-CH2=CHCH2C&-
nature of Ar-, compounds of greater cardioselectivity and lesser cardiac depressant activity have been sought. Schwender and co-workers studied the effects of aromatic substitution7 and carbonyl-containing analogs8 on the 8-blocking properties of bunalol (la) and concluded that no enhancement of cardioselectivity could be obtained. Tolamolol (2) - was the most cardio-
OCH2CH (OH)CH 2NHCH (R) (CH2) no
-2
(R=H, n=1, RV=4-CONH2)
selective member of a series of @-blockers and clinical studies indicated negligible myocardial depression9 Practolol (Ib) was effective in treating supraventricular tachyarrhythmias in patients who could not tolerate
.
-68
Sect. I1
-
Pharmacodynamic Agents
Clarke, Ed.
propranolol because of i t s bronchoconstrictor a c t i v i t y . 1 ° The (-) - isomer of p r a c t o l o l r e t a i n s t h e 6-blocking a c t i v i t y and, u n l i k e p r o r a n o l o l , l i t t l e The a b s o l u t e anti-arrhythmic a c t i v i t y i s r e t a i n e d i n t h e (+) -enantiomer.lf c o n f i g u r a t i o n of ( + ) - p r a c t o l o l w a s determined t o be (R) by t o t a l s y n t h e s i s . 1 2 H 93/26 ( l c ) and H 87/07 ( l a ) were r e p o r t e d t o possess considerable c a r d i o s e l e c t i v i t y and H 87/07 had i n t r i n s i c a c t i v i t y s i m i l a r t o a l p r e n o l o l ( l e ) and p r a c t 0 1 o l . l ~ N o improvement i n anti-arrhythmic a c t i v i t y over prop r a n o l o l w a s seen f o r 5-2395 (lf)14, K-4423 (Lg)15, BFE-60 (1h116, o r YB-2 (li) 17. Greater potency and less c a r d i a c depressant p r o p e r t i e s than prop r a n o l o l were a s c r i b e d t o CS-359 (1j)18 and D-32 (1k).19 N o improvement over p r a c t o l o l could be obtained f o r a r e v e r s e amide (11) o r c y c l i z e d r e v e r s e amide (l m ) .20 A s e r i e s of N-substituted-2-amino-l-(thienyl)ethanols were evaluated a s $-blockers and found t o be less p o t e n t than propranolol a s i n h i b i t o r s of t h e tachycardic response t o i s o p r o t e r e n o l . 21 A s e r i e s of h e t e r o c y c l i c propanolamines had 6-blocking a c t i v i t y , t h e most p o t e n t ( 3 ) having t e n times t h e a c t i v i t y of propranolol on t h e guinea p i g a t r i a l
ArCH2CH (OH) CH2NHC (CH3)3
3 (Ar=6-phenanthridinyl) C l i n i c a l e v a l u a t i o n of b u n i t r o l o l (Koe 1366)23 and t i m o l 0 1 ~shows ~ no c l e a r advantages over propranolol f o r i n c r e a s i n g e x e r c i s e t o l e r a n c e o r t r e a t i n g arrhythmias. I n dogs, p i n d o l o l (LB 46, _In) produced no depression of v e n t r i c u l a r function and increased myocardial performance, while K8 592 depressed myocardial performance and propranolol produced an even g r e a t e r depression i n v e n t r i c u l a r function. 25 Thus , s u b t l e changes i n s t r u c t u r e confer s u b t l e changes i n pharmacological p r o f i l e whose s i g n i f i c a n c e i s n o t r e a d i l y understood. Vaughan-Williams has suggested t h a t p - s u b s t i t u t e d phenyl groups tend t o make a 6-blocker more c a r d i o s e l e c t i v e , s i n c e t h e orthos u b s t i t u t e d isomer of p r a c t o l o l has no c a r d i o s e l e c t i v i t y , whereas 2-oxprenolol The r o l e ( l o ) w a s h i g h l y c a r d i o s e l e c t i v e and more p o t e n t than p r a c t o l o l . 26 o T metabolism i n d e l i n e a t i n g t h e p r o f i l e of a c t i v i t y of a 6-blocking agent i s not c l e a r , b u t evidence f o r s e p a r a t i o n of 6-blocking a c t i v i t y and a n t i arrhythmic a c t i v i t y v i a m e t a b o l i t e s e x i s t s . l 9
-
Quaternary Ammonium Derivatives Quaternization of arnine-containing a n t i arrhythmic agents has sometimes r e s u l t e d i n improved a c t i v i t y with a r e duction i n c e r t a i n s i d e e f f e c t s . UM-272 ( 4 ) , t h e dimethyl d e r i v a t i v e of propranolol , e x h i b i t s n e i t h e r t h e 6-blocking p r o p e r t i e s nor t h e l o c a l a n e s t h e t i c p r o p e r t i e s of propranolol. UM-272 is e f f e c t i v e a g a i n s t d i g i t a l i s and myocardial i n f a r c t i o n arrhythmias i n t h e dog. 27 128 I n a d d i t i o n , UM-272 was shown t o s i g n i f i c a n t l y i n c r e a s e v e n t r i c u l a r f i b r i l l a t i o n threshold29 1 3o and reduce t h e occurrence of v e n t r i c u l a r f i b r i l l a t i o n following a c u t e occlusion of t h e l e f t a n t e r i o r descending coronary a r t e r y and i t s subsequent de-occlusion. Miura and co-workers have found t h a t t h i s compound e x h i b i t s e l e c t r o p h y s i o l o g i c a l e f f e c t s s i m i l a r t o propranolol on i s o l a t e d canine
Chap. 8
Antiarrhythmic, Antianginal Agents
Adelstein, Dean
3
Purkinje fibers and rabbit atria.31 Quaternized lidocaine (methyl lidocaine) exhibited the therapeutic efficacy of lidocaine without the CNS side effects of the latter.32 On the other hand, the dimethyl ammonium quaternary salt of bunalol exhibited only weak anti-arrhythmic activity with pronounced myocardial depression. 33
Bretylium tosylate, a neuronal blocking agent, has generated interest as an anti-fibrillatory agent.34 Counsel1 and co-workers have synthesized a series of bretylium analogs and found that 2-iodobenzyltrimeth lammonium iodide showed high uptake by myocardial tissue. 35 Ring-labeled I3lI bretylium analogs (RIBA) were useful as myocardial scanning agents.36 Miscellaneous Agents - Clinical studies indicate that disopyramide phosphate (?a, R=CONH2, Norpace@) was effective in treating both atrial and ventricular arrhythmia~.37-~0Adelstein found that replacement of the amide by heterocyclic rings (rb, 5c) did not significantly enhance activity.41 Cusic and co-workers found that substitutions on phenyl or pyridyl groups did not improve the therapeutic index of disopyramide.42 Aprindine (6) is a new anti-arrhythmic agent that is 20 times more potent than lidocaine as a local anesthetic. Clinical studies have shown that aprindine is a long-acting, orally effective anti-arrhythmic agent which compares favorably with intravenous lidocaine in preventing ventricular arrhythmias after acute myocardial infarction.43
-5
a) R=CONH2 b) R=5-tetrazolyl c) R=2-methyl-1,3,4-oxadiazol-5-yl
6 -
Diphenidol (1) was recently shown to effectively protect dogs against digitalis into~ication.~~ Its mechanism of action was found to result from a shortening of the atrium-to-His bundle conduction time and a suppression of ventricular automaticity. Studies on a group of patients exhibiting digitalis-induced arrhythmias confirmed the animal data and strongly suggest a clinical role for diphenid01.~~ A similar type of action has been suggested for the steroidal antialdosterone agent, potassium canrenoate.46
DCAA (17,21-di-monochloroacetyl ajmaline) abolished digitalis-induced arrhythmias in the majority of dogs and significantly reduced mortality rate. No adverse effect on myocardial function was 0bserved.~7
Sect. I1
70 -
-
Clarke, Ed.
Pharmacodynamic Agents OH
Verapamil (iproveratril) was shown to be an anti-arrhythmic agent several years ago48 I 49 and Singh and Vaughan-Williams postulated that this drug acts by a unique mechanism of action; namely, blockade of the calciumcarrying depolarizing current.50 Recent studies have confirmed the electrophysiological characteristics and effectiveness of verapamil both in vitro and in ouabain intoxicated dogs. In each case, the effect of verapamil was readily reversed by administration of calcium, thus lending support to Singh and Vaughan-Williams ' hypothesis.51 52 In isolated guinea pig atria, BL-3676 and BL-3677 (isomers of 5-endobenzoyloxy-N- (dimethylaminopropyl)-bicycle (2.2.1) heptane-2 ,3 - e dicarboxylic acid hide HC1) increased stimulus threshold but did not alter the resting membrane potential.53 In dogs, W36095 (2-amino-2',6'-propionoxylidide) was an orally effective, long-acting anti-arrhythmic agent with little depressant effect on the cardiovascular system. 54 CRD-401, the benzothiazepine derivative 2, reversed epinephrine-induced arrhythmias in guinea pigs, but failed to reverse arrhythmias due to twostage coronary ligation.55 LL-1530 (10) , whose structure is reminiscent of propranolol, was not a &blocker and was found to have a better therapeutic index and anti-arrhythmic activity of longer duration than quinidine in dogs56 and was effective clinically with minimal myocardial depressant effects.57 OCH2CH (OH)CH2C (=NOH)NH2'HC1 I
10 -
CH CH NMe2.HC1 2 2 PGF7, is reportedly effective in reversing ouabain-induced arrhythmias in ~ a t s ~ ~ - aanclinical d study verified the therapeutic effects.59 The mechanism of action has not been studied. ANTIANGINAL AGENTS Angina pectoris is the result of a disparity between myocardial oxygen demand and oxygen supply. Factors which determine oxygen demand include heart ratelarterialpressure, preload, ventricular volume and mass, and contractility.60 Oxygen supply is determined by the caliber of the coronary arteries, arterial p02, oxygen carrying capacity of the blood, affinity of hemoglobin for oxygen and viscosity of the blood. 61
Chap. 8
Antiarrhythmic, Antianginal Agents
Adelstein, Dean
2
Current a n t i a n g i n a l therapy i s aimed a t reducing oxygen demand and/or i n c r e a s i n g oxygen supply. The n i t r i t e s and organic i n t r a t e s reduce preload by causing venous pooling of blood. These agents have a l s o been shown t o r e d i s t r i b u t e myocardial blood flow, thereby improving oxygen d e l i v e r y t o ischmic areas even though t o t a l coronary blood flow i s n o t increased. 62163 Nitrates continue t o b e drugs of choice i n a l l e v i a t i n g a n g i n a l pain, b u t c o n t r o v e r s i e s e x i s t as t o the e f f i c a c y of long-acting n i t r a t e s . 6 4 A r e c e n t study i n d i c a t e d t h a t cutaneous absorption of n i t r o g l y c e r i n from an oiniment base may be d i s t i n c t l y s u p e r i o r t o t h a t of s u b l i n g u a l o r o r a l n i t r a t e s i n angina pro phyla xi^,^^ suggesting t h a t t h e search f o r more e f f e c t i v e n i t r a t e s might be b e t t e r served by exploring new r o u t e s of administrat i o n f o r nitroglycerin.
Propranolol reduces oxygen needs through a 6-adrenergic blocking a c t i o n which r e s u l t s i n reduced h e a r t r a t e , blood p r e s s u r e and c o n t r a c t i l i t y . 66 ,67 Propranolol a l s o decreases t h e a f f i n i t y of hemoglobin f o r oxygen a t low oxygen tension.68 This increased P50 r e s u l t s i n a greater r e l e a s e of oxygen i n hypoxic t i s s u e s . A study comparing t h e e f f e c t i v e n e s s of propranolol, oxprenolol, and p r a c t o l o l i n anginal p a t i e n t s suggests similar a n t i a n g i n a l a c t i v i t y f o r a l l t h r e e drugs. 69
The p r e v i o u s l y described dimethyl quaternary analog of p r o p r a n o l o l , UM-272, i s n o t a 6-adrenergic blocker. Like propranolol, UM-272 was r e c e n t l y shown t o reduce t h e ST segment s h i f t s observed i n canine e l e c t r o c a r d i o grams d u r i n g c a r d i a c ischemia. 70 This f i n d i n g i n d i c a t e s t h a t 6-adrenergic blockade per se may n o t be necessary f o r e f f e c t i v e a n t i a n g i n a l a c t i o n . The e f f e c t of UM-272 on t h e hemoglobin-oxygen d i s s o c i a t i o n curve has n o t been studied. L 8040 (ll), an analog of t h e coronary v a s o d i l a t o r amiodarone, w a s shown t o b e e f z c t i v e i n decreasing myocardial oxygen consumption by an adrenergic blocking e f f e c t , b u t n o t by competitive 6-blockade. 71 Cardiac o u t p u t w a s n o t reduced.
Br
Chlorthalidone (Hygroton@), a d i u r e t i c , has an a n t i a n g i n a l e f f e c t u n r e l a t e d t o i t s hypotensive e f f e c t . 72 A double-blind study of perhexilene @exid@) i n angina p a t i e n t s showed improved e x e r c i s e t o l e r a n c e . 7 3
Although t h e s e a r c h f o r b e t t e r a n t i a n g i n a l drugs continues, t h e r e is no consensus of opinion regarding t h e most d e s i r a b l e mechanism of a c t i o n .
72 -
Sect. I1
-
Pharmacodynamic Agents
Clarke, Ed.
The controversy over t h e a c t i o n s of n i t r o g l y c e r i n and propranolol continues and, t h e r e f o r e , t h e r e l a t i v e importance of s e l e c t i v e d i l a t a t i o n of coll a t e r a l coronary a r t e r i e s compared t o reduction i n myocardial oxygen needs p e r s e has n o t been e s t a b l i s h e d . While propranolol appears e f f e c t i v e i n t h e p r o p h y l a c t i c therapy of angina p e c t o r i s , concern has been r a i s e d over t h e apparent "rebound" i n c r e a s e i n anginal a t t a c k s a f t e r c e s s a t i o n of therapy. Therefore, f u r t h e r i n v e s t i g a t i o n i s needed before propranolol i s accepted a s t h e s i n e qua non of a n t i - a n g i n a l drugs.
--
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G. W. Adelstein and R. R. Dean, Ann. 9. , 1972, R. V. Heinzelman, ed., p. 63, Academic Press, New York, 1973. P. F. C r a n e f i e l d , A. L. W i t , and B. F. Hoffman, C i r c u l a t i o n 47, 190 (1973). B. F. Hoffman and P. F. Cranefield, Am. J. Med. 37, 670 (1964). D. H. Singer, R. Lazzara, and B. F. Hoffman, Circ. R e s . 21, 537 (1967). S. Weidmann, J. Physiol. 213 (1955). B. F. Hoffman, P. F. Cranefield, and A. G. Wallace, Mod. Conc. Cardiov. D i s . 35, 103 (1966). C. F. Schwender, R. E. Pike, and J. Shavel, Jr., J. Med. Chem. 2, 254 (1973). C. F. Schwender, R. E. Pike, B. R. Sunday, and J. Shavel, J r . , i b i d , 16, 585 (1973). J. Augstein, D. A. Cos, A. L. Ham, P. R. Lemming, and M. Snarey, 16, 1245 (1973). P. Van Durme, L. Bossaert, P. Vermeire, and R. Rannier, Am. Heart J. 86, 284 (1973). G. J. K e l l i h e r and J. Roberts, Eur, J. Pharmacol. 20, 243 (1972). J. C. Danilewicz and J. E. G. Kemp, J. Med. Chem. 16,168 (1973). B. Ablad, E. Carlsson, and L. Ek, L i f e S c i . I , 107 (1973). M. Lauble, G. Cheymol, P. Mouille, J. C. G i l b e r t , and H. Schmitt, Arch. I n t . Pharmacodyn. Ther. 201, 323 (1973). M. Bergamaschi, V. Mandelli, R. Tommasini, C. Turba, and M. M. Usardi, 667 (1972). Boll. Chim. Farm. Y. Maruyama, T. I s h i h a r a , I. H i r a k i , M. Nozaki, and F. Takenaka, Jap. J. Pharmacol. 23, Suppl. 35 (1973). H. Kato, Y. Noguchi, K. Nakao, and K. Takagi, H ISuppl. 39 (1973). T. Oshima, S. Krimakura, H. Koike, and K. Nakayama, E l497 (1973). A. Izumi, M. Mieda, S. Nishida, and Y. Hiramatsu, Suppl 37 (1973). G. Shtacher, M. Erez, and S. Cohen, J. Med. Chem. 16, 516 (1973). C. C o r r a l , V. Darias, M. P. Fernandez Tome, R. Madronero, and J. d e l 882 (1973). Rio, H I R. F. Meyer, C. D. S t r a t t o n , S. G. Hastings, and R. M. Corey, H I 16, 1113 (1973). W. Klempt and F. Bender, Arzneim.-Forsch. 23, 1064 (1973). J. A. Franciosa, E. D. F r e i s , and J. Conway, C i r c u l a t i o n 48, 118 (1973). R. D. Smith and C. B. Nash, Arch. I n t e r n . Pharmacodyn. Ther. 203, 151 (1973). E. M. Vaughan-Williams, Schweiz. Med. Wochenschr. 262 (1973). F. J. Kniffen, D. P. Schuster, and B. R. Lucchesi, C i r c u l a t i o n , Suppl. 11, 46, 177 (1972).
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28. D. P. Schuster, B. R. Lucchesi, N. L. Noven, M. N. Mimnaugh, R. E. Counsell, and F. J . Kniffen, J. Pharmacol. Exp. Ther. 213 ( 1 9 7 3 ) . 29. F. J. Kniffen, D. P. Schuster, and B. R. Lucchesi, J. Pharmacol. Exp. Ther. 260 ( 1 9 7 3 ) . 30. F. J. Kniffen and B. R. Lucchesi, Am. J. Cardiol. 142 (1973). 475, 31. D. S. Miura, A. B. Hodess, and M. R. Rosen, Fed. Proc. a b s t r . #1491 (1974). 32. R. A. G i l l i s , F. H. Levine, H. Thibodeaux, A. Raines, and F. G. S t a n d a e r t , C i r c u l a t i o n 47, 697 ( 1 9 73). 33. H. R. Kaplan, J. Barker, D. Dugan, J . Fox, and E. Giardino, Pharmacologist 192 (1973). 34. G. Sanna and R. Arcidiacono, Am. J. Cardiol. 32, 982 (1973). 35. R. E. Counsell, T. Yu, V. V. Ranade, and A. Buswink, J. Med. Chem. 16, 1038 (1973). 36. E. A. C a r r , Jr., R. E . Counsell, and M. C a r r o l l , C l i n . Pharmacol. Ther. 14, 132 (1973 ) . 37. D r e i f u s , Am. J. Cardiol. 1 2 9 (1973). 38. R. B. Kalmansohn and R. W. Kalmansohn, Cardiov. R e s . , V I World Congr. Cardiol. , 179 ( 1 9 7 0 ) . 39. L. Vismara, J. Lies, R. MaSSumi, R. Z e l i s , D. T. Mason, and E. A. Amsterdam, C l i n . Res. 456 (1973). 40. B. B e f e l e r , A. C a s t e l l a n o s , Jr., D. E. Wells, M. C. Vagueiro, B. K. Yeh, and R. J. Myerburg, Am. J. Cardiol. 31, 119 (1973). 41. G. W. Adelstein, J. Med. Chem. 16, 3 0 9 (1973). 42. H. W. Sause, J. W. Cusic, and K . .P Yonan, Abstr. Papers, Am. Chem. SOC. 165 Meet. MEDI 1 2 ( 1 9 7 3 ) . 43. F. Hagemeijer and P. G. Hugenholtz, Am. Cardiol. 33, 142 (1974). 44. W. J. Mandel, H. Hayakama, J. K. Vyden, M. Carvalho, W. W. Parmley, and E. Corday, Am. J. Cardiol. 2, 67 (1972). 45. A. F e l i p e and S. Cecgna, Am. J. Cardiol. 33, 130 (1974). 915 (1972). 46. B. K. Yeh, P-K Sung, and A. K. Saha, CircTRes. 47. M. Dalmastro, T-W Lang, S. Rubins, C. C o s t a n t i n i , and S. Merrbaum, Am. J. Cardiol. 133 (1974). 48. K.. I. M e l v i l l e , H. E. S h i s t e r , and S . Huq, Can. Med. Assoc. J. 90, 76 (1964). 49. C. Hanna and J. R. Schmid, Arch. I n t e r n . Pharmacodyn. Ther. 1 8 5 , 228 (1970). 50. B. N . Singh and E. M. Vaughan-Williams, Cardiovasc. Res. 6, 109 ( 1 9 7 2 ) . 51. R. M. King, D. P. Zipes, A. N i c o l l , and J . Linderman, A m . J . Cardiol. 33, 1 4 8 (1974). 52. R. Rosen, J. P. I l v e n t o , and C. Merker, Am. J. Cardiol. 33, 166 ( 1 9 7 4 ) . 53. M. S. Strauch. R. W. Gardier, and P. B. Hollander, Pharmacoggist 192 (1973). 54. G. Gerstenblith. J. F. Spear, and E. N. Moore, C l i n . R e s . 419 (1973). 55. K. Yamada, T. Shimamura, and H. Nakajima, Jap. J. Pharmacol. 23, 3 2 1 (1973). 56. J. D u t e i l , J. Nadaud, M. Henry, E. ASSOUS, and R. Gompert, Therapie 28, 703 (1973). 57. R. T r i c o t , P. E. Valere, and A. C a s t i l l o Fenoy, 28, 7 2 1 (1973). 3, 895 (1973). 58. W. F o e r s t e r , H. J. Mest, and P. Mentz, Prostaglandins -
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2,
46,
C.
22,
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201,
72. 73.
Pharmacodynamic Agents
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33,
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Chapter 9. Antithrombotic Agents J. Stuart Fleming and John E. MacNintch Bristol Laboratories, Syracuse, New York 13201
The medical problem of thrombosis encompasses three distinct physiological processes, platelet aggregation, coagulation and fibrinolysis. Drugs capable of influencing one or more of these processes are potentially useful from either a prophylactic or a therapeutic standpoint in various clinical situations. This review will attempt to highlight developments during the past year in this very active field of medicinal research. Platelet Aggregation Inhibitors A large number of compounds capable of inhibitin in vitro platelet aggregation (PA) have been reported in recent years.1-2 Some of these compounds, such as aspirin, dipyridamole and sulfinpyrazone were being used clinically for other purposes. Others are new compounds which were either developed particularly for their effect on PA or which possess this potential in association with another pharmacologic activity, e.g. the nonsteroidal anti-inflammatory agents. In either case, the demonstration of in vitro activity cannot be considered sufficient to qualify the compound as an antithrombotic agent. It should also be capable of inhibiting thrombosis in vivo in one or more of the available laboratory models. Many compounds possess potent in vitro activity but fail to be effective in vivo ’ either due to poor absorption, rapid metabolism or the presence of undesirable side effects.
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Aspirin conAspirin and Other Non-Steroidal Anti-Inflatmatory ARentS tinues to receive the greatest attention of the compounds in this category. In 1971, Smith and Willis reported that aspirin interferes with prostaglandin production in human platelets.5 Recently, it has been reported that both aspirin and indomethacin administered to human subjects abolished platelet prostaglandin production as well as the second wave of epinephrine induced PA,6 Furthermore, the duration of the effect on prostaglandin production matched the duration of the effect on PA. It was suggested that the nonsteroidal anti-inflammatory agents exert their antihemostatic or antithrombotic effects through disruption of platelet prostaglandin production. It has also been demonstrated that aspirin and PGEl produce supra-additive effects on PA and experimental thrombosis,7,8 although conflicting evidence exists concerning in vivo potentiation. The implication is that platelet levels of two or more prostaglandins may act to modulate platelet adhesiveness. Additional experimental evidence has also been reported which substantiates the effectiveness of aspirin in preventing arterial thrombosis. The compound was active in a variety of animal models where mechanical, chemical, electrical or laser injury was used to induce arterial thromboais.10-14 In addition, studies in cats have demonstrated aspirin to be effective in reducing the incidence of thrombosis on catheters placed in the inferior venae cavae.15
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Sect. I1 - Pharmacodynamic Agents
Clarke, Ed.
Three new synthetic anti-inflammatory agents have been reported which possess activity against collagen induced PA. Flurbiprofen (1)was effective in vitro in human, rat and rabbit platelet rich plasma (PRP) and in rats and rabbits following in vivo dosing.16 It produced significant inhibition of collagen induced PA in human PRP following a single oral dose of 10 mg/kg and had a duration of action in rats of nearly two days. Flurbiprofen did not affect platelet cyclic AMP levels but was capable of inhibiting platelet release of serotonin. It was effective in several other in vivo animal models which included the protection of mice from lethal thromboembolism resulting from collagen infusion. Sudoxicam (2) was also found to be a potent inhibitor of collagen induced PA both & vitro and in viv0.17 This compound demonstrated activity in humans following oral administration of 50 mg/kg and in animal models of thrombosis. The third agent, ditazol (2)was somewhat less potent in rabbit PRP but was also reported to produce some inhibition of ADP induced PA at high concentrations.18 Bleeding time in mice was prolonged at daily doses of 100 mg/kg P.o., but PA was only slightly inhibited in human PRP after subjects had received the compound for two days at a dose of 800 %/day.
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Dipyridamole and Related compounds A variety of pyrimidopyrimidines and thienopyrimidines have previously been shown to inhibit the primary phase of ADP induced PA and to demonstrate activity in animal models of thrombosis. In addition, dipyridamole has been shown to normalize enhanced platelet consumption associated with a variety of clinical conditions, and it may be more effective when used in combination with an anticoagulant or another inhibitor of PA such as aspirin.19 There have been several attempts to clarify the mechanism of action of these agents but no clear cut theory has yet evolved.20,21 It would appear that these agents have some degree of phosphodiesterase inhibitory activity and that they are capable of potentiating the inhibitory activity of adenosine on PA. In view of the rather weak activity of dipyridamole, the conflicting reports of its clinical effects and the limiting side effects of more potent analogs, a good candidate for extensive clinical evaluation probably has not yet emerged from this series of compounds.
-
Adenosine Derivatives Adenosine and certain of its derivatives were among the first compounds described which were capable of inhibiting PA. Adenosine is not useful clinically due to its severe cardiovascular side effects and the fact that it is rapidly inactivated in vivo. 2-Chloroadenosine was found to be a powerful inhibitor of PA but was very toxic.22 2-Methylthioadenosine 5'-monophosphate was shown to be less toxic but also was considerably less potent.23 In searching for potentially useful analogs, a series of N6-substituted adenosine8 and N6-substituted adenosine 5 ' -monophosphates have recently been synthesized.24
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Compound 4 is an example of one of mc6H11 the more active members of this series. Studies in rabbits demonstrated that this N+N N p ) agent was also active following an intradN,q ) C6H11SkN N venous infusion of 8 mglkg. Additional 2-thioadenosine compounds were reported which also were effective in v i t r ~ . ~ ~ Compound 2 is an example of one of the HO OH HOOH more active members of this series. However, no in vivo data were reported for these compounds and it is not known if either series possesses activity in animal models of thrombosis.
-4 Hmd -
-
-
It has been reported that high concentrations of Miscellaneous Agents penicillin G are capable of inhibiting ADP, collagen and thrombin induced PA as well as the platelet release reaction in human PRP and in suspensions of washed platelets from rabbits or pigs.26 Clofibrate and related compounds have been used extensively as lipid lowering agents in patients with various forms of hyperlipemia and atherosclerosis. In addition to lowering serum lipid levels, these compounds have been reported to affect all three aspects of the problem of thrombosis being considered in this review. Two recent reports further describe the ability of these compounds to decrease PA in hyperlipemic patients. 27,28 A series of substituted 3,4-pentadienyldiamines was reported to inhibit ADP induced PA both in vitro and in vivo.29 Compound 5 was one of the more potent members of the series and was effective in guinea pigs administered 30 mg/kg p.0. for four days. However, 100 mg/kg for four days was lethal. The lower dose also had a slight effect on ADP induced fall in guinea pig platelet count. A series of N-diethylaminoethylaryloxyacetamides were also reported. Compound L was one of the more active members of the series and inhibited both collagen 72H5 and ADP induced PA in vitro.30 However, this agent did not demonstrate activity in (CH3)2C=C=CHCCH2NH(CH2)3NH2 I rabbits after an intravenous dose of 10 6 C4H9 mg/kg. The compound B-(2-p-chlorophenylthiazole-4yl)acrylic acid (WY-23,049) (8) 000CH2CmcH2cH2NEt2 has been reported to inhibit ADP-induced I thrombocytopenia in rats and to significantly prolong clotting time.31 The comC10-7;N SACH=CHCO2H 8 pound AY-16,804 (9) was found to have & I vitro activity in rat PRP and was nearly equipotent to sulfinpyrazone but markedly 9 less active than PGEl in vivo.32 &(CH2 16C02H
-
. 1
-
-
-
General Considerations The spectrum of potential clinical uses for an effective compound in this area is continuing to broaden. During the past year a number of reports have appeared which tend to substantiate the role of PA in the hyperacute rejection of tissue transplants. It has been known for some time that antigen-antibody complexes are capable of aggre-
78
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gating blood platelets and that intravascular coagulation has been shown to play a major role during the hyperacute rejection of allografts. Recently Claes has shown that during such a rejection process involving dog kidney, labelled platelets aggregate and are removed as blood perfuses the transplanted organ.33 Furthermore, it was demonstrated that this platelet accumulation in the allograft could be abolished with the use of platelet anti-aggregation substances such as cyproheptadine or dextran and that transplant rejection was postponed in treated animals.34 Similarly, it has been shown that PA plays a role in hyperacute rejection of heterotropic cardiac allografts in presensitized dogs.35 Once again, an anti-aggregation agent, sulfinpyrazone, proved effective in postponing rejection. A novel model for the study of allograft rejection was reported in which the rejection process was followed microscopically in a segment of kidney allgrafted into the ear chamber of a rabbit.36 When a whole kidney was allografted into an animal with an established ear chamber allograft, both grafts rejected simultaneously within seven days. It has been suggested that although platelet participation is dramatic in the case of hyperacute rejection, PA may play a more subtle, though important,role in more chronic forms of rejection. It was found that cyproheptadine added to the therapeutic regimen had a beneficial effect on the post-transplant course of rejection crisis in human renal transplant patients.37 It was also reported that heterologous antithymocytc globulin and antilymphocyte globulin, which are often used as part of the therapeutic regimen in transplant patients, have platelet activating activity in vitro and may have thrombogenic properties when administered in vivo. 3g-Thus, an anti-aggregation agent may be useful in post-transplant therapy in counteracting the thrombogenic properties of these globulins, as well as preventing both antigenantibody-induced hyperacute rejection and more subtle platelet involvement leading to the endothelial thickening associated with chronic forms of reject ion.
A number of rather limited trials have been reported with agents such as aspirin, dipyridamole, sulfinpyrazone and cyproheptadine in a variety of clinical conditions. An example is a rather dramatic effect seen with aspirin and dipyridamole in a case of thrombotic thrombocytopenic purpura.39 Variable results with these agents have also been previously reported in transient cerebral ischemia,40,41 platelet consumption following prosthetic heart valve s u r g e r y , 4 2 ~and ~ ~ postsurgical venous thrombosis.44 Certainly results from clinical studies of a larger scale, such as those currently underway in the United States and Canada, will have to be completed before a valid assessment of the potential clinical utility of these agents can be made.45~46 Anticoagulants Anticoagulant therapy in recent years has involved the use of either heparin or the oral anticoagulants, coumarin and indandione derivatives, on venous thrombosis. The only newer agents with clinical potential appear to be several snake venoms or venom fractions which act either through defibrinogenation or inhibition of prothrombin activation. In view of the growing body of laboratory and clinical evidence
Chap. 9
Antithrombotic Agents
Fleming, MacNintch
2
which implicates platelet aggregation as the initiating event in most forms of arterial thrombosis, the emphasis of anticoagulant therapy is focusing more and more on conditions such as postoperative venous thrombosis and pulmonary embolism. Currently, the use of subcutaneous heparin in low doses is receiving a great deal of attention. Numerous studies and opinions have been published in the past two years regarding this form of therapy. Gallus et al. reported a prospective study of 350 high risk surgical and medical patients.47 The thrombosis rate was lower in those patients treated with the lov-dose heparin regimen, regardless of whether they had elective surgery, emergency hip surgery or were suspected of having myocardial infarction. Overall, the rate of thrombosis in heparin-treated patients was 3.6 X as compared to 21.5 X in the untreated group. While there have been some studies which have not demonstrated a beneficial effect of 8ubcutaneous heparin in low doses, the predominant opinion is that venous thrombosis and pulmonary embolism can be delt with effectively by combining the latast diagnostic techniques such as 1125 fibrinogen scans with the proper use of currently available anticoagulant agents .48 The anticoagulant properties of several snake venoms have previously been reported.4 The venom of Ankistrodon acutus contains a procoagulant principle as well as an anticoagulant fraction, both of which have been purified and studied. It has recently been found that the anticoagulant factor inhibits prothrombin activation by interfering with the interaction between prothrombin and its activation factors.49 Fibrinolytic Agents Potential for the application of fibrinolytic therapy lies in two distinct areas. One area involves the concept of curative therapy directed primarily toward dissolution of thrombi formed in cases of vascular obstruction. The other area i s based on the concept that the endogenous fibrinolytic system of the body is responsible for clearing fibrinous deposits from the circulation as soon as they are formed and this concept involves prophylactic therapy as accomplished by chronic enhancement of the endogenous fibrinolytic activity. Evidence for diminished fibrinolytic activity in patients suffering from diseases which have a thrombotic component has been recently reviewed. 50 With respect to clinical effectiveness, success has been restricted to the curative aspect and has involved the intravenous infusion of direct activators which convert plasminogen to plasmin. 51 The greatest success has been achieved with streptokinase, a potent activator which is believed to exert its effect through activation of both circulating plasminogen and intrinsic thrombus plasminogen by formation of an activator complex with the plasminogen molecule itself. The current literature contains an excellent comprehensive review of the clinical pharmacology, mechanism of acticm and therapeutic uses of streptokinase. 52 Streptokinase, however, has been faulted on three major points: (A) its induction of pyrogenic side effects, (B) it@ induction of a refractory state with respect to fibrinolysis because of the ability of this agent to act as an antigen and (C) its low se-
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lectivity in causing activation of gel phase plasminogen within thrombi in preference to soluble phase plasminogen in the general circulation which New deoften leads to hyperplasminemia and a hemorrhagic state.52 velopments include the synthesis of compounds, notably substituted benzylamines for the purpose of potentiation of urokinase fibrinolytic activity, 53 and the development of a streptokinase-plasminogen mixture (molar ratio 2:l to 1:2) as an activator of the fibrinolytic system which is not affected by natural inhibitors in the blood.54 The principle behind the latter concept is that the plasminogen masks the antigenic groups of streptokinase, consequently the mixture can be injected without risk of reaction even if the patient has antibodies against streptokinase resulting from prior contact with streptococci or prior injection with streptokinase. Clinical 8uccess in clot dissolution without the concomitant problems of pyrogenicity and antibody formation with associated development of resistance and titration problems has been achieved with urokinase. The high cost of urokinase, which is now approximately $ 1000 per patient per day, precludes this agent from becoming the drug of choice. Unless current attempts to reduce the cost of urokinase production are successful, the future for acute fibrinolptic therapy lies in the preparation of a synthetic plasminogen activator. However, because of the enzymatic nature of the conversion of plasminogen to plasmin, development of a low molecular weight direct activator of a nonenzymatic nature is essentially precluded. Although the search for agents which might reduce the level or block the action of natural inhibitors of plasmin or of plasminogen activation continues, as yet no agent with an effective efficacy/toxicity ratio has been found.55 The greatest efforts have been directed towards the search for low molecular weight agents which induce the release of endogenous plasminogen activator. The goal is to obtain a non-toxic, orally effective agent capable of producing either a modest but prolonged increase in the level of circulating plasminogen activator for prophylactic purposes or a strong, relatively prolonged fibrinolytic response of sufficient magnitude to be effective acutely in dissolving preformed thrombi. Since no concrete evidence presently exists that such indirect activation of the endogenous fibrinolytic system is either powerful enough or prolonged enough to dissolve preformed thrombi, the greatest potential for indirect activators of fibrinolysis undoubtedly involves the prophylactic approach to the problem. It has been suggested that a $-type of adrenergic receptor may be wholly56-58 or partially59 involved in the fibrinolytic response to infusion of such agents as epinephrine. However, substantial evidence also exists that this is not the case and thata- and $-blocking drugs do not inhibit the fibrinol tic response to agents such as serotonin, bisobrin to exercise-induced fibrinolytic responses63 or to and epinephrine, fibrinolytic responses induced by venous stasis.64 Fibrinolytic responses induced in humans by oral or parenteral administration of the B-receptor have been reported. 5 8 ~ 6 5 ~ 6 6 agonist salbutamol (lo) This not only suggests the possibility of a new lead for orally OH HOCH2 active non-toxic fibrinolytic agents but also H /cH3 HO~-{-CH2-N-C-a3 may serve to shed some light on the implication \ of $-receptors in the endogenous fibrinolytic H CH3 sequence. Salbutamol, a synthetic saligenin 10
60-6q
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Chap. 9
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Fleming, MacNintch
analogue of isoprenaline, is a selective $2 receptor stimulant, which acts selectively on smooth muscle of bronchi, uterus and arteries in preference to heart and intestinal muscle. Consequently, salbutamol produced a fibrinolytic response without the problems of tachycardia associated with the administration of $1 receptor stimulants such as epinephrine and isoprenalineS8, 66 and provides evidence that plasminogen activator responses may be $2 receptor mediated.58 However, it has been recently reported in humans treated chronically with orally administered salbutamol (4 mg/kg qid) that a profound depression of the plasminogen activator, factor VIII and pulse rate responses to epinephrine infusion occurred.66 In addition, there was no evidence of significant change in the resting levels of these parameters indicating a lack of prophylactic fibrinolytic potential for prolonged salbutamol administration at this treatment level. Promising results have been reported con. q O C H 3 cerning the induction of fibrinolytic actiJ CH2 -CHz vity in humans and experimental animals by -CH2-CH2 OCH3 11 the bis-tetrahydroisoquinoline EN-1661 bisobrin) (11) and related compounds. 67s 6 These investigat ions have led to the synthesis of other isoquinoline deriv a t i v e ~ ’and-a ~ series of 1,w-diphenyl-l,@-alkane diamines of comparable general activity.71 However, it has been demonstrated that the fibrinolytic activity induced by bis-6,7-dimethoxy isoquinoline derivatives is reduced significantly with prior administration of the antihistaminic promethazine.70 It is postulated that this type of fibrinolytic agent acts by inducing the release of histamine which in turn is able to activate the fibrinolytic system by mediating the release of plasminogen activators localized in the vascular endothelium. 70s72-74 The fibrinolytic activity elicited by dimethaxpisoquinoline derivatives and related compounds appears to be limited in duration and effect and not adequate for an effective thrombolytic action in cases of massive vascular occlusions. 55 m3°J3& CH-0
6
The well known association of anti-inflammatory and fibrinolytic activities continues to be reported as indicated by evidence of comparisons of fibrinolytic activity of flufenamic acid with niflumic acid, mefenamic acid, azapropazone dihydrate and some trifluromethyl analogues of azapropazone in the hanging plasma clot test.75 A number of dlcl-4-isobutyl(cyc1ohexen-1-y1)alkanoic acids and their derivatives, some of which have anti-inflammatory properties, were reported to have fibrinolytic activity in the standard hanging plasma clot teat.76 The most active compound (l2) was reported to decrease fibrin monomer levels after intravenous infusion into rabbits and to decrease the H euglobulin clot-lysis time in rat blood after daily iC4Hg~-Co0H oral administration for prolonged periods with no evidence of tachyphylaxis. Results in this area are 12 CH3 sometimes confounded by the fact that elevated plasma fibrinogen levels observed in the inflammatory state can exert a marked influence on the fibrinolysis values obtained by standard clot-lysis method^^^,^^ and may mask the true fibrinolytic activity. This frequently makes results difficult to interpret as observations of increased fibrinolytic activity may be secondary to decreases in plasma fibrinogen content. It should also be emphasized here that Baillie and Sim79 maintain
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that the effects of acidic anti-inflammatory agents (such as those of Von Kaulla) on enzyme systems are accomplished as a result of their combination with substrate protein rather than by the enzyme itself. Consequently, the enzyme activating effect of such compounds in the hanging clot system should be interpreted with caution since the possibility exists that neither plasmin nor plasminogen are directly involved. These authors reported that flufenamic acid and 3-bromo-4-tertiarybutyl benzoic acid were not only highly active in the hanging clot test but also were both potent inhibitors of plasmin. They also found a positive linear correlation between fibrinolytic and antiplasmin activities (rs-0.81, p<.05) using this test
.
The literature contains equivocal results concerning the beneficial effects of prolonged clofibrate treatment of fibrinolysis.80-83 The most recent study involved treatment of elderly arteriosclerotic patients with a combination of two clofibrate derivatives, diisopropylamine p-chlorophenoxyisobutyrate and 2-dimethylaminoethanol p-chloro-phenoxyisobutyrate (GerobrekR) for a period of 30 days. A statistically significant increase in fibrinolytic activity was observed along with a significant reduction of platelet aggregation and a potentiation of the anticoagulant activity produced in patients receiving an indirect anticoagulant.28 Although studies have suggested that clofibrate exerts a protective effect against sudden death and myocardial infarction unrelated to its lipidlowering action,B4 the effect of GerobrekR in elevating fibrinolytic activity is in all probability secondary to hypolipemic effects. It is well documented that in humans, low density of lipoproteins and chylomicrons inhibit the fibrinolysis of plasma euglobulin in vivo.85-87 In the same context, treatment of hyperlipemic patients orally with a combination of nicotinic acid and the synthetic heparin-like polyanion S P 5 4 R has been reported to reduce the hypercoagulable state by increasing fibrinolytic activity, reducing platelet aggregation and reducing both cholesterol and triglyceride levels in the blood over a 9-week period.87 Increased fibrinolytic activity associated with decreased blood lipids in humans was also reported subsequent to oral diethanolamine treatment .a9 Compounds causing enhancement of fibrinolytic activity over extended periods of time by increasing concentrations of circulating plasminogen activator still fall mainly into the group of anabolic steroids such as ethylestrenol and stanozolol and the glucose-lowering agents such as tolbutamide and phenformin. Treatment with orally administered phenformin in conjunction with either ethylestrenol or etanozolol continues to give evidence of prolonged improvement of overall fibrinolytic activity ae evidenced by the results of a nine month study in patients with cutaneous vasculit is having impaired fibrinolytic a~tivity.9~The oral ant idiabetic was reported to drug gliclazide (S1702) (2) H O H induce fibrinolytic activity in dogs and rats after both oral and parenteral administration. c H / 3-10s o 2 - ~ - : - ; - N ~ g1 Infusion of prostaglandin Eg into female 13 patients for the termination of pregnancy was reported to increase plasma antithrombin concentration and increase fibrinolytic activity. 92
-
Chap. 9
Antithrombotic Agents
Fleming, MacNintch
83
Heparin-serotonin complexation to form an entity with anticoagulant activity and lytic activity different from that of plasmin with respect to the fact that it lysed unstabilized fibrin clots in the presence of EACA has been reported.93 Evidence of a protective action of vitamin C in deep vein thrombosis has also been reported.94 A potential area of interest which has as yet had no clinical application is the approach of rendering a thrombus fibrin more susceptible to fibrinolysie, and is treated in a comprehensive review on factor XI11 inhibit ion.95 -.-I_ I t 1 3 I \Ireiic e
s
1. J. Y . S c h o r , Ann. Rrporte in bled. Chem., 1 9 6 9 , C . K. Caln, Ed., Academic P r e s s , New York, N. Y., 1 9 7 0 , p 2 7 7 . 2 . L. J. C Z U b B , Ann. R cport s i n Mcd. Chem., 1 9 7 0 , C . K. Gain, Ed., Academic P r e s s , New York, N. Y . , : 9 7 1 , p. 6 0 . 3 . L. J . Czuba, Ann. Report s i n Med. Chem. 1 9 7 1 , R . V. Heinzetman,Ed., Academic P r e s s , New York, N. Y . , 197.2. p. 70. 4 . R . G . Herrmann and W. 8. L a c e f i e l d . Ann. R eports i n Med. Chem., 1 9 7 2 , It. V Heinzelman, Ed., Asndrmic Prefin, New York, N . Y . , 1 9 7 3 , p. ? 3 . 5 . J. B. S d t h and A . L. Willis, Nature New Dlol., 231, 2 3 5 ( 1 9 7 1 ) . 6 . J. J . Kocais, J . Hernandovich, M. J. S i l v e r , J. R. Smith and C . Ingernun, P r o s t a g t a n d l n s , 3 , 141 ( 1 9 7 7 ) . 7. Rall, G. C. Breveton, M. Fulwood, D. M. I r e l a n d and P. Yatea. Bioc he mJ . , 1 2 0 , 7 0 9 ( 1 9 7 0 ) . 8 . J . S. FLcrnlng, J . 0. Buchanan, S . P. King, R. T. C o r n i s h and M. E. Bierwagen i n " F l a t e l e t s and T1-rombosia", Ed. A. S c r i a b t n e and S. Shorry, Untv. Pa rk P r r r s , I n Pre e n. 9 . M. G . Emsser, Biomedicine, 19, 9 0 ( 1 9 7 3 ) . 10. M. L. Dylcen, R. L. Campbell, J . W l l e r , 11. Peue r, T. Horner, R. King, 0 . Kolar. E. Solow and F. H. .Tnncn, S t r o k a , 5, 387 ( 1 9 7 3 ) . 11. M. G . Bnusser and C. L e r r u b l e r , La Nouv. P r e s s e Med., 23, 1687 ( 1 9 7 3 ) . 1 2 . I . B. Kova'cs, I.. Csal ay and r . C&gg, Microvasc. Rer., 6 , 1 9 4 ( 1 9 7 3 ) . 1 3 . J. P, Mayer, 0 . L. Harmond, Ann. Surg.. E , 108 ( 1 9 1 3 ) ; 1 4 . C , 8. M O ~ C ~ O S K. , L a h i r l . M. Lyons, A. 8. Welsse, II. A. O lde w orte l and T. J . Regan, Am. Heart J . , n-6, 61 ( 1 9 7 3 ) . 1 5 . I . I . K r i c b e f f , ti. B. Zucker, T. B. Tschopp and A. K o t o d j i e z , Diagn. Radiology, 1 0 6 . 4 9 ( 1 9 7 3 ) . 16. E. B. Nlshfzawa, D. J. Wynalda, D. E. Suydam and B. A. Molony, Thromb. Res., 3, 577 ( 1 9 7 3 ) . 1 7 . J . W. C o n s t a n t i n e and I. M. P u r c e l l , J. Pharm. Ex?. The r. , 187, 653 ( 1 9 7 3 ) . 18. L. C a p r ln o , F. B o r r e l l f and R. F a l o h c t t i , Arzneim. F o r e r h . , 23, 1777 ( 1 9 7 3 ) . 1 9 . J . F. Mcstard and M. A. Packharn, Blochem. Pharm., 22, 3151 ( 1 9 7 3 ) . 2 0 . M. C . ltozenherg and C. M. Walker, Brit. J . Haematology, 24, 409 ( 1 9 7 3 ) . 2 1 . P. L. R i f k i n and M. B. Zuckcr, Throinbos. Di at he a . haemorrh., 2,694 ( 1 9 7 3 ) . 2 2 . G. V. I?. Born, A. J. Honour and J. R . A. M i t c h e l l , N a ture , 202, 9 5 ( 1 9 6 4 ) . 2 3 . F. Michal, M. H. Maguire and C. Cough, Nature. 222, 1073 ( 1 9 6 9 ) . 24. K. Kikupawa, K. I i z c k a end M. Ichfno, J . Med. Chem., l6, 358 ( 1 9 7 3 ) . 2 5 . K . Klktiaawcl, I I . S u e h i r o and M. Ichi no. J . Med. Chem., .l6, 1381 ( 1 9 7 3 ) . 2 6 . J. P. Carenclvc, M. A. Packham, M. A. Guccione and J. F. Mts ta rd, Proc. SOC. Exp. B t o l . Med., 1 4 2 , l5Y ( 1 9 7 3 ) 2 7 . A . C a r v ~ I I i o , R. V a i l l a n c o u r t , R. C a b r a l , R . W. Colman and R. S. Lees, C i r c u l a t i o n , 48, I V - 1 5 (1973). 2 8 . T. L. S p r r a f l c o , G. F r n n d o l i and G. Turazza, Arznetm. Pors c h. , 23. 2 3 6 ( 1 9 7 3 ) . 2 9 . K. D . HacKrnzie, T. R . Dlohm and J. M. C r i s a r , J . Med. (:hem., 688 (1973). IT.. Farmaso. 28. 511 ( 1 9 7 3 ) . 3 0 . F. Dc.'di-chi, $. F , T amgnure and F. Dorato. 7 1 . I). L. renirtirl and 11. K. Alburn, C t r c u l a t L o n , 48, I V - 1 5 2 ( 1 9 7 3 ) . 3 ? . C . R. Yuirhead. T'lrOmbOR. D i n t h e s . h a e n o r r h . , 30, 138 ( 1 9 7 3 ) . 3 3 . G . C l a r s . Acta C h t r . Scan:l., 139, 1 7 3 ( 1 9 7 3 ) . 3 h . G . C l n e s . Bcla C h l r . Scaiid.. 127 ( 1 9 7 3 ) . 3 5 . H. M. Sharma, J. Rocerrsweig, S. C h a t t e r f e e , S. Moore and M. I..dechamplatn, Am. J. P a th o l o g y , 125 (1Y73). 3 6 . J. B. Hobha and W . J. C l t f f , J . Exp. Med., 137, 7 7 6 ( 1 9 7 3 ) . 3 7 . L. Burrows, M. Ilaimov. L. A l e d o r t , E. L e i t e r , C. Nirmul, H. Shanzur, R. Taub and S . Cla bman, T r a n s p l a n t a t i o n P roc., 157 ( 1 9 7 3 ) . 3 8 . J . Thnnns, P. Thoman, R. M u r c r . J . Caul and D. M. Hum. Organ T r a n s p l a n t a t i o n , l3, 288 (1972). 3 9 . J. Amir and S. Krousn, B1ood. 42. 27 ( 1 9 7 3 ) .
.
c.
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I?,
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SO. G. Evans, Can, Med. Assoc. J., E, 463 (1973). 4 1 . B. Boneu, B. Guiraud and D. Fernet, Nouv. Presse. 863 (1972). 4 2 . H. S. Weily and E. Genton, Circulation, 42, 967 (1970).
L
43. L. A. Harker and S. J. Slichter. New Eng. J. Hed., 283, 1302 (1970). 4 4 . J. R. O'Brien, V. Tulevski and M. Etherington, lancet, L, 399 (1971). 45. Drug Research Reports, 2,1 (1972). 4 6 . H . J. M. Barnett, Can. Med. Assoc. J., 138, 462 (1973). 4 7 . A. S. Gallus. J. Hirsh, R. J. Tuttle, R. Trebilcock, S. E. O'Brien, J. J. Carroll, J. H. Minden and S. M. Hudecki, New Eng. J. Md., 285. 545 (1973). 48. J. J. Skillman, Surgery, 75, 114 (1974). 49. C. Ouyang and C. M. Teng, Toxicon.. 2,287 (1973). 50. c. M. R. Prentice and J. F. Davidson. Clinics in Haematology, 2. 159 (1973). 5 1 . G. P. McNicol and J. A. Davies, Clinfcs in Haematology, 2, 23 (1973). 52. R. N . Brogen, T. M. Speight and G. S. Avery. Drugs, 2, 3 5 (1973). 53. R. P. Johnson and J. Harold Short, Abbott, U. S. Patent 3726969-5, 10 April 1973; D e w a n t 23852U-B. 54. A. Hemburger. H. Ronneberger and M. Schick, Behringwerke, German Patent DT-214886)-Q, 5. April 1973; D e m e n t 21388U-B. 55. M. verstraete, Drugs, 5, 353 (1973). 56. H. Yarnazaki, T. Sand. T. Odakura, K. Takeuchi, T. Nasmura. S. Hosaki and T. Shimamto, Thromb. Diach. haemorrh. 26, 251 (1971). 57. J. G. Kral, B. Ablad, G. Johnason and KOrSPn-BengtSen, Eur.J. Clin.Pharrrmcol., 3 1 4 4 (1971). 5 8 . A . M. A. Gader, A. R. Clarkson and J. D. Cash, Thromb. Res., 2, 9 (1973). 59. J. D. Cash, D. G. Wooafield and A. G. E. Allan, Brit. J. Haematol., l8, 487 (1970). 60. 0. Ponari, A. Giannini. M. Squeri and A. G. Dettori, Coagulation, 2, 249 (1970). 61. Y . Sasaki and S. Takeyam, Jap. J. Pharmacol., 22 (Suppl.), 120 (1972). 375 (1964). 62. A. R. Transer and H. Smellie, Clin. Sci., 63. R. J. Cohen, S. E. Epstein, L. S. Cohen and L. H. Dennis. Lancet, 2, 1264 (1968). 6&. 0. Ponari. E. Civardi, A. Elegha, M. P h i , R. Pot1 and A. G. Dettori, Brit. J. Ilaem., 463 (1973). 65. J. D. Cash and A. Gader, 111. Congrerr on Thrombosis and Haemostasis, Warhington, D. C., 1972, Abstr. p. 280. 66. A. M. A. Gader, S. Parker, G. K. Crompton and J. D. Cash, Thrombosis Res., 2, 137 (1973). 67. L. J. Flieaner, Jr.. J. M. Schor. M. J. Myers and I. J. Pachter, J. Med.Chem., &,580(1971). 68. Chemical Control of Fibrinolysis Thrombolysis, ed.by J. M. Schor, Wiley-Interscience, New York, N . Y., 1970. 5 9 . P. Rajagopalan, 164th National ALS Meeting, New York. N. Y., 1972. 70. P. Markwardt, H. P. Kliicking, J. H. Hauptwnn and G. Paust, Thrombosis Res., 2, 383 (1973). 71. L. J. Fliedner, Jr., M. J. Myers, J. M. Schor and I. J. Pachter, J.Med.Chem.. 16,749 (1973). 72. M. H. Cho and W. Choy. Thrombos. Diathes. haemorrh. (Stuttg.), II, 372 (1964). 73. R. Holemans and R. D. Langdell, Proc. SOC. Exp. Biol. (N.Y.), 115, 584 (1964). 7 4 . R. Holemans, American J. Physiol., 208, 511 (1965). 75. J. T. Wagner, U. Jahn and W. Buerlimann, Arzneim. Porsch., 911 (1973). 76. M. Vincent, C. Remond, P. Desnoyers and J. Labaume, J. Med. Chem., l6, 710 (1973). 77. J. A. Hickman, Brit. J. Haem., 20. 611 (1971). 78. J. A. Hickman, I. C. Gordon-Smith, P. F. Whitfield and S. J. Godfrey, J. Clin. Path., 189 (1973). 351 (1972). 79. A. J. Baillie and A. K. Sim, Thrombos. Diath. haemorrh. (Stuttg.), 80. S. C. Srivastava, M. J. Smith and H. A. Dewar, J. Atheroscler. Res., 3, 640 (1963). 81. E. H. Jepson and D. C. 0. J6ae6, J. Atheroscler. Res., 3, 554 (1963). 62. A. Gibelli, G. Frandoli. P. Giarola and P. Denicola, Hemostase, 285 (1966). 83. R. C. Cotton and E. G. Wade, Lancet, 1, 263 (1969). 8 4 . i. R. Kransno and G. J. Kidera, J. Amer. Hed. Assoc., 219, 845 (1972). E 5 . S. F. Chopin and E. L. Beard, Thrombosis Diath. naemorrh. (Stuttg.), 29. 286 (1973). b6. J. fidsiatwicz. 2. Skrzydlevski and M. Bielecki. Lxperentia, 274 (1967). 87. 2 . Skrzvdlweski, S. Niewiarwski and J. Skrzydlewska, Atherosclerosis Res., 5 273 (1966). @a. G. Turazza,,P. L. Spreafico and G. Frandoli, Arz. Porsch., 23, 654 (1973). 89. D. C. itoehm, Clin. Res., 240 (1973). 40. 8 . Dodman, W. J. Cuncliffe, B. E. Roberts and R. Sibbald, Brit. Mea. J., II, 82 (1973). 31. P. Desnoyers, J. Labaume, H. Anstett, M. Herrera, J. Pesquet and J. Sebastien, A r m . Forscn.. 22, 1691 (1972). 92. P. K . H w i e , A. A. Calder, C. D. Forbes and C. M. R. Prentice, J.Clin.Path., 2 3 3 5 4 (1973). 93. B. A. Kudrjashov, T. M. Kalishevskayo and L. A. Lyaprna, Vopr. 3ed. Khirn., l9, 269 (1973). 44. C. R. Spittle and U. K. Wakefield, Lancet, & 199 (1973). 95. The BioloRical Role of the Clot Stabilizing Enzymes: Transglutamase and Factor XIII, K. Laki, Ed., AM. New York Acad. Sci., 202. 1-348 (1972).
a,
a
-
a
a,
a
z.
Chapter 10:
Pulmonary and A n t i a l l e r g y Drugs
Ralph E. Giles and David J. Herzig Warner-Lambert Research I n s t i t u t e , Morris P l a i n s , N . J. This review covers p a p e r s p u b l i s h e d i n t h e p e r i o d 1971-1973 w i t h a few o l d e r c i t a t i o n s when t h e y were needed f o r t h e a p p r o p r i a t e d i s c u s s i o n of a concept. The main emphasis of t h e review is t h e p r e s e n t o r p o t e n t i a l therapy of asthma o r r e v e r s i b l e airways d i s e a s e . A s e c t i o n i s devoted t o emphysema, an " i r r e v e r s i b l e " airways d i s o r d e r . Asthma i s a t y p e of r e v e r s i b l e , o b s t r u c t i v e pulmonary d i s e a s e due t o widespread narrowing of t h e b r o n c h i a l airways and edema of t h e b r o n c h i a l mucosa, caused by s p e c i f i c a l l e r g i c and/or n o n s p e c i f i c i r r i t a t i v e s t i m u l i ls2. A s p e c i f i c immunoglobulin, IgE, i s produced i n t h e a t o p i c o r allerg i c i n d i v i d u a l , i n response t o t h e a p p r o p r i a t e exposure t o a n t i g e n s . An important predisposing f a c t o r t o the asthmatic a t t a c k is t h e acute sensit i v i t y of t h e b r o n c h i a l tree t o pharmacologic a g e n t s , i r r i t a n t s and spec i f i c allergens. Therapy of r e v e r s i b l e airways d i s e a s e i n c l u d e s symptomatic r e l i e f of t h e a t t a c k , c o n t r o l of s p e c i f i c c a u s a t i v e f a c t o r s as w e l l as g e n e r a l i z e d care of t h e p a t i e n t . Drugs are employed t o relax and d i l a t e b r o n c h i o l e s (beta-sympathomimetics and p h o s p h o d i e s t e r a s e [PDE] i n h i b i t o r s ) , reduce i n flammation ( s t e r o i d s ) , l i q u e f y mucus ( a c e t y l c y s t e i n e ) , d e c r e a s e mucous membrane c o n g e s t i o n (alpha-sympathomimetics) and p r e v e n t mediator release (cromolyn sodium). I n i t i a l c l i n i c a l s t u d i e s of a n t i c h o l i n e r g i c s i n a s t h matics and b r o n c h i t i c s are under way.3-5 Beta Adrenoceptor Agonists - Beta a d r e n o c e p t o r s have been d i v i d e d i n t o s u b c l a s s e s based on t h e r e l a t i v e a c t i v i t i e s of v a r i o u s sympathomimetic m i n e s on d i f f e r e n t t a r g e t t i s s u e s . 6 For example, beta-1 r e c e p t o r s a r e found i n t h e h e a r t and beta-2 r e c e p t o r s are found i n b r o n c h i o l a r and o t h e r smooth muscle. The p o t e n t beta adrenoceptor s t i m u l a n t i s o p r o t e r e n o l , a common agent f o r t h e t r e a t m e n t of asthma, i s n o n s e l e c t i v e and may cause u n d e s i r a b l e c a r d i a c s t i m u l a t i o n a t doses which r e l a x b r o n c h i o l a r smooth muscle. Besides d i r e c t e f f e c t s on b r o n c h i a l smooth muscle, t h e s e compounds i n h i b i t mediator release from s k i n 7 o r p a s s i v e l y s e n s i t i z e d human lung' and a l s o i n h i b i t a n t i g e n - s t i m u l a t e d h i s t a m i n e s y n t h e s i s i n human leucocytes.
Chemical m o d i f i c a t i o n s of t h e catecholamine s t r u c t u r e by a ) r e p l a c e ment of t h e 3-hydroxyl group, b ) replacement of t h e 3,h-dihydroxy configu r a t i o n by a 3,5-dihydroxy c o n f i g u r a t i o n , c ) attachment of a l a r g e r subs t i t u e n t on t h e a l k y l n i t r o g e n and d) a l t e r a t i o n of t h e ethanolamine s i d e c h a i n have y i e l d e d compounds w i t h g r e a t e r b r o n c h o s e l e c t i v i t y , i o n g e r d u r a t i o n of a c t i o n and g r e a t e r o r a l a c t i v i t y t h a n i s o p r o t e r e n o l . D i f f e r ences i n d u r a t i o n of a c t i v i t y and o r a l potency a r e probably r e l a t e d t o a l t e r a t i o n s of t h e 3,4-dihydroxy c o n f i g u r a t i o n ; l a c k of t h e 3,4-dihydroxy c o n f i g u r a t i o n p r e c l u d e s metabolism by catechol-0-methyl t r a n s f e r a s e (COMT)
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and a p p e a r s t o i n t e r f e r e w i t h s u l p h a t e c o n j u g a t i o n i n t h e major m e t a b o l i c pathways f o r i s o p r o t e r e n o l .
9
both
Compounds r e p o r t e d t o b e more s e l e c t i v e and l o n g e r a c t i n t h a n i s o proterenol include s a l b u t a m o l ~ ' - ' k e r b u t a l i n e , l5-I6 Th 1165a, K7-1 salmefahexoprenaline,21 trimeto s o t e r e n 0 1 , ~r~i m i t e r o l , 2 6 c a r b u t e r o l , 27-29 and p y r b u t e r o l . 39 I s o e t h a r i n e , a l s o more b r o n c h o s e l e c t i v e than i s o p r o t e r e n o l , h a s been t e s t e d u s i n g a s u s t a i n e d release system t o p r o l o n g d u r a t i o n . 31 The b r o n c h o d i l a t o r a c t i v i t y of a group of monofluorom e t h a n e s u l f o n a n i l i d e s was r e c e n t l y r e p o r t e d ; 32 u n f o r t u n a t e l y t h e b e s t a g e n t i n t h i s series caused i r r e v e r s i b l e e y e changes i n c a n i n e s .
R i m it e r o 1
H
A common c l i n i c a l problem of t h e o r a l o r p a r e n t e r a l r o u t e of adminis t r a t i o n of t h e newer b e t a - s t i m u l a n t s , such as s a l b u t a m o l and t e r b u t a l i n e , adrenoce t o r s of t h e i s d i g i t a l tremor.16,3?his e f f e c t i s mediated beta-2 s u b c l a s s . I n c i d e n c e of tremor may b e as h i g h as 40%. No subs t a n t i a l s e p a r a t i o n between b r o n c h o d i l a t o r and tremorogenic a c t i v i t y h a s been r e p o r t e d . 3 3 A d e c r e a s e i n t h e t e n s i o n and d e g r e e of f u s i o n of incomp l e t e t e t a n i c c o n t r a c t i o n s of t h e slow c o n t r a c t i n g s o l e u s muscle of t h e c a t i s used as a l a b o r a t o r y model p r e d i c t i v e of tremor i n man.35
"
Use of p r e s s u r i z e d a e r o s o l s c o n t a i n i n g beta a d r e n o c e p t o r a g o n i s t s was i m p l i c a t e d i n i n c r e a s e d d e a t h s among a s t h m a t i c s i n England. 36 However, S t o l l e y r e c e n t l y showed a s t r o n g c o r r e l a t i o n between c o u n t r i e s having an e l e v a t e d d e a t h r a t e i n a s t h m a t i c s and c o u n t r i e s marketing a s t r o n g prepar a t i o n of i s o p r o t e r e n o l . 37 However, i n t h e U.S. , where t h e s t r o n g prepar a t i o n w a s n o t marketed, t h e d e a t h r a t e from asthma d e c r e a s e d d u r i n g t h e y e a r s 1958-1969 w h i l e t h e number of p r e s s u r i z e d a e r o s o l s s o l d i n c r e a s e d 60% between 1964 and 1968.38
Chap. 1 0
Pulmonary, A n t i a l l e r g y Drugs
G i l e s , Herzig
87
P r o s t a g l a n d i n s - P r o s t a g l a n d i n s are found i n n e a r l y a l l t i s s u e s ; PGE2 i s t h e dominant p r o s t a g l a n d i n i n b r o n c h i a l t i s s u e , w h i l e PGF2, p r e d o m i n a t e s i n l u n g parenchymal t i s s u e . 39-40 However, p r o s t a g l a n d i n s are n o t s t o r e d as s u c h i n t i s s u e . A l a r g e number of s t i m u l i can t r i g g e r p h o s p h o l i ase A which c l e a v e s a r a c h i d o n i c a c i d from c e l l membrane p h o s p h o l i p i d s . 41-e2 A r a c h i d o n i c a c i d i s r a p i d l y c o n v e r t e d t o p r o s t a g l a n d i n s by t h e enzyme p r o s t a g l a n d i n s y n t h e t a s e . Thus measurement of t i s s u e l e v e l s of p r o s t a g l a n d i n s may b e i n d i c a t i v e of s y n t h e t i c a b i l i t y a t t h e t i m e of measurement r a t h e r than b a s a l t i s s u e values. A e r o s o l a d m i n i s t r a t i o n o f PGEl o r PGE2 p r o d u c e s b r o n c h o d i l a t a t i o n i n a n i m a l s 4 3 and man44 w i t h minimal c a r d i o v a s c u l a r s i d e e f f e c t s . 45-46 BY t h i s r o u t e PGEl o r PGE2 w a s 1 0 t i m e s more p o t e n t on a w e i g h t b a s i s t h a n i s o p r o t e r e n o l , b u t had a s l o w e r o n s e t of a c t i o n ; 4 7 d u r a t i o n o f a c t i v i t y w a s i n t h e r a n g e of i s o p r o t e r e n o l and t h e r e f o r e c o n s i d e r a b l y less t h a n t h e l o n g a c t i n g b e t a - 2 s t i m u l a n t s . A l i m i t i n g s i d e - e f f e c t of t h e a e r o s o l a d m i n i s t r a t i o n of PGEl o r PGE2 i s l a r y n g e a l i r r i t a t i o n and coughing. 46 Furthermore, some a s t h m a t i c s have developed an a t t a c k of asthma a f t e r i n h a l a t i o n o f PGE1, p o s s i b l y due t o i r r i t a n t e f f e c t s i n t h e a b s e n c e of a b r o n c h o d i l a t o r r e s p o n s e . 48 While PGEl h a s been r e p o r t e d t o i n c r e a s e CAMP l e v e l s i n i s o l a t e d l u n g t i s s u e , f u r t h e r mechanism s t u d i e s are i n o r d e r . 4 9 P G F ~ Bc a u s e d b r o n c h o d i l a t a t i o n i n t h e c a t and g u i n e a p i g . 5 o PGF2, produced b r o n c h o c o n s t r i c t i o n i n man. 51 Human a s t h m a t i c s were 8000 t i m e s more s e n s i t i v e t o t h e b r o n c h o c o n s t r i c t o r e f f e c t s of PGF2, t h a n were normals and o n l y 10 times more s e n s i t i v e t o h i s t a m i n e - i n d u c e d bronchospasm.52 I t h a s been s u g g e s t e d t h a t t h e endogenous PGF/PGE r a t i o may b e c r i t i c a l i n a s t h m a t i c s , b u t t h i s awaits d e f i n i t i o n . 5 3 C e r t a i n a n a l g e s i c , a n t i i n f l a m m a t o r y d r u g s such as a s p i r i n , indomethac i n , f l u f e n a m i c a c i d and mefenamic a c i d i n h i b i t p r o s t a g l a n d i n s y n t h e s i s . 54 A s p i r i n and t h e fenamates a l s o a n t a g o n i z e d t h e d i r e c t c o n s t r i c t o r e f f e c t s of PGF2a, b u t n o t t h e r e l a x a n t e f f e c t s of P G E z , ~on ~ i s o l a t e d human bronc h i a l t i s s u e . 5 4 Indomethacin and a s p i r i n reduced t h e b a s a l t o n e of i s o l a t e d t r a c h e a , d e c r e a s e d t h e e f f e c t of s m a l l d o s e s and i n c r e a s e d t h e e f f e c t of l a r g e d o s e s of h i s t a m i n e o r a ~ e t y l c h o l i n e . ~These ~ latter s t u d i e s suggest a modulating i n f l u e n c e f o r t h e p r o s tag lan d in s i n t r a c h e a l smooth muscle. High m o l e c u l a r w e i g h t a n i o n i c p o l y e s t e r s of p h o s p h o r i c a c i d and p h l o r e t i n , c a l l e d p o l y p h l o r e t i n p h o s p h a t e (PPP) have a n t a g o n i z e d t h e c o n s t r i c t o r e f f e c t s of PGF2, on human b r o n c h i . Yet n e i t h e r t h e d i l a t o r r e s p o n s e t o P G E z ~n o~ r t h e c o n s t r i c t o r r e s p o n s e t o c a r b a c h 0 1 ~were ~ a l t e r e d , s u g g e s t i n g s e l e c t i v e antagonism. PPP a l s o i n The d i f f e r e n t h i b i t s a l k a l i n e p h o s p h a t a s e , h y a l u r o n i d a s e and u r e a s e . m o l e c u l a r w e i g h t c o n s t i t u e n t s of t h e m i x t u r e of PPP polymers have been s e p a r a t e d t o y i e l d t h e P G - i n h i b i t o r y component i n t h e low m o l e c u l a r w e i g h t f r a c t i o n s and t h e enzyme i n h i b i t o r y a c t i v i t y i n t h e h i g h molecul a r w e i g h t f r a c t i o n s . 59 Asthmatics have f a i l e d t o show an improvement i n airway r e s i s t a n c e a f t e r 5 mg d o s e s of PPP were i n h a l e d . 1 8
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P r o s t a g l a n d i n s i n h i b i t e d immunologic release of h i s t a m i n e from human l e u c o c y t e s 0 and human l u n g f r a g m e n t s ; 6 1 t h i s i n h i b i t i o n w a s a s s o c i a t e d w i t h i n c r e a s e d CAMP levels. PDE I n h i b i t o r s - CAMP i s r a p i d l y metabolized by a PDE which h y d r o l y t i c a l l y c l e a v e s t h e 3'phosphate bond t o y i e l d a 5 ' phosphate e s t e r of adeno s i n e (AMP). A similar r e a c t i o n o c c u r s f o r cGMP. CAMP r e l a x e s smooth muscle such as i s o l a t e d t r a c h e a . 6 2 The d e m o n s t r a t i o n of a Ca4-dependent i n c r e a s e i n cGMP i n smooth muscle i n response t o c h o l i n e r g i c a g e n t s , h i g h K+ c o n c e n t r a t i o n s , and h i s t a m i n e s u g g e s t s a r o l e of t h i s n u c l e o t i d e i n smooth muscle c o n t r o l . 6 3 However, d i b u t y r l cGMP and 8-bromo-cGMP r e l a x e d t r a c h e a l smooth muscle. 64 A b i p h a s i c c o n t r a c t i o n and r e l a x a t i o n have a l s o been r e p o r t e d .
Theophylline and o t h e r m e t h y l x a n t h i n e s are PDE i n h i b i t o r s and are f r e q u e n t l y used t o t r e a t asthma. I n a comparative s t u d y u s i n g f r o g eryt h r o c y t e c y c l i c n u c l e o t i d e PDE, t h e o p h y l l i n e ( 4 ~ 1 0 - ~ Mwas ) a potent inh i b i t o r of t h e h y d r o l y s i s of CAMP b u t w a s a much less e f f e c t i v e i n h i b i t o r of t h e h y d r o l y s i s of c G M F ' . ~ ~ Compounds c a p a b l e of i n h i b i t i n g PDE' s from v a r i o u s s o u r c e s have been t e s t e d f o r b r o n c h o d i l a t o r e f f i c a c y . A t r i a z o l o p y r a z i n e ( I C I 58,301) and a t r i a z o l o p y r i m i d i n e (ICI 63,197) p r o t e c t e d g u i n e a p i g s from t h e l e t h a l e f f e c t s of h i s t a m i n e ( r e s p e c t i v e ED50 v a l u e s were 0 . 5 and 0.05 mg/kg).67 Propranolol did not block the antibronchoconstrictor e f f e c t s i n v i t r o but d i d i n v i v o . The a p p a r e n t blockade i n v i v o could be due t o antagonism of r e f l e x l y - r e l e a s e d catecholamines which c o n t r i b u t e t o t h e p r o t e c t i v e response.
S t u d i e s u t i l i z i n g r a t e r y t h r o c y t e PDE l e d t o t h e d i s c o v e r y of 4(3butoxy-4-methoxybenzyl)-2-imidazolidinone (RO 20-1724; IBM).6 8 T h i s a g e n t w a s 5000 t i m e s more p o t e n t than t h e o p h y l l i n e i n t h e e r y t h r o c y t e p r e p a r a w a s about 400 times more p o t e n t t h a n t i o n , w i t h an 150 of 0 . 1 pM. IBM t h e o p h y l l i n e i n r e l a x i n g t h e i s o l a t e d guinea p i g t r a c h e a and i t s e f f e c t S i m i l a r r e s u l t s were n o t e d u s i n g i s o l a t e d w a s n o t blocked by p r o p r a n o l o l . w a s a v e r y poor PDE i n human t r a c h e a and b r o n c h i . I n t e r e s t i n g l y , IBM h i b i t o r i n dog t r a c h e a l smooth muscle o r i n whole guinea p i g t r a c h e a . Thus i t s mechanism o f t r a c h e a l r e l a x a t i o n cannot be e x p l a i n e d i n terms of
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PDE i n h i b i t i o n . The p u r i n e compound i n o s i n e h a s a l s o been r e p o r t e d t o i n h i b i t PDE and i n h i b i t b r o n c h o s p a s t i c r e s p o n s e s i n guinea p i g s . 6 9
A n t i c h o l i n e r g i c s - I n b o t h animals and man, s t i m u l a t i o n of e p i t h a l i a l i r r i t a n t r e c e p t o r s i n t h e airways by a n t i g e n i c and n o n a n t i g e n i c y h y s i c a l o r pharmacological a g e n t s r e s u l t e d i n r e f l e x b r o n c h o c o n s t r i c t i o n . This b r o n c h o c o n s t r i c t i o n could be blocked by a t r o p i n e i n humans71 and by a t r o p i n e o r vagotomy i n a n i m a l s , 72 i n d i c a t i n g a p a r a s y m p a t h e t i c m e d i a t i o n . Vagal blockade reduced t h e i n c r e a s e i n pulmonary r e s i s t a n c e caused by h i s t a m i n e ( i n j e c t e d i n t o t h e pulmonary c i r c u l a t i o n ) from 800% t o 100%.7 3 B r o n c h o c o n s t r i c t i o n caused by a n t i g e n i n h a l a t i o n i n a s t h m a t i c s h a s been blocked by a t r o p i n e . These experiments have c h a l l e n g e d t h e t r a d i t i o n a l concept of mediator-induced d i r e c t muscle c o n t r a c t i o n as t h e primary e v e n t i n bronchospasm. Atropine h a s h i s t o r i c a l l y been viewed as dangerous t o a s t h m a t i c s because of t h e p o t e n t i a l of d r y i n g e f f e c t s and mucous p l u g s . I n one a c u t e t r i a l , i s o p r o p y l a t r o p i n e (Sch 1000) was found supeAcute r i o r t o salbutamo1,‘l w h i l e i n a n o t h e r t h e reverse was r e p o r t e d . ’ s t u d i e s may n o t be t h e most a p p r o p r i a t e means t o e v a l u a t e t h e problem of mucous p l u g s . It h a s r e c e n t l y been r e p o r t e d t h a t a few s e l e c t e d p a t i e n t s were t r e a t e d w i t h a e r o s o l i z e d a t r o p i n e f o r up t o 6 months w i t h o u t observi n g mucous problems. 7 4 Emphysema - Emphysema i s an anatomic a l t e r a t i o n of t h e l u n g c h a r a c t e r i z e d by an abnormal enlargement of t h e a i r s p a c e s d i s t a l t o t h e t e r m i n a l , nonr e s p i r a t o r y b r o n c h i o l e , accompanied by d e s t r u c t i v e changes of a l v e o l a r walls. S i n c e B r ~ w n - S e q u a r d ~f i~r s t d i s c u s s e d t h e p r o d u c t i o n of e x p e r i m e n t a l emphysema i n t h e l a t e n i n e t e e n t h c e n t u r y , r e p o r t s of v a r i o u s exp e r i m e n t a l procedures t o induce t h i s c o n d i t i o n have appeared i n t h e l i t e r a t u r e . Emphysema-like c o n d i t i o n s have been produced by phosgene, 76 c i g a r e t t e smoke, 7 7 t r a c h e a l l i g a t i o n and ph t o h e m a g g l u t i n i n , 7 8 p a p a i n , adm i n i s t e r e d i n t r a t r a ~ h e a l l y ~o’ r by polymorphonuclear-enriched l e u k o c y t e homogenates, 8 1 and cadmium c h l o r i d e a e r o s o l . 82 U t i l i z a t i o n of p r o t e o l y t i c materials, such as p a p a i n , t o produce an e x p e r i m e n t a l emphysema-like s t a t e may be of p e r t i n e n t v a l u e i n l i g h t of r e c e n t e v i d e n c e t h a t a d e f i c i e n c y of an a n t i p r o t e o l y t i c f a c t o r , a 1 - a n t i Many i n v e s t i g a t o r s t r y p s i n , i s a s s o c i a t e d w i t h p a n l o b u l a r emphysema. have i n d i c a t e d t h a t papain-induced emphysema i s similar morpholo i c a l l y , p a t h o l o g i c a l l y and p h y s i o l o g i c a l l y t o human emphysema. 79 3 8 o 9 8 4 - 8 F Cadmium i s of s p e c i a l i n t e r e s t because of i t s p r e s e n c e i n c i g a r e t t e smoke. P r o g e s t e r o n e a n t a g o n i z e d t h e development of emphysema induced by i n t r a t r a c h e a l l y - a d m i n i s t e r e d papain and t r a c h e a l l i g a t i o n B 8 and by papain a e r o s o l i n male r a t s , 8 9 9 9 0 as w e l l as t h e development of an emphysema-like c o n d i t i o n produced by phytohemagglutinin and t r a c h e a l l i g a t i o n . 7 8 D i m e t h i s t e r o n e and m e g e s t r o l acetate were e f f e c t i v e a g a i n s t an emphysemal i k e c o n d i t i o n produced by papain a e r o s o l and t r a c h e a l l i g a t i o n . 88 Medroxyprogesterone acetate p r e v e n t e d t h e development of an emphysema-like l e s i o n caused by t h e m i c r o b i a l p r o t e a s e , thermolysin. 91 Reasons f o r i n i t i a l l y s t u d y i n g p r o g e s t e r o n e i n e x p e r i m e n t a l emphysema i n c l u d e d t h e obs e r v a t i o n s t h a t a ) t h e r e w a s a lower i n c i d e n c e of pulmonary emphysema i n
90 -
S e c t . I1
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Pharmacodynamic Agents
C l a r k e , Ed.
females t h a n i n males, 92 b ) female emphysematous p a t i e n t s improve d u r i n pregnancy when t h e blood t i t e r o f p r o g e s t a t i o n a l hormones i s e l e v a t e d . 9 g The mechanism of t h e b e n e f i c i a l e f f e c t of p r o g e s t e r o n e i s unknown. S i n c e p r o g e s t e r o n e and o t h e r p r o g e s t a g e n s are e f f e c t i v e i n s e v e r a l t y p e s of emphysema-like c o n d i t i o n s , i t seems u n l i k e l y t h a t t h e b e n e f i c i a l e f f e c t i s due t o antagonism of papain-induced p r o t e o l y s i s . S t u d i e s t o a ) d e f i n e t h e dose r e s p o n s e r e l a t i o n s h i p of p r o g e s t e r o n e i n e x p e r i m e n t a l emphysema and b ) e v a l u a t e t h e r e l a t i o n s h i p between p r o g e s t a t i o n a l a c t i v i t y and "antiemphysema" a c t i v i t y seem a p p r o p r i a t e . C o r t i c o s t e r o i d s - C o r t i c o s t e r o i d s have a number of s i d e e f f e c t s t h a t can r e s t r i c t t h e i r u s e i n asthma. The most troublesome of t h e s e i s t h e supp r e s s i o n of normal a d r e n a l f u n c t i o n and t h e development of Cushingoid symptoms. Recently beclomethasone-17,21-dipropionate h a s been used c l i n i c a l l y w i t h r a t h e r good r e s u l t s as a replacement f o r o r a l s t e r o i d therapyB4-?Z p a t i e n t s w i t h moderately severe c h r o n i c b r o n c h i a l asthma t h a t were n o t on o r a l s t e r o i d s , beclomethasone a e r o s o l (100 pg, 4 times a day) improved l u n g f u n c t i o n tests and reduced b r o n c h o d i l a t o r usage w i t h o u t a s i g n i f i c a n t e f f e c t on plasma c o r t i s o l l e v e l s . 9 8 The l a c k of s u p p r e s s i o n s e e n w i t h t h e a e r o s o l may b e due t o t h e l o c a l i z e d a p p l i c a t i o n and l a c k of a b s o r p t i o n . I n p a t i e n t s a l r e a d y on maintenance o r a l s t e r o i d s , a e r o s o l beclomethasone reduced b u t d i d n o t r e p l a c e p r e d n i s o l o n e P 6 This l i m i t e d response may be due t o a ) t o o low a dose of a e r o s o l s t e r o i d o r b ) o t h e r n e c e s s a r y s y s t e m i c a c t i v i t i e s of c o r t i c o i d s d i s c u s s e d below t h a t cannot b e r e p l a c e d by t o p i c a l a p p l i c a t i o n .
Although p a r t of t h e e f f e c t of s t e r o i d s i n asthma may b e due t o t h e well-known a n t i i n f l a m m a t o r y a c t i o n and s t a b i l i z a t i o n of lysozomal memb r a n e s , t h e r e a l s o appear t o be e f f e c t s of t h e s e compounds on t h e immune system. The s t i m u l a t i o n o f i n v i t r o immunoglobulin s y n t h e s i s by epinep h r i n e on p e r i p h e r a l l e u c o c y t e s from normal humans could be augmented by 10-6M h y d r o c o r t i s o n e , w h i l e lymphocytes from a s t h m a t i c s d i d n o t respond t o e p i n e p h r i n e o r t o augmentation w i t h h y d r o c o r t i s o n e . loo
'',
C o r t i c o s t e r o i d s have a l s o been shown t o augment t h e e f f e c t of c a t e cholamines on b r o n c h i a l t i s s u e , such t h a t g u i n e a p i g s , c h r o n i c a l l y treated w i t h h y d r o c o r t i s o n e , were more s e n s i t i v e t o i s o p r o t e r e n o l . Lungs from such guinea p i g s t o o k up l e s s e p i n e p h r i n e and n o r e p i n e p h r i n e i n v i t r o w i t h o u t changing catecholamine metabolism. l o 1 , I o 2
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S p e c i f i c I n h i b i t o r s of Anaphylactic Mediators - Slow r e a c t i n g s u b s t a n c e of a n a p h y l a x i s (SRS-A), a c o n s t r i c t o r of b r o n c h i a l smooth muscle, has been shown t o d i f f e r from any of t h e o t h e r known p h y s i o l o g i c a l l y a c t i v e a g e n t s (MW of 350-450; p I = 4 . 7 ) . Diethylcarbamazine i n h i b i t s t h e release of SRS-A from rat and human t i s s u e s b u t i t h a s n o t been e f f e c t i v e i n amelior a t i n g b r o n c h i a l c o n s t r i c t i o n i n a s t h m a t i c s , perhaps because of t o x i c l i m i t a t i o n s . FPL 55712 a c t s i n v i t r o as a h i g h l y s p e c i f i c a n t a g o n i s t o f SRS-A-induced c o n t r a c t i o n s of g u i n e a p i g ileum, b e i n g s e v e r a l t i m e s less a c t i v e a g a i n s t o t h e r c o n t r a c t o r s . l o 4 FPL 55712 a l s o r e v e r s e d an ongoing SRS-A-induced c o n t r a c t i o n of guinea p i g ileum. The c l i n i c a l u t i l i t y of t h i s compound i s unknown. A n t i h i s t a m i n e s , which u s u a l l y e x e r t t h e i r e f f e c t s by b l o c k i n g h i s t a mine a t end-organ r e c e p t o r s , are e f f e c t i v e i n u r t i c a r i a and a l l e r g i c r h i n i t i s b u t a r e of q u e s t i o n a b l e e f f i c a c y i n asthma. Troublesome s i d e e f f e c t s of t h e c l i n i c a l l y u s e f u l a n t i h i s t a m i n e s i n c l u d e b o t h CNS stimul a t i o n and d e p r e s s i o n . R M I 9918 h a s been r e p o r t e d t o be an e f f e c t i v e H I s p e c i f i c a n t i h i s t a m i n e i n r a t s , mice and guinea p i g s w i t h no o b s e r v a b l e CNS a c t i v i t y . 1 0 5 Anaphylactic I n h i b i t o r s - Cromolyn sodium i n h i b i t s t h e release of a l l e r g i c m e d i a t o r s such as h i s t a m i n e and SRS-A from s e n s i t i z e d t i s s u e s , b u t does n o t i n t e r f e r e w i t h t h e combination of a n t i g e n and a n t i b o d y . Like i t s p r e c u r s o r a z a t a d i n e , a z a n a t o r maleate (Sch 15,280) h a s s i g n i f i c a n t a n t i m e d i a t o r a c t i v i t y . O 6 I t also i n h i b i t s rat r e a g i n i c p a s s i v e cutaneous a n a p h y l a x i s (ID50=3 mg/kg p . 0 . ) and i n v i t r o a n a p h y l a c t i c h i s t a m i n e release from r a t p e r i t o n e a l m a s t c e l l s ( I C 5 0 = lO-'M). This drug a l s o r e l a x e s guinea p i g t r a c h e a l smooth muscle ( n o t mediated v i a b e t a a d r e n o c e p t o r s ) and p r e v e n t s a n a p h y l a c t i c b r o n c h o c o n s t r i c t i o n i n s e n s i t i z e d rats. Other " a n t i - a l l e r g y " compounds shown e x p e r i m e n t a l l y t o i n h i b i t a l l e r g i c r e a c t i o n s i n a wide range of i n v i t r o t e s t models i n c l u d e t h r e e xanthones, (AH 6556) (AH 7079) (AH 7 7 2 5 ) ~ 0 ~ s e r i of e sxanthone-2 c a r b o x y l i c a c i d s a n t a g o n i z e d p a s s i v e cutaneous a n a p h y l a x i s i n t h e r a t . a There h a s been g r e a t i n t e r e s t i n s t u d y i n g mechanisms f o r p r e v e n t i n g a l l e r g i c r e a c t i o n s a t t h e l e v e l of t h e antigen-antibody t a r g e t c e l l . When i n c u b a t e d w i t h cromolyn sodium i n v i t r o r a t p e r i t o n e a l mast c e l l s and human l u n t i s s u e s become s t o i c h i o m e t r i c a l l y a u t o d e s e n s i t i z e d t o The c l i n i c a l i m p l i c a t i o n s of t h i s o b s e r v a t i o n are uncromolyn. log-K1l known. Chrysotherapy h a s been used f o r a r t h r i t i s and some r e l a t e d d i s e a s e s , and r e p o r t s e x i s t of some v a l u e i n asthma. SK&F-39162 a p p e a r s t o be a s p e c i f i c i n h i b i t o r of a n a p h y l a c t i c h i s t a m i n e release i n r e a g i n i c a l l y sens i t i z e d animals. Y e t t h e compound h a s no d i r e c t antagonism t o known m e d i a t o r s . 112 It i s n o t known whether t h i s compound h a s t h e same l i a b i l i t i e s of o t h e r g o l d s a l t s i n humans, such as kidney and s k i n l e s i o n s .
92
1. 2. 3. 4.
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- Pharmacodynamic Agents
Clarke, Ed.
W.B.
Sherman, Asthma, I n &cil-Loeb Textbook of Lbdicine, ed. by F.E. iieeson, and k . ?hDermott, pp. 53E539, H.B. Saimdzrs Co., PhilaBeluhia, 1973. A.W. ~ r a ~ k l a n dI,n Clinical Aspects of h m o l o g y , ed. P.G.K. LelL and R.A. Coombs, F.A. CQ,, Philarlslphia, 1968.
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U d o r s u o , 9.6. V i l j a n e n , and A. l l u i t t u r i , %end. J. of Clin. and k h . I n v e s t , 31, Suppl. 13,16 (1973). 6. Z L : . Lands, A. Arnold, J.P. lIcAuliff, F, P. Ludumaand %& &own, N i t i r e (London) 214, 597 (1967). 7. A s s e m and A.V. X i c h t e r , & r u n o b g y , a 729 (19-1). Z.S.A. Assem and H.Q. S c h i l d , Nature (London! 224, lG2e (1959). 63. Assem and J.J.I. Feigenbaum Br. 3. P h a r x c o l . 46, 519 (1372). 9. 2.S.K. 10, 1i.D. domay, I. Minatoya, A.M. Lands and J.;'. S h e k o s l n r J . Fharm. Sci. 57, 1135 (1968) 11. d.2. Liles, and J.;r. M i l l q J. Pharmacol. Exp. Ther. 156 201 (1967). 12. P.S. Davics, C.P. fhrgan, !d.i. Conolly, J.\ E a t t e r s o F L s J . Sandler and C.T. Dollery Fed. ROC.28, 797 (1969). &lm, J.B. iGm, D. Jack and G.?. Levy, S it. J. P h : r a c o l . 35, 1 4 13. f.A. (1969) 14. C.L. S n i d e r and R. Laguardie, J. J.A.MbA. 221, 682 (1972). 15. .!1 Persson and T'. Ollsson, Acta Med. S c a n d ~ s u r p l . 512 1 (1970). 16. P. Glass and !..J. h l f a n o , C u r r . Ther. Res. 15, 141 m 3 { . 17. S.R. O'Donnell, 6ur. J, Ejharm. 12, 35 (1970)T F i n k e l , J. Pharmacol. Exp. Ther. 472 l o . H.S. L i l e s , J.C. Williams and (1973). 19. Y,. J o r d e and YI. K e i s t e n , E-kd. Klin. 68, 961 (1973). Kededy and C.H. Dash, Acta m a r g o 1 27, 22 20. M.C.S. (19721. 21. J.C. Vermak, h1.A. deKooE- and J.1,. J o u b a r t , S . A f r o ZPd. Z. 46, 1999 (1972). 22. 1. Iwasawa and A . Kiyomoto, Jap. J. rharmacol. l7, 143 (1967)T 23. h. Yamamoto, Asian Med. J. 13, 165 (1970). 24. F.K. Lish, J.k. L e i k e l a n d r 3ungan, J. Fharmacol. Sxp. Tier. 149, 161 (1965). 25. J.P. L r i f f i n and P. I'urner, J. Clin. Pharrxtcol. 280 1971 26. H.C. Bowman and 1.W. Rodger, B-. J. Fharmacol. 574 119721. 27. J.R. Viardell, Jr., D. C o l e l l a , C. Kaiser and S . T o a s , Xbs. of Yolunteer Papers, 5 t h I n t l . Congress on Pharmacology, San Francisco, C - d i f . , July 2x28, 1972, p. 246. 28. J.2. Wardell, Jr., D.F. Colella, A. S h e t z l i n e and F.J. Yowler, J. Pharmacol. bxp. Ther., 189, 167 (1974). 29. C. Kaiser, D.F. C o l e l l a , IL.S. Schwartz, 3. Carvey and J.R. Yardell, Jr. J. hkd. Chem. 17,49 (1974).
5.
m.X.
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s,
.
8:
.
Chap. 10
Pulmonary, Antiallergy Drugs
Giles, Herzig
93
225, 521 (1973). 30 United S t a t e s hdopted lJa~ii?s,J.A.M.A. Lees, =to J. Dis. Shest. 67, 6 1 (1973). 31. 3.N. Sinha, G.W. Allen and C.N. J. Lled. ;hem. 2, l.!&urran and J.2. Robertson, 32. 2.H. B a n i t t , K.E. Soyne, K.P. 116 (1974). 33 U. Jack, In, Asthma: Physioloty, I-minopherirmecology and Treatment, ed. by K . T . Austin anTL.34. Lichtenstein, Academic &ess, New York, p. 351 (1973). , 1367(m). 34 Soh. Spstein, J.A. Biirnaxd and T.T. Zsoter, h e r . Rev. Resp. X s . 9 %man and lL7+.Nott, Br. J. Fharmacol. 38 37 1970). 35 9i.C. 36. F.3. 6peizer and 3. Doll, Bit. l&d. J. 3 247t1968 37 P.D. Stolley, Am. ilev. Hesp* 92s. 105, E85 (1972). 5 O m 7 1 ) . 38 N.C. H a r r i s , Ann. Allergy 29, 2 K a r b , ld. Sandler 3.D. Williams, Er. J. Pharmacol. 31, 340 (1967). 39 .:.!.S Cuthbert, In Frostaglandbw: Pharnxicoloty and Thera e u t i c z ed. by M.F. 40. !.F. Ckthbert, Heinemann !led. aook, London, pp. 251-2e2 (19727. 41 J. ifakaiio and J.X. Kessinger, Froc. Soc. Exp. 3.01. ibd. 133, 1314 (1970). ., Plemrn 42. J. Nakano, I n Prostaglandins ed. by PbTiJ, Ramell and J.3. ‘sE;z;w, vol. l p. 239 (1975). Press, l?ew=k, 43. 1d.2. Hosenthale, A. Dervinis, A.J. Begany, M. Lapidus and i.1.1. G l u c h n , ixperentia, 26, 1119 (1979. 44. &F. C u t h b e r c Proc. Ray. SOC. l&d. 6 4 , s (1971). 45. L1.9. Rosenthale, A. Delervinis and J. E s s a r i c h , J. Fharmacol. Exp. Ther. 178, 541 (1971). 46. 1vl.F. Cuthbert, Bit. >led. J. 4, 723 (1969). 47 A.P. Smith, and 1ul.F. Cuthbert, kit. Med. J. 2, 212 (1972). 48 A.P. Smith, I n Asthma: Physiolody, h u n o p h a r m c o l o g y and Treatment, ed. by K.F. 267 (1973). ATJstin and Liahtenstein, Acaienic h-PraSS, Kew York, Baird and S.W. Sutherland, I n F r o s t a g f h i i n Syclrosium of the 49 R.N. &tcher, C.2. Worchester foundation f o r d x p e r b n t a l Biolo,yTed. by F.K. Famiell and 5.3. ShaA’, Interscience Publishers. New York (1967). 50. id.$. Rosenthale, A. D e r h n i s and MiI. G i u c b n , Prostaglandins 3, 767 (1973). 51. A.P. Smith and bl.F. Cuthbert, *it. !dear J. 3, 212 (1972). N. Tvanborg, Brit. Med. J. 2, 193 (1973). 52. &A. Mathe; P. riedqvist, A. 53.
.. . .
f.
a
.. .
.
Lx.
54. 55 56 57 58
.
59. 60
61. 62. 63.
J. Grphek, I..^-.-.
68 0
63. 70 71. 72. 73. 74. 75. 76 77. 78.
Douglas,
(Lond) 219, 864 (1968). Bouhuys’;-mat. New Biol.,
(Mf3l.
z, P4
hthe’; K. Strandbsrg and A. Astrom, Nature (Lond) 230, 215 (1971). K.E. ERkins, J.9, bBller and S.M.M. K a r i m , J. PhermacorExp. Ther. 176, 441 (1971). K.E. Eakins, Ann. N.Y. &ad. Sci., le0, 386 (1971). Lob:. Lichtenstein and C. Henney, 11,Tostaglavdins i n Cellular 8iolofy, ed. by P.W. Hatnwell, Plenum Fublishing %, N.Y., p. 293 (1972). Austin, J. A’lergy and Clin. k.1. Tauber, Id. K a l i n e r , D.J. Stechschulte and K.F. Irmunol. 51, 106 (1973). P.Y. Moore, L.C. I o r i o and J.M. hlci.lanus, J. Pharm. Pharmacol. 20, 36@ (1966). G. Schultz, J.C. Hardman and 3.W. Sutherland, In Asthma: Physixogy, Inn-inoLichtenstein, hcademic pharmacoLogy ai;rl Treatment ed. by X.F. AustinTnd L.! Press, Inc., N.Y., p. 123 11973). L. bzaduykisSzadiiski, and I’. Berti, Phasmacol. Pes. Connun. 4 , 53 (1972). A.J. Lerris, J.S. Douglas and A. r’ouhuys, J. Pharm. Pharmacol. 75, 1071 (1973). 0M .. Rosen, Arch. Bioc’ilea. Siophys. 139, U 7 (1973). L E O Davies, J. Pharm. Pharmacol. z5el (1973). H. Sheppard, I n Asthma: FLLysiolofy,Z-mnophwmacoloy’y and !Creatm#>nt, ed. by K.F. Austir. and L.M. Lichtaastein, Acadellic Press, p. 235, N.Y. (1973). A. B e r t e l l i , C. 3ianchi and L. b a n i , J. Pharm. Pharmacol. 25, 60 (1973). J.G. Widdicomband Go:!. Sterliw’, Arch. Intern. lied. 126, 3 n (1970). 3.G. Simonsson, F.’:. Jacobs and J.A. Nadel, J. CZin. I n v e s t . 46 182 (1967). U.V. Gold, G.F. Kesslm and 1.Y.C. Yu, J. Appl. Physiol. 33, (1972). 1A.A. DeKock. J.A. Iiadel. S. M . H.J.Y. Colebatch and C . x Olsen. J. A.. m l .physiol. 21; 185 (1906): J.A. N a d e r I n A s t h n a : Physiology, hmnopharmacoloFy and Treatment, ed. bj K.’!. Austin and LT- Licbtenstein. Academic Press. N.Y.. D. 3. 1973. C.2. &own-Sequard, C.?. Soc: €601. 37, 354 [1085): J.R. Clay, and H.C. flossink, m h n l . 78 544 (1964). J.A. Zernandez, h e r . €rev. fieL2:hiis. 93, 7811966). S. I t o and D.% Aviada, J. Pharmacol. ,xp. - + e r . 161, 197 (1968). A.A.
.
64 65. 66 0 67
J.S.
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n6
Sect. I1
94 -
.
- Pharmacodynamic Agents
Clarke, Ed.
79 P. Gross, E.A. E f i t s e r , E. Tolker, Id. Babyak and 1:. Kaschak, Arch. 3nviron. h e a l t h 11, 50 (1965). ao. h.2. C i s s , !!.P. k i n k e l and 2.. Leeds, R.oc. 5oc. 4xp. Biol. ? i d . 134 157 (1970). 81.. V, -0 B Ilhss D.X. l'eranzg, f lieinhavm and P. 'imhel, h e r . R e c f i e s p . ?is. 104, 5% 719711. S n i d e r , J.A. Hayes, A.L. Korthy and L.P. Lowis, Amer. i{ev. Reap. D S s . 100, 820
.
n 40 11973).
133. C.3. Laurell, and S. k i k s s o n , Scand. J. Clin. Lab. I n v e s t . 15, 132 (1963). Aviado, &oh. dnviron. H e z t h , 15, 332 (1367). 84. P. Palecek, 1.1. Palecekovs and Arch. Z w i r o n . Heanh 35., 332 85 I.P. G o l d r i n g , I. Gresnburg and L T i . Ratner.,
.
86 0
87 88. 89. 90.
(1967)
f.'UahDakOm, J.C. Hog A.J. Wooloock, A,?, fm us, P.T. bhcklem and Wdd. f h w l b e c k , h r . Rev. &sp. Dis, 102, 77P, (19707. 458 (1971). E.J. Caldwell, J. Appl. Physiol. D.M. Aviado, and G.R. MDXinnq$ P h a r a Res. Corn. 1, H.S. Giles and J.C. \ i i l l i a m s , Lne Pharmacologist 17, ii.i. G i l e s , 5.2. l r i l l i a m s and M.F. E'inkel, k o c . ?&.
H,
(1973) 91. 2. Ivjandl, S. Keller, T. Xosannah and C. Blackwood In: Pilmonary 3Fphyaem and P r o t e o l y s i s , ed. by C. Mittman, pp. 439-447, A c a d k c Press, New York, 1972. StuartHarris
92. C.H.
and T. Sanly, O x o n i c Ih-ooohitis .?mphysema and C n r PulTonale.
J o b 'Aright and Sons, Ltd. 3 i s t o 1 , h g l a n d , 1957. Bickerman, Fulmonary Emp\Ayserna, The Viilliams and b i l k i n s 93. A.L. Barach and K.A. Co., Baltir?ore, 1956. Clark, Lancet, I, 1361 (1972). 94. T.H.H. Rhalla, S.N. Singha1 and A.L. B:t,lPr, Bit. Meed. J. 95. S. Lal. D,t& Harris, 311, 3j4 (1972). 96 -- $""ran, P.J. Cooper, !I,?. Home and T.ti,B, Crant, Brit. :led. J. 2, 205 (lY/JJ* Ashurst, V. Kirton, Y+.T. SiTpson, HalL-Smith, R.A. h b i n P.J 37 I.w, Caldwel1, S.P. G.h. V i i l l i a m s , &it. J. Dermatol. 80, 11. 13.966). S i n c l a i r , C. Skinner and K.'$;.V. 98. J.W. Laddie, G.R. P e t r i , I.W. Heid';-d.J.kI. Pa-, Lancet, I691 (1973). ii.s, Smith and E. h!iddleton, J. Allergy Clin. I~munol., 52, 13 A.ASherman, 99 N,? $. LIYISlr
m.
.-
-
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100.
his.
k t h , N.A.
Sherman and E. I'iddleton,
J. A l l e r g y Clin.
Impunol.,
2, 328
(1973). 101. L.Q. Pun, M.W. McCulloch and 1d.J. Rand, Em. J. Phnrmacol. 22, 162 1973). 102. &A. Idathe', and B. Levine, J. A l l e r g y Clin. h u n o l . , 53, 1% (19741. 103. k.P. &a 'e, H.C. Murphy, M.L. Karnovsky and K.Y. Austen, J. Lmunol. E, 760 (1978. 104. 5. Augstein, J B. Famner, !.B Lee, I.. Sheard and %To T a t t e r s a l l , Mature New Biol. 245, 215 11373). 105. C.R. m s o l v i n g N.L. ~.$.uro and A.A. C a r r , T!ie D h a r m c g l o g i s t , 15, 221 (1973). 106. Lor,. Smith, S. f o z z i and J. P e t i l l o , J. Allerp i l i n . +mnmol. 84 (1974). 107. E.S.K. Assem, Int. Arch. Allergy 45, 708 (19733. 108. J.R. P f i s t e r , Rob. F e r r a r e s i , I . n a r r i s o n , 1t.H. Rooks, A.P. Doszkovski, A. ?2n Horn and J.H. F r i e d , J. Bded. Cham. 15, 1033 (1972). lC9. 2.J. Kusner, 3. Dubnick, and D.J. G e i g , J. Fharmacol. Expo Ther., 2 4 , 41 (19731. 110. 3 . S . Thomson and D.P. Zvans Clin. gxp. I m u n o l . 13, 537 (1973). 111. H.G. Johnson, and C.R. van &out, Proc. SOC. Cxp. n o l . h:ed., 143, 427 (1973). lPr L.W. Chakrin, 3.T. Walz, A. b!isher, J, Mengel, D. Yo.mg, V. m o r n e and J.R. Wardell, The Pharmacologist l5, 220 (1973).
z,
Section 111
- Chemotherapeutic Agents
George H. Warren, Wyeth Laboratories, Inc., Philadelphia, Pa.
Editor:
Chapter 11. Antibiotics
F. Leitner and C. A. Claridge, Bristol Laboratories, Syracuse, N. Y.
-
General The molecular basis of antibiotic action was discussed in a monograph1 and at a symposium.2 The proceedings of several symposia were published, each devoted to a single antibiotic3'5 or drug combination.6 A collection of papers on the clinical evaluation of amoxicillin7 and a preliminary report on the pharmacokinetic properties and therapeutic efficacy of pivampicillin8 appeared. An issue of "Seminars in Drug Treatment"9 contains reviews on adverse effects of antibiotics98 and the treatment of various bacterial infe~tions.9~'~A therapeutic guide to selection of systemic antibacterial agents was prepared.10 A review appeared on the nephrotoxicity of antibiotics and the therapy of infections in patients with impaired renal function.1l The synthesis of B-lactams12, the allergenicity of penicillinsl3, the biological properties of semisynthetic penicillinsl4, and the chemical and biological properties of ansamycinsl5 were reviewed. Several antibiotics16a-d and antibacterial agents16esf are included in the first two volumes of a new serial publication16 on physical, chemical, and biological properties of selected drugs. The concentration of antibacterial drugs in interstitial fluid was determined by a new experimental technique.17 The molecular properties of R-factorsl8-20 and the biochemical mechanisms of resistance to antibiotics21 were discussed.
-
Infectious resistance to antibiotics Plasmid-borne antibiotic resistance was demonstrated in Streptococcus faecalis22-24 and Bordetella bronchisepand evidence for its conjugal transfer was adduced.24,25 The curing of an R-factor from Escherichia by trimethoprim was achieved.26 R-factors carrying multiple copies of a penicillinase gene were identified.27 An R-factor mediated resistance to penicillins not involving p-lactamase occurred under anaerobic conwas described.28 R-factor transfer in E. ditions simulating those prevailing in the mammalian gastrointestinal tract.2b Transfer of R-factor in the human gut from an ingested donor strain to resident organisms was detected, but on1 when the host was treated with therapeutic doses of a relevant antibiotic. 50 The authors confirmed the identity of the plasmid in the donor and recipient strains by DNA/DNA hyb r i d i ~ a t i o n .The ~ ~ transfer of a plasmid carrying a €3-lactamase marker from Klebsiella aerogenes to Pseudomonas aeruginosa occurred in the burns of a patient .-32
-
a
-
An intense effort of late to devInfections caused by obligate anaerobes elop anaerobic methodology €or use in the clinical laboratory resulted in numerous publications on the c ~ l l e c t i o n ~and ~ - transportation33-36 ~~ of specimens, the c ~ l t u r e ~ sand - ~ i~ d e n t i f i ~ a t i o n ~of ~ r or ~ ~ enisms, and the determination of their antibiotic susceptibility profile.82-47 Several lab-
96 -
Sect. I11
- Chemotherapeutic
Agents
Warren, Ed.
oratory manuals48-50 appeared on this subject and part of the proceedings of the Second International Symposium of MicroecologyS1 was devoted to the discussion of anaerobic techniques. Two drugs were tested against infections with anaerobes. Clindamycin 2-phosphate, recently approved for parenteral use, was highly effective against various infections caused by Bacteroides, such as septicemia, peritonitis and abscesses, including a brain abscess.52-54 During acute meningitis, the drug reached bactericidal concentrations in the CSF. 54 Metronidazole was bactericidal for all 77 strains of Bacteroides fragilis against which it was tested, at concentrations obtainable in the serum following normal therapeutic dosee.55,56 Initial clinical experience with this drug in treating deep-seated anaerobic infections showed promise. 57
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The effect of FL 1060 (la), a 60-amidinopenicillanic acid, on @-Lactams was intensely investigated. The morphological changes elicited b E,. this compound differ from the classical penicillin-induced response.58t58 The three enzymes sensitive to penicillins, i.e., peptidoglycan transpeptidase, D-alanine carboxypeptidase, and endo eptidase, were not inhibited at bactericidal concentrations of FL 106060~6, but the formation of peptidoglycans covalently bound to the cell wall or to lipoproteins was impaired.60 A number of pivaloyloxymethyl 6-W1-cyanoamidinopenici11anates (lb) were prcpared.62 The antibacterial activity of the corresponding free acids, obtained by enzymatic hydrolysis of the esters, resembled that of structurally analogous penicillins.
P
V (&) was resistant to Bacillus cerand moderately active against gram-positive organisms.63 Several 2-acetoxymethyl penicillins ( 2 a . 9 6 4 and 2-spirocyclopropyl cephalosporins (3)65 were prepared. Various chemical procedures were developed for the 7(6ya-methoxylation of p-lactam antibiotics.66-72 Conversion of penicillins to cephalosporins was achieved by new synthetic routes. 73-76 Some [2,3]-fused tricyclic cephem derivatives (&-Q were synthesized.77,78 Compound 4c displayed significant antibiotic activity, 78 Racemic cephalothin, 7-methoxyce halothin, and cefoxitin were obtained by total synthesis of a novel type.yy-81 A nuclear analogue of a 7-methylcephalosporin prepared by total synthesis, lacked antibiotic activity.82 A carbon analogue of penicillin
eus p-lactamase
(z),
The asymnetric incorporation of valine into penicillins N and V and cephalosporin C was demonstrated by incubating the producing cultures with 3-chiral ~ a l i n e s84 . ~ ~The Pa-methyl of the penicillins and the cephalosporin 3-methylene derive from the same methyl group of the amino acid. The activity of selected cephalosporins against Mycobacterium tuberculosis H37Rv was correlated with their structure.85 Two other studies relating activity to structure were undertaken, one on 7(R)-mandelamidocephalosporanic acid&, the other on a-sulfopenicillins.8~ AmoxLcillin was effective and well tolerated in a variety of infections: chronic respiratory diseases caused by Haemphilus influenzae i n children88; urinary tract infections due to gram-negative bacilli or S,.
Chap. 11
Antibiotics
-H b
NC-N=CR-NH-
Leitner, Claridge
97
-H
a
-CHiOCOC(C;H,S
COOH
3
5
faecelis89,90; uncomplicated gonorrhoea (when administered in combination with probenecid).9l The serum concentrations of ticarcillin after parentera1 administration to patients suffering from neoplastic diseases were comparable to those obtained with cerbenicillin.92 However , the serum inhibitory activity against p. aeruginosa in vitro was greater with ticarcillin. Given by iv infusion, ticarcillin was effective in 18 of 20 patients with Pseudomonas infections, even in the presence of severe granulocytop e n i a y
Sect. I11
98 -
-
Chemotherapeutic Agents
Warren, Ed.
Cefamndole was more active in vitro than cephalothin, cephaloridine or cephalexin against Haemophilus and gram-negative bacilli. Moreover, its spectrum covered many organisms resistant to the other cephalos orins, such as strains of Enterobacter and indole-positive Proteus species. 84 Cefoxitin is highly resistant to degradation by most 0-lactamases from gram-negative organisms.95,96 In human volunteers the mean serum concentrations of cefoxitin reached peak values of 10.9 and 22.5 Bg/ml after im administration of 0.5 and 1 g, re~pectively.9~About 90% of the administered dose was recovered in the urine. Cefazolin was approved for use in the U.S. Recent reports confirm the high efficacy and tolerance of this compound in the treatment of a variety of severe infections, including staphylococcal and bacillary endo~arditis.~7-99
-
Amikacin (BB-K a), when compared to other aminoglycosides, Aminoglycosides shows a broad spectrum of activity100 that includes a number of strains of E. Klebsiella, Enterobacter, Pseudomonas and Serratia resistant to tobramycin, gentamicin and kanamycin.101 It may present no advantage & vitro over gentamicin.102 However, in a study involving amikacin, gentamicin, tobramycin, sisomicin and butirosin, determination of an inhibitory index (ratio between mean peak serum levels and concentration required to inhibit 213 of each species) showed that the index for amikacin was the largest (with the exception of 11. aeruginosa, where all agents were equally effective).l03 S ectinomycin was the least active when compared to these aminoglycosides~~4 and its major value may be in the treatment of gonorrhoea in patients allergic to penicillin.105 Pharmacokinetic studies on amikacin and tobramycin were reported. 106-108
e,
Mixtures of a penicillin with tobramycin or gentamicin were synergistic in vitro against enterococci109,110 and a spectrum of gram-negative clinical isolates.111 A similar combination therapy with a penicillin or cephalosporin and these aminoglycosides has been reported efficacious in gram-negative infections of cancer patients. 112 However, there were two reports of acute renal failure associated with combined gentamicin-cephalothin therapy in patients with normal renal function.113,114 The chemical procedures for the synthesis of 4"-deoxygentamicin C115 , configurational and positional isomers of amikacinll6 butirosin B117, 5"amino-5"-deoxybutirosin~~~, 3 ' ,4' -dideoxybutirosin Bli9 , 3 -deoxykanamycin B (tobramycin)l*O, 6'-amino-6' -deoxylividomycin B, 6'-deoxy-6'-methylaminolividomycin B and 6 ' -deoxy-6 ' (2-hydroxyethylamino)lividomycin B12 1 have been described. l-N-(L-T'-Amino-a-hydroxybutyryl) derivatives of kanamycin B, 3' ,4'-dideoxykanamycin B122, lividomycin A123 and 3' ,4'-dideo~yneamine12~ were also synthesized. The total syntheses of dihydrostreptomycinl25 and of kasugamycinl26, the latter through the methyl N,N' -diacetyl-a-d-kasugaminide, were described. Degradation of butirosin yielded l-N-aminohydroxybutyrylneamine, a compound having antimicrobial activity. Substitution of the aminohydroxybutyric acid in butirosin with other acyl groups produced a new 8 eries of active compounds. 1 2 7
-
New active aminoglycosides were produced biosynthetically from mutant cultures of Bacillus circulans (butirosin)128 and Streptomyces ribosidificus
Antibiotics
Chap. 11
Leitner, Claridge
99 -
(ribostamycin). 129 These mutants could form antibiotic only when Z-deoxystreptamine (2-DOS) was added exogenously. Substitution of the 2-DOS with streptamine and streptidine in the former culture and with 1-N-methyldeoxystreptamine, streptamine and 2-epi-streptarnine in the latter culture yielded new antibiotics. A similar mutant of Streptomyces kanamyceticus (kanamycin) yielded two new kanamycin analogues by the addition of 1-N-methyldeoxystrep tamine and 2 epi - 8 treptamine. 129 The successful incorporation of str eptidine by the B. circulans mutant and 1-N-methyldeoxystreptamine by 2. &sidificus and g. kanamyceticus is in contrast to the production of hybrimycins, where a rigid requirement for an unsubstituted 1,3-diamino function on the aminocyclitol was reported.130 The scope of enzymes which inactivate aminoglycosides has been widened with the reports that neomycin-kanamycin which phosphorylates the 3'-hydroxyl phosphotransferase I (from &. group of the kanamycins, also phosphorylates the 5"-hydroxyl group of lividomycin A.131 An enzyme in p. aeru inosa was found to phosphorylate only the 5"-hydroxyl group of ribostamycin 3-N-Carboxymethylation of ribostamycin was induced by the producing culture (5. ribosidificus)l33, where the acetic acid is introduced via the methyl group, the first biochemical report of this reaction. Gentamicin acetyltransferase 11 catalyzes the acetylation of the 2'-amino group of the aminohexose ring of gentamicin, tobramycin, kanamycins and related antibiotics.134 Another new enzyme, discovered in E. & resistant to gentamicin, acet lates the 3-amino group of the 2-deoxystreptamine moiety of gentamicin C1.1$5 Antibiotic inactivating enzymes have also been discovered in actinomycete cultures.136 Two antibiotics related to the butirosins were reported as minor components in that fermentat ion. 137
-
w),
+
The elucidation of the structures of the members of the nebramycin complex has continued. Factor 2 (now called apramycin) has the unique structure (g)l38, factor 4 ( 7 a ) is 6"-O-carbamoyl kanamycin B, factor 5 is kanamycin B (7b), factor 5' is 6"-O-carbamoyl tobramycin (7c) and factor 6 (E)is tobramycin. Factor 5 and tobramycin are not normally detected in fermentation broths but arise from the acid- or base-catalyzed hydrolysis of their carbamoyl derivatives.139
Sect. I11 - Chemotherapeutic Agents
.-100
Warren, Ed.
The structure-activity relationships among the aminoglycoside antibiotics, based on bacterial inhibition and misreading of polypeptide synthesis, was discussed.140
A thin layer chromatographic assay for the separation and quantitation of the members of the gentamicin complex was detailed.141 In addition, biological142 143 and radioimmune assays144 for the measurement of members of this group were reported. A rapid assay for serum levels of amikacin and all aminoglycosides with a free 6'-amino group using the kanamycin acetylstrain carrying thc R-factor for this enzyme transferase from an E_. was de~cribed.1~5
-
Rifampicin appears to have imnunosuppressive effects in some Ansamycins tubercular patients when used in conventional doses.146 However, it is still a highly useful drug in the treatment of tuberculosis i n combination with ethambutoll47 or streptomycin plus isoniazidl48, but long term treatment of chronic infections such as trachoma with rifampicin as the sole drug appears undesirable because of the emergence of resistant mutants. 149 Rifampicin action was shown to be caused by its tight binding to RNA polymerasel50, but no genetic effects were shown in Drosophila or in human leukocyte chromosomes i n vitro.151 Rifamycin biogenesis probably occurs through a condensation of propionate and acetate units.152
-
Chloramphenicol is considered the drug of choice for Chloramphenicol treatment of typhoid fever, but recently R-factor mediated resistant strains of Salmonella typhi have appeared in epidemics in Mexico1S3, Los Angelesl54 and Viet Nam.155 In staphylococci, chloramphenicol resistance has been shown to be caused by acetyltransferases of 4 distinct types, each existing as a tetrameric protein with a molecular weight of 80,000 and an identical subunit size of 20,400.156 Studies on the biosynthesis of chloramphenicol by Streptomyces v s zuelae, based on a carbon-by-carbon degradation of the antibiotic using labeled glucose were consistent with the shikimate pathway of aromatic biosynthesis. 15$ The structure-activity relationships of 37 chloramphenicol derivatives were studied. The trifluoro derivative is 1.7 times more active than the parent.158 against g.
-
Lincomycin A detailed pharmacokinetic study of clindamycin 2-phosphate i n man was reported.159 Both lincomycinl60 and clindamycin161 were shown to cross the placental barrier in pregnant women in therapeutic concentrations. Intrathecal administration of lincomycin was successful in treatment of 86% of patients with meningitis of gram-positive and gram-negative origin.162 Reports on the successful use of clindamycin 2-phosphate in a variety of infections continue to mount.163-167 An oral dosage form, clindamycin 2palmitate, was re orted to be well tolerated by man168 and useful in pediatric practice. 169, 170 2-Deoxylincomycin has the same spectrum as the parent but only 1% of the activity. Its P-anomer and 2-0-methyllincomycin were only marginally active.171 However, 7-0-methyllincomycin has the same antibacterial spec-
Antibiotics
Chap. 11
Leitner, Claridge
101
trum as lincomycin and 1.3 to 1.8 times the in vitro activity. It showed equivalence to lincomycin in mouse rotection tests against Staphylococcus aureus on subcutaneous inje~tion.17~A series of lincomycin 2-monoestersl73 and 2,7-di(alkyl carbonate) esters174 was synthesized. Good antibacterial activity was observed with monoesters of chain lengths from C 4 to C16. All of the 2,7-diesters were inactive in vitro although many were hydrolyzed by the esterase in rodent serum but not by human serum. N-Demethyllincomycin was produced by Stre tom ces lincolnensis in fermentations to which sulfonamides were added
TrK-y-
New celesticetins, members of a class of antibiotics related to the lincomycins, have been produced by mutants of Streptomyces caelestis. NDemethylcelesticetin and N-demeth 1-7-O-demethylcelesticetin were less active than celesticetin itself.lq6 A procedure for separation by gas chromatography of closely related members of this family as the trimethylsilyl ethers was described. 177
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Macrolides Chemical modifications of erythromycin A yielded the 8-hydroxy derivative, having one-half the antibacterial activity of the parent compound but 500-600 times greater acid stability.178 A similar derivative of erythromycin B was also less active.179 In vitro and in vivo antibacterial activities of 9(S)-erythromycylamine (the C9 amine counterpart of 9(S)-dihydroerythromycin), of a series of aliphatic and aromatic aldehyde condensation products of 9(S)-erythromycylamine, and of 9(R)-erythromycylamine were reported. N-Benzylidene-9(S)-erythromycylamine was absorbed better than erythromycin after oral administration in mice and dogs, but gave lower blood concentrations in human subjects.180 Chemical modifications of the 17-membered macrocyclic antibiotic lankacidin group showed that some of the lankacidin C monoesters (particularly the propionate) had little in vitro but good in vivo activity, with wide tissue distribution. 181* 182-&ridomycin has also been described183 and modified in a similar manner, with the 9-propionyl derivative being the most effective for in vivo use.184 The structures of megalomicins A, B, C1 and C2 have been elucidated along with the description of a new dimethylamino sugar, 2,3,6-trideoxy-3-dimethylamino-D-lyxo-hexopyranose, c o m n to all of them. 185-187 The biosynthetic pathway to the platenomycins was studied through the use of blocked mutants and cosynthesis experiments.188
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Peptide antibiotics The structures of a m p h ~ m y c i n ' ~and ~ the enduracidinsl9U were elucidated, and the complete chemical synthesis of etamycin was accomplished. 191 A new ring closure method involvin diethyl phosphite was reported useful for the synthesis of valinomycin. lgl The 3-hydroxypicolinic acid fraction of this antibiotic was shown to originate biosynthetically from L-ly~ine.19~The total cell-free biosynthesis of bacitracin A was accomplished in a reaction mixture containing urified enzyme from Bacillus licheniformis, ATP, Mg*, and 10 amino acids.164 This synthesis was not affected by blockers of ribosomal protein synthesis. Edeine D, one of the complex of 4 edeine antibiotics, was shown to contain glycine, 2-hydroxy-3-aminopropionic acid, 2,6-diamino-7-hydroxy-
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azelaic acid, 2 ,6-diamino-A7-azelaic acid, 2,3-diamino ropionic acid, spermidine and B-phenyl-P-alanine, a rare amino acid. 185 A new spectrophotometric assay more suitable than the microbiological assay for measurement of the gramicidin and tyrocidine components of tyroA gas chromatographic procedure for the assay of thricin was described. colistin was 0ut1ined.l~~ Polymyxin given prophylactically by aerosol was found to reduce significantly the incidence of colonization of the upper respiratory tract by nosocomial gram-negative bacteria in post-operative patients and in patients wi th respiratory failure .I98 Endurac id in, administered im, was effect ive against urinary tract and dermatological infections, but not against chronic osseus infections, caused by methicillin-resistant S. aureus. 199
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Doxycycline was reported effective in treatment of urinary Tetracyclines tract inf ections200, chronic trachoma201, typhus202, and respiratory tract i n f e ~ t i o n s . 2 ~Minocycline, ~ likewise, was useful in the treatment of tetracycline-resistant staphylococcal infections204,205 and urinary tract infections.206 It was proposed that minocycline is effective against tetracycline-resistant staphylococci because of its ability to penetrate the cells more readily and to reach inhibiting concentrations at sensitive reaction sites .207 Increas ed incidence of tet racycline-resis t ant pneumococci has been reported.208 A tetracycline-novobiocin combination proved syner istic in the treatment of melioidosis caused by Pseudomonas pseudomallei.2 9 Doxycycline, and presumably other tetracyclines, were shown to interfere with the bactericidal effect of human complement in vivo.210
8
The total syntheses of anhydrochlorotetracycline211 , d,l-7-chloro-6-deoxyand d, 1-4tetracycline, d, l-7-chloro-6-demethyl-6-deoxytetracycline~~~, amino-7-chloro-2-N-methylcarbamyl-2-decarbamyl-4-dedimethylamino-6-deoxytetracycline213 were reported. A new fluorometric assay for oxytetracycline in blood and plasma using thiol reagents to prevent decomposition was described. 214
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The structures of a- and $ - l i p ~ m y c i n ~oleficin2", ~~, and Miscellaneous manumycin21/, polyene antibiotics active against gram-positive organisms, were elucidated. Two pigments with growth inhibitory activity for positive bacteria were isolated: roseoflavin from a Stre tom cea2lfz; AB-64 from a variant of Actinomadura roseoviolacea. 2 The structure of the former was fully and that of the latter partially determined. Oxamicetin, closely related to amicetin, was isolated from Arthrobacter oxamicetus sp. n. and its chemical and biological properties were described.220,221 The compound is moderately active against a variety of organisms, including acid-fast bacteria. A study on kinamycins, correlating activity to structure, was done.222 The total synthesis of nybomycin and deoxynybomycin was accomplished.223
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REFERENCES
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104 50.
51. 52. 53. 54. 5s. 56. 57 58. a
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. 93.
94. 95. 96. 97. 98.
99. 100. 101. 102.
103. 104. 105. 106.
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V.K. Si-wn, E.U. Hosinger 6: V. M l e r c z 7 , AAC, 3.445,(1973) X.C. h'nber, S.R. Kestenfelder & P.O. ?udsen, AAC, 2,469,(1973) R.D. L i t k c , C . Reganry, J.T. Clarke h U . M . M . Kirby, MC, 4,5b4,(1973) R. Moellering, Jr., C . jlennersten Q A . J . Veinstein, M C , 3.526,(1973) 111. J. Klasterskg, A . Henri & L. Vandenborre, Am. J. Ned. S c i . , =,13.(1973) 112. J. Klastersky, R . Canpel h D. Daneati, Cancer, 2,331,(1973) 113. J.P. P i l l a s t r c , R . Lauwnler, C. €iumbert, D . Dubols, J. Metayer, A . Dclpech, J. Leroy & Y. Robert, B r i t . Mcd. J.. 2,346, (1973) 114. D. Kleinknechc, D . Caneval h D. Groz, Lancet, 1,1129,(1973) 115. A.K. Kmllams, H.P. Verruy, D.P. Crow, G. Detrs, M. Tanabe C D.H. Yasuda, J. A n t i b i o t . ,
107. 108. 109. 110.
116. 117. 118. 119. 120. 121. 122. 123. 124. 125. 126, 127. 128. 129.
UO
.
131. 132.
u3. 134. US. 136. 137.
138. 139. 140.
141. 142. 143.
144. 145. 146. 147. 148. 149. 150.
151, 152. 153. 154. 155. 1%. 157.
158. 159. 160. 161. 162. 163.
26,782, (1973) Naito, S. U b g a w a , Y. A b e , S. T d a . K. PUjiSdWa. T. Hiyaki, 8 . Koshiyama, H . O h k m 6 8 . Kawaguchi, J. Antlbiot.. 26,297,(1973) E. Akita, I . Horluchi 6 S . Yasuda, J . Antibiot, 26,365,(1973) T.P. Culbertson, G.R. khtson & T.H. Hnakell, J. A n t i b i o t . , 26,790,(1973) D. Ikeda, T. Tstrchlya, S. :'u?rawa, H . Ihezawa h H. Ilanoda, J. A n t i b i o t . , 26,307,(1973) I. Takagi, T. Hiyoke, T. Tsuchiys, S. Lkezawa C H. W t a w a , J . A n t l b i o t . , 26,403,(1973) I . Wetanabe. T . fsuchign. S. U z a w a 6 H. Ihrezawa, J. Antibiot., 25,802,( 1 f i 3 ) S. Kondo, K. Iinrrsa, 11. I n s a m t o , K. U c d a 6 H. Wzawa, J. Antiblot., g , 4 1 2 , ( 1 9 7 3 ) I. Watanabe, 'I. Tsucbiya, S. Wezawa & H. llmezawa, J. Antibiot., 211,31O,(i973) S. Umezawa, D. Ikeda, T. Tsuchiya 6 H. Vsczawa, J. Antlbiot.. 2&,304,(1973) S . I b u w a , 2. Tsuchiya, T. YdasSaki, H . Sano h 1. T a b h a s h i , JACS, 96,920.(1974) S. Yasuda, T . Ogaoawara. S . KJvaDlta, I. Iwataki 6 T. Natsrrmoto, Tetrahedron, %,3141,(1973) R. Tsukiura, K. Fujlsawa, !4. Konlshi. K. Saito. K. Numata, R. Ishlkava, T. Mlyakl, K. Tomita 6 8. Kawaguchi, J. A n t i b i o t . , 26,351.(1973) X.D. DePuria & C.A. Claridge, Abst. Ann. Meeting Am. Soc. Uicrobiol., Mami Beach, P l a . , 6-11, 1973 U. Kojima 6 A . Satoh, J. A n t i b i o t . , 26,784,(1973) Y.T. S h i e r , S, Ogaua, M. Hickens 6 K.L. Rinehart, Jr., J. Antibiot., 26.551,(1973) 8. Uwzava, H. Tamamoto, X. Yagisawa, S. Kondo, T. Takeuchi h Y.A. Chabbsrt, J. A n t l b i o t . , 26,407, (1973) Kida, S . I g a r a s i , T. O h t a n i , T. Asako, K. l i r a g a & S. n i t s u b a s h i , AAC, &92,(1974) S. Omoto, S. Inouye, M. Kojinm h T. N i i d a , J. Antibiot.. 26,717,(1973) Y. Chevereau, P . J . L . Daniels, J. Davies 6 F. LeGoffic. Blochem., l3,538,(1974) 8 . Ilhmzrwa. H. Tagisawn, I. Hatsuhashl, R. NaagaMW, R. Yamamto. S. Kondo, T. Takeuchi 6 Y.A. Chabbert, J . A c t f b i o t . , 26,612.(1973) A. Benveniste 6 J. Cavies, Proc. Nat. Acad. Sci. USA, 70,2276,(1973) R. Tsukfurr, K . S s i t o , S. Kokaru, H. Konishl h It. Kowaguchi, J. Antlbiot., 26,386,(1973) S. O'Connor 6 L.K.T. Lam, Abst. 165th ACS Meeting, Dallas. Texas, A p r i l 8-13, 1973 K . I . Koch, F A . Davis 6 J . A . Rhoades. J. Antibiot., 26,745,(1973) R. Eenveniste h J. Davies, AAC. &,402,(1973) U.L. Wilson, C. Richard & D.W. Rcqhes, J. Fharm. S d . , 62,282,(1973) D.V. Alcid 6 S.J. Seligrcan, AAC, 2.559,(1973) X.E. Lund. D . J . Blarevic 6 J . H . Fatsen, AAC, 4,%9,(1973) W.A. Mahon, J. Ezer h T.W. Wilson, AAC, 2,585,(1973) N.J. Hnas 6 J . DEVieS, AAC. 4,497,(1973) P. Xukerjee, S . Schuldt 6 J.E. Kasik, AAC, 4.607,(1973) Anonymous, Brit. Ned. J.. 4,568, (1973) Anonymous, Lancet, 1,1331, (1973) A. Kcshishyan, L. HanM & E. Javetz, Nature, =,173,(1973) U.I. L i l l & C.R. Haitriinn, Eur. J. Bio:hem., 38,336,(1973) E. Vogel 6 G . Obe, Zxperiantia, 29,124,(1973) R . J . White. E. M a r t i n e l l i . G.G. Gallo, G. Lancini 6 P. Beynon. Nature, =,273,(1973) R.N. Lawrence, E. C c l d s t e i n 6 P.D. Hoeprich, J A M , 3 , 8 6 1 , (1973) C . Overturf, K . I . Ydrton 6 A.W. Hathien, Jr., Nev Eng. J. Med., 289,463,(1973) T. Butler, N.C. Linh, K. Arnold & M. Pollack, Lancec, 2,983, (1973) L.C. Sands C W.V. Sbaw, AAC. 2,299,(1973) W.P. O'Neill, R.F. Nystrom, K.L. Rinehart. Jr. 6 D. G o t t l i e b , Biochem., 2 . 4 7 7 5 , (1973) C. Reosch, K. NakaaUtO, M. Gorin, P. Denlsevlch, E.R. Garrett, S.H. Henurn-Ackah & C.H. Won, J. Hed. Cham., l6,917.(1973) R.U. DeIiaan, C.H. Mettler, D. Schellenberg h W.D. Vandenbosch, J. Clin. Phsrmacol., 2 , 1 9 0 , (1973) W.H. DuigMn, J. Andrevs & J.D. W i l l l a s r , Brrif. ?fed. J . , 2.75,(1973) A. Philipson, L.D. SaabPth 6 D. Charles, New E n g . J. Med. 2&1219,(1973) I. H i y a u k i , Artne1m.-Porsch., Z3,940,(1973) D. Mildvan, U. Ravreby, B.R. )(eyers 6 S . Z . HirschmPn. H t . Slnnl, J. Mad., 40,7M, (1973)
106 164. 165. 166. 167. 168. 169. 170. 171. 172. 173. 174. 175. 176. 177. 178. 179. 180. 181. 182. 183. 184. 185. 186. 187. 188. 189. 190. 191. 192. 193. 194. 195. 1%. 197. 198. 199. 200. 201. 202. 203. 204. 205. 206. 207. 208. 209. 210. 211. 212. 213. 214. 215. 216. 217. 218. 219. 220. 221. 222. 223.
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W. Sshumer, R.L. Nichols, B . Killer, E.T. %met 6 G.O. HcDorsrld, Arch. Surg., =,578,(1973) A.H. Pontifex & D.R. HcNHuqht, ChA J . , ~ ~ , l O 5 , ( 1 9 7 3 ) V. B a l s c h n d a r , P.J. C o l l i p p h B . J . RLeinq, Clin. Fled., 80,(4),2&,(1973)
A.T. Edmirdson, Ann. Surg., %.637,(1973) A.A. P o r i s t , R.U. DcKaan 6 C.H. W t z l e r , J . Pharmecakin. Biopharm., 1.89, (1973) R.T. P f e i f e r , K . M . CeHaan, W.D. Vandentbsch 6 D. Schtllenberg. Clin. Hed. , 80,(5),21. (1973) W.C.L. Csrr. Curr. Ther. Res., 1',,630,(1573) B. Bannister, JCS Perkin I. p . 3025,(1972) B. Ennnister, JCS Perkin I , p . 1676,(1973) W. Moroaovich, A.A. Sfnkule, P.A. HacKellar 6 C. Levis, J. Pharm S c i . , 62,1102.(1973) A.A. Sinkula & C. Lewis, J . Phai-m. S c i . , J2,1757,(1973) A.D. Argoudclir, L.E. Johnaon h T.R. e k e , J. Antibiot., 26,429,(1973) A . D . Argoudelis, J.H. C o a t s , P.C. LEmaw h O.K. Sebek, J. Antibiot., &7.(1973) T.F. Erodasky C A.D. Argoueelir, J. Antrbiot ., 2 . 1 3 1 , (1973) K. K r w i c k i h A . Zamojski, J. A n t i b i o t . . ?fi,575,(1973) R. Krovicki & A. Zamo]skl, J. A n t i b i o t . , 22,587,(1973) E.H. Hassey, B.S. % i t c h e l l . L.D. Martin h h . Gerzon, J. Hed. Chem., ~ ~ , 1 0 5 , ( 1 9 7 4 ) S. Earada, T. Yaeulzaki, K. Aatano, K. Tsuchiya 6 T. Kishi. J. A n t i b i o t . , 26,647,(1973) S . Harada, S. Tanayarm 6 T. Kiohi, J. A n t i b i o t . , 26,658.(1973) 8 . Ono, T. Rasegnwa, E. Efqashide 6 M. Shibata, f. Actibiot, 26,191.(1973) H. Kondo, K. Ishifuji, K. Tsucniya, S. Goto h S. Kuwahara, M C , &,156,(1973) A . K . Mallams, JCS Perkin I, p. 1369,(1973) R.S. J a r e t , A.K. M 8 l h a S h H. Reinann, JCS Perkin I, p. 1374,(1973) R.S. Jaret, A.K. Mallams h H.P. Vernay, JCS Perkln I, p. 1389.(1973) T . Furumai, Y. Seki, K. Takeda, A . Kinurraki 6 H. Suzuki, J. Antibiot., 26,708,(1973) M. Bodanszky, G.P. S i g l e r 6 A . 3odansrky. JACS, 9S,2352,(1973) A. I v a s a k i , S. Horli, M. h s a i , K. Nftuno, J. Uayaoagi & A . niyaka, Chem. Phara. Bull., 2, 1184, (1973) J.C. Sheehan h S.L. k d i r , JACS, 95,875,(1973) H. Rothe 6 W. Kreiss. Angev, Chem. I n t e r n a t . E d i t . , lZ,lO12,(1973) D . J . Hook & L.C. Vining, JCS Chem. Corrm., p. 185, (LE73) P. Pfaender, D. Spccht, G. lieinrich, E. Schwarr, E. Kuhnle h M.M. Simlot, PEBS Letters, 32,100, (1973) Wojciechovska, J. Ciarkarski, H. Chmra 6 E. b r o w s k i , Experientfa, 28.1423, (1972) E.. Ivashkiv, Biotech. Bi0er.g.. lS.821, (1973) A.A. Al-Khayyat & A.L. Aronson, Chemotherapy, l9.75, (1Y73) S . G r e e n f i e l d , D. Teres, L . S . Burhnell, J. Hedley-khyca h D.S. Peingold, J. Clin. Invest., 52,2935, (1973) H. Peromct, E. Schoutcns h 1 . Yourascowsky. Chemotherapy, l9,53.(1973) C.E. Cox, C l i n . )led., 8f),(10),30,(1973) I. Hoshivara, H.B. Ootlcr, L. Ram, F. C i g n e t t i , V.R. Colermn & E. Jawetr, JAHA, 224.220, (1973) J. Huys, J. Kayihigi, P. Preyens h G . Vanden Berghc, Chemotherapy, l8,314,(1973) G.A. Pankey, C l i n . Ned., 80, (9),12,(1973) H. Lades, C l i n . Xed., 80, (9),31. (1973) A . Hoagland h L.G. Smith, Clin. Ued., 60,(4),22,(1973) P. Veysoier, Y . Bouasougant & C. Mariel, Path. Biol., 2l,59,(1973) N.A. Kuck C H. Forbes, AAC, 2,662,(1973) K.V. Copalakrishna & P . I . Lerner, Amr. Rev. Resp. Die., ~ , 1 0 0 7 , ( 1 9 7 3 ) 0. Calabi, J. M d . H i c r o b i o l . , 5,293,(1973) A. Forsgren h H. Cnarpe. Nature New Blul., 3 + , 8 2 , ( 1 9 7 3 ) R. Muxfoldt, H. Capp, J.E. Kaufran, J . Schneider, P.E. Hanren, A . .%saki h T. Geiscr, Angew Chm. I n t e r n a t . E d i t . , &497.(1973) W. Martin, H. Gartung, H. t i r h c h 6 W. Ddrckhcirrrr, T e t r a . Lett., p. 3513,(1973) H. Urbach, H. Bartunq. W. E a r t i n h W. Durckheinrr, T e t r a . Lett., p. 4907,(1973) B . S c a l e s ti D . A . Assinder, J . %am. S c i . , 62,913.(1373) K. Scbabacher 61 A . b e c k . T e t r a . Lett., p . 2691.(1973) G. Horvith, J. Gyimesi h 2. H6:hesfalvi-Va~na. T e t r a . Lett., p. 3643.(1973) K. Schrdder h A . Zeeck. T e t r a . Lett., p. 4935,(1973) S. Otani, H. Takatsu, M. Nakano, S, Kasai, R. Hiura h K. t!atiui, J. Antibiot., 27,88,(1974) A . Tanura, R. Puruta, H. Kotnni h S. Naruto, J. A n t i b i o t . , 26,492,(1973) M. Konishi, M. Ximeda, H. Tsukiura. H. Ysmmoto, T. IIoshiya, T. n i y s k i , K. Pujioam, 8 . Koshiyama 6 H. Kawaguchi, J. A n t i b l o t . , 26,752,(1973) H. Konishi, M. F a r u i s h i , T . Tsuno, B. Tsukiura h H. Kawaguchi, J. Antiblot., 26,757,(1973) S. h u r a , A . Nakagawa, H. Yameda, T . Hat., A . Purusaki 6 T. Watanabe, Chem. Pharm. l u l l , 21.931, (1973) E M . Forbis h K.L. R i n e b r t , Jr., JACS, 95,5003,(1973)
Chapter 12. Antifungal Agents Smith Shadomy, Medical College of Virginia, Richmond, Virginia
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Reviews The changing clinical setting and etiology of fungal endocarditis was reviewed, with particular reference being made to the changing pattern of this disease as seen in narcotic addicts and following cardiac surgery.192 The renal toxicity of antibiotics, including that of amphotericin B, also was reviewed. The therapeutic indications, pharmacology, and mode of action of amphotericin B y nystatin and candicidin were the subject matter of a new text on fundamentals of ~hemotherapy.~ Methods - A standardized procedure for in vitro susceptibility testing with 5-fluorocytosine (Ancobon) which ermitted both more accurate and more reproducible results was described.! Two new experimental in vivo models were reported; one dealt with cladosporiosis in mice treated with 5-fluorocytosine6 and the second with experimental urinary tract infections in rats In the former model, 5-fluorocytosine procaused by Candida albicans tected experimentally infected animals against cerebral disease caused by Cladosporium trichoides but did not clear the tissues of infection; in the albicans were treated successfully with latter model, infections due to 5. ~-~amphotericin B, and the model appears useful in the study of anticandidal agents.
.
Improvements were reported in cultural techniques available for the detection of candidemia in man by use of a lysis-filtration procedure.' Improved serologic diagnosis of fungal infections was the subject of several reports. A slide latex agglutination test for use in sporotrichosis was described as being specific, sensitive and rapid.9 Similarly, an immunodiffusion test capable of detecting approximately 80 per cent of proven cases of blastomycosis was reported .lo Reevaluation of a previously reported medium for identification of dermatophytic fungill revealed that several nondermatophytic fungal pathogens could be misidentified as dermatophytes thus possibly resulting in the selection of totally inappropriate therapy.i2 A method for coupling antibiotics, including amphotericin B, to cellulose and cellulose carbonate to produce active, insoluble agents was described.l3
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Clinical Experience Amphotericin B continued to be the drug of choice in treatment of serious or systemic fungal infections. It was reported earlier that while either 2-hydroxystilbamidine or amphotericin B was effective in treatment of noncavitary pulmonary blastomycosis, the latter drug was the more effective agent in treatment of this infection in those cases with cavitary disease or with systemic organ involvement.I4 Amphotericin B also was described as useful in the treatment of canine blastomycosis.15 A brief comparative study of the pharmacology of amphotericin B failures in treatment of cryptococcal meningitis, histoplasmosis and blastomycosis was reported.16 The authors of this study attribute the higher failure and relapse rates observed in patients with cryptococcal disease treated with am-
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photericin B to the lower ratios of in vitro minimal inhibitory concentration (MIC) to serum drug level as compared with similar ratios recorded in the other, more successfully treated infections. Continuous bladder irrigation with amphotericin B was reported to be useful and without toxicity in management of candidal urinary bladder infections.I7 Low nontoxic doses of amphotericin B (10-355 mg given over 4-18 days) were reported as efficacious in treatment of mucocutaneous and systemic Candida infections exclusive of meningitis and endocarditis.I8 There was continued interest in the clinical use of clotrimazole (Bay b 5097), a compound previously described as being broad-spectrum and effective vitro, but without effect orally in vivo against systemic fungal pathogens.l’ssz6 In one study, topical clotrimazole was reported to be well tolerated and superior to a drug-free vehicle in treatment of dermatophytic infections.21 In contrast, it also was reported as being ineffective and associated with gastrointestinal side effects when given orally in the treatment of mycetoma due to Madurella mycetomi and paranasal aspergilloma, although it was said to be effective topically in treatment of candidal paronychia.22 Other investigations reported late in 1972 described limited but successful results with clotrimazole in treatment of urinary tract infections due to Candida albicans23 and chronic mucocutaneous candidiosis24 in infants and children. Miconazole, a 8-substituted l-phenethyl-imidazole (R 14889, 1-{2,4dichloro~-[(2,4-dichlorobenzyl)oxy]phenethyl~imidazole) with broad spectrum antifungal activity25 was evaluated both. in normal individuals and clinically. It was reported as being effective topically in treatment of both dermatophytic infections26 and locally in treatment of vaginal candidiasis.26,27 Pharmacological studies showed that oral miconazole, 3 g/day, produced no hematologic, biochemical or cardiac abnormalities and was capable both of reducing the fungal flora of sputum and feces and of producing moderate blood levels; it was not absorbed through skin or mucous membranes.28 Studies continued with two other topical agents for use in treatment of dermatophytoses. Tolnaftate (Tinactin), as a 1 per cent powder, was found effective in both prevention and treatment of tinea pedis caused by Candida, Trichophyton and Epidermophyton.29 Haloprogin (Halotex), used topically as either a 1 per cent solution or as a cream, was found to be more effective than a placebo30 and comparable to tolnaftate3l in the treatment of experimental dermatophytic infections (Trichophyton rubrum) and naturally occurring human infections. Additional experience was gained both in the clinical use of and pharmacology of 5-fluorocytosine, Oral 5-fluorocytosine was found beneficial in treatment of both meningeal and pulmonary aspergillosis although relapses occurred in some patients with chronic cavitary disease and some resistance to the drug was observed.32 It was reported to be effective in treatment of urinary tract Candida infections and without side effects at dosages of 100 mg/kg/day in children with congenital anatomical abnormali-
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ties.33 It also was reported effective in treatment of pulmonary Candida infections.34 Pharmacological studies revealed a direct relationship between the status of renal function, as measured by serum creatinine clearance rates, and retention of the drug; guidelines for determination of appropriate dosages in patients with impaired renal function were presented35 Contrarily, hepatic function failed to influence 5-fluorocytosine serum levels, either in experimental animals with CC14-induced hepatic insufficiency or in a patient with postnecrotic cirrhosis.36 Preliminary data were reported on the successful use of combinations of amphotericin B and 5-fluorocytosine in treatment of human cryptococC O S ~ S . This ~ ~ mode of therapy, based on the earlier demonstration of a synergistic action between the two drugs in vitro against yeasts,38 was validated experimentally in animals. In one study in mice infected with Candida albicans, CD50 dosages of amphotericin B were reduced in the presence of an otherwise inactive dose of 5-fluorocytosine.39 An additive effect for the two drugs was observed in mice infected with Cryptococcus neoformans .40 New Antifungal Agents - New sources of antifungal agents were investigated. One of these, the passion fruit, Passiflora spp., yielded an active agent following aqueous extraction of cut rind."l This agent, with UV absorption max. at 340, 358 and 375 to 377 nm, was active against a variety of fungi including both yeasts and dermatophytes.42 Antifungal compounds also were detected in cowpea tissues following infection with tobacco necrosis virus43 and in cuticle of the coffee berry.44 Polifungin, a complex of tetraene antibiotics similar to nystatin was isolated from Streptomyces noursei var. polifungini ATCC 21581.45 This complex was best recovered by paper chromatography at pH 8.2 and was developed with a chloroform-methanol-ammonia solvent system; it appeared to consist of three fractions. Another heptaenic antibiotic, cryptomycin, was isolated from a new species of actinomycete, Actinomyces bulgaricus.46 This antibiotic, consisting of a complex of 6-7 heptaene antibiotics, was highly active against yeasts and filamentous fungi including C. neoformans. Preliminary data were reported on U-42,126, an antifungal antibiotic produced by fermentation of %. sviceus.47 This compound was active in vitro against fungi and in vivo against L1210 leukemia in mice. Actinomyces voloviolaceus spp. nov., isolated from a soil sample, was found to produce a polyenic heptaene identical to component A3 of the levarin complex.48
s-
Various synthetic antifungal compounds were examined. A series of polyhalophenyl esters of p-sulfamoylcarbanilic acid were tested against yeast and fungi and were found to be active against C. albicans, Penicillium notatum and Aspergillus f u m i g a t ~ s . ~The ~ most active compounds, in order, were the pentachloro, tribromo and triiodophenyl esters. N-trisubstituted methylimidazoles related to clotrimazole and 2 congeneric compounds with 2-pyrrylmethylidenimino and 2-pyrimidinyl amino groups substituted for the imidzaole nucleus were tested;50 three compounds were found
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active against a variety of pathogenic fungi in vitro and one was active C. neoformans.
-in vivo against C. albicans, A. fumigatus and
An alkaloid, 6,6'-dihydroxythiobinupharidine isolated from Nupar, was described.51 It was reported as being active against several of a variety of human fungal pathogens, including Histoplasma capsulatum, Blastomyces dermatitidis, Trichophyton mentagrophytes, Microsporum gypseum, g. canis, C. neofonnansand 5. albicans.51 Optimal conditions were reported for fermentation of A9145, a water soluble antifungal containing adenine.li2 Production was stimulated by cottonseed oil, adenine, various nucleosides, and tyrosine. The antifungal properties of 2-alkenoic acids and 2-bromoalkanoic acids were studied .53 Antifungal activity of these compounds and of analogous alkanoic and 2-fluoroalkanoic acids were affected by chain length, pH and presence or absence of absorbents. The relative antifungal activity of the compounds was: 2-alkenoic acids > 2-fluoroalkanoic acids > 2-bromoalkanoic acids > alkanoic acids. Biological Studies - One of the more important areas of study with antifungal agents involved the synergistic action of amphotericin B with other antifungal or even antibacterial compounds. This developed from earlier -in vitro studies with amphotericin B and 5-fl~orocytosine~~ in which combinations of the two drugs were found to be additive or synergistic. Amphtericin B was found to potentiate the antitumor activity of 5-fluorouracil and of chromomycin A3 on RNA synthesis in transformed fibroblastic cells while polymyxin B potentiated bleomycin 2 in its action on both DNA and RNA syntheses.55 Combinations of amphotericin B and rifampin were found to be fungicidal for yeast-phase cells of 11. capsulatum at low concentrations, whereas either drug alone was only fungistatic and only at higher concentrations.56 RNA synthesis was inhibited by the combination of the two drugs. Combinations of amphotericin B and 5-fluorocytosine were additive or synergistic in vivo in mice infected with 2. albicans and C. neoformans. 39 3 40 The effect of polyenes of membranes of susceptible and resistant fungi was the subject of several investigations. Mutants of 2. albicans resistant to nystatin, amphotericin B and filipin were isolated following exposure to N-methyl-N'-nitro-N-nitrosoguanidine; they apparent1 were stable and differed from the parent yeasts primarily in morphology57 Changes in cell membrane chemistry were studied in these organisms; new sterols were detected in membranes of nystatin-resistant mutants of 5. albicans and Saccharomyces cerevisiae, and there also were increases in ergosterol levels ' 8 in the polyene-resistant mutants. Three compounds, SKF 525-A (B-diethylaminoethyldiphenylpropyl acetate), SKF 3301-A (2,2 diphenyl-l-dimethylaminoethoxyl]-pentane) and SKF 16467-A (N-dimethylaminoethyl-a, a-diphenyl valeramide, HC1) which inhibited cholesterol biosynthesis were inhibitory for both the polyene-resistant mutants and the parent y e a ~ t s . 5 ~ Electron micrographic studies of yeast phase cells of E. capsulatum and 5. dermatitidis exposed to amphotericin B and hamycin revealed degenerative changes in both plasma membranes and mitochondria followed by plasmolysis within 6 hours of exposure.60 Mutants of Neurospora crassa with blocks in
.
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111
the earlier stages of ergosterol synthesis were resistant to various polyene antibiotics.61 Lyposomes were used as models for the study of the action of polyene antibiotics.62 It was noted that changes in permeability were related to the composition of membrane phospholipid fatty acid chains, and that the distribution of double bonds in the sterol nucleus determined the selective toxicity of the polyenes for natural sterol-containing membranes. These observations were confirmed in studies on natural memb r a n e ~ . Similar ~~ differences in binding affinity of amphotericin B and another polyene, vitamin A, were measured in a study of the enhancement by these compounds of the specific actions of rifampin fusidic acid and 1,3bis (2-chloroethyl)-l-nitrosourea on mouse L cells.b4 Additional data regarding 5-fluorocytosine were provided. In one study, it was noted that premature reading of inhibitory endpoints could be misleading in terms of missed resistant strains; percentages for primary resistance to the drug in several species of pathogenic yeasts also were reported.65 Cytosine, uridine and adenine were found to be antagonistic for both 5-fluorocytosine and 5-fl~orouracil,6~ thus supporting the suggested pathway and mode of action for 5-fluorocytosine; i.e. intracellular deamination of the former with incorporation of the latter in 5fluorouridylic acid. Data regarding 5-fluorocytosine resistance in C. Eformans suggested the existence of a similar pathway in this latter organism.6' Several other studies were concerned with the effect of antibacterial and antifungal agents on nucleic acid synthesis. For example, nalidixic acid was noted to cause a transient inhibition of RNA, DNA and protein synthesis in cells of 5. cerevisiae.68 Lomofungin was found to have a specific inhibitory effect on synthesis of both r osomal and messenger RNA preIt also was inhibitory for cursors in protoplasts of Saccharomyces spp." purified Escherichia DNA-dependent RNA polymerases.70 The sterol binding activity of the polyene filipin was studied.71 Two forms of the drug were demonstrated; an "active form" which binds with cholesterol and an "inactive form" which does not bind with cholesterol. Inactive filipin was converted to the active form by incubation at 50' for 2 hours. An alcoholic extract of bark of the walnut tree, Juglans regia, was found to contain a fungistatic agent active against many common dermatophytic pathogens.72 The stabilities of nystatin and amphotericin B in the presence of phosphate buffers were studied and it was found that both drugs were stable at a pH of 5 to 8; it also was noted that nystatin was more active at a lower temperature (30-25'C) while amphotericin B was more active at a higher temperature (41OC) .73 Intravenous solutions of this drug in 5% dextrose were found to be stable for up to 24 hours when exposed to light at 25OC; addition of hydrocortisone or heparin had no effect on stability.74 5-Fluorocytosine was found to increase survival in immunosuppressed mice injected with C. albicans.75
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Warren, Ed.
References
1. D. Kaye, "Infectious Disease Reviews, William J. Holloway , Ed. , Futura Publishing Co., Mount Kisco, New York, 2, 99 (1973). 2. D. Kaye, "The Medical Clinics of North America", J. Katz and D. Kaye. Eds., W.B. Saunders Co. 5J:4,941 (1973). 57:4, 1045 (1973). 3. P. Kovnat, E. Labovitz and S.P. Levison, 4. W.B. Pratt, "Fundamentals of Chemotherapy," Oxford University Press, New York (1973). 5. E.R. Block, A.E. Jennings and J.E. Bennett, Antimicrob. Ag. Chemother., 5 , 392 (1973). 2, 95 (1973). 6. E.R. Block, A.E. Jennings and J.E. Bennett. 2, 474 (1973). 7. G.J. Miraglia and K.J. Renz, 8. R.A. Komorowski and S.G. Farmer, Amer. J. Clin. Path. 3,56 (1973). 9. S.O. Blumer, L. Kaufman, W. Kaplan, D. McLaughlin and D.E. Kraft, Appl. Microbiol., 26, 4 (1973). 244, 10. L. Kaufman, D.W. McLaughlin, M.J. Clark and S. Blumer, (1973). 11. D. Taplin, N. Zaias, G. Rebell, and H. Blank, Arch. Dermatol., 2, 203 (1969). 12. I.F. Salkin, Appl, Microb., 26, 134 (1973). 3, 575 (1973). 13. J.F. Kennedy and H.C. Tun, Antimicrob. Ag. Chemother. 14. J.F. Busey, Amer. Rev. Resp. Dis., 105, 812 (1972). 15. W.J. Stubbs, Can. Vet. J., 2, 125 (1973). 16. B.T. Fields, J.H. Bates and R.S. Abernathy, Amer. Rev. Resp. Dis. 105, 997 (1972). 17. G.J. Wise, S. Wainstein, P. Goldbern - and P.J. Kozinn, J. Amer. Med. ASSOC., &, 1636 (1973). 18. G. Medoff, W.E. Dismukes, R.H. Meade, 111, and J.M. Moses, Arch. Intern. Med. 130, 241 (1972). 19. S . Shadomy, Infec. Immun., 5, 143 (1970). 20. S . Shadomy, Antimicrob. Ag. Chemother., -1970, 169 (1971). 21. L.M. David, N.K. Veien, J.D. Schmidt, W. Murtishaw and E.B Smith, Curr. Therap. Res., IS, 133 (1973). 22. E.S. Mahgoub, Sabouraudia, 10,212 (1972). 23. R.J. Holt and R.L. Newman, Develop. Med. and Child Neurology 14 (Supp. 27), 70 (1972). 24. W. Meinhoff and D. GGnther, Arch. Derm. Forsch. 242, 293, (1972). 25. J.M. Van Cutsem and D. Thienpont, Chemother., 17,392 (1972). 26. A.A. Botter, Mykosen, 15,179 (1972). l5, 35 (1972). 27. M. Thiery, B.M. Mrozowski and H. Van Kets, 28. J. Breigmans, J. Van Cutsem, J. Heykauts, V. Schuermans and D. Thienpont, Europ. J. Clin. Pharmacol. 5 , 93 (1972). 29. P. Charney, V.M. Torres, A.W. Mayo and E.B. Smith, Int. 3. Dermatol., 2, 179 (1973). 30. H.W. Herman, Arch Dermatol., 106,839 (1972). 106,837 (1972). 31. R. Katz, and B. Cohn, 32. G.W. Atkinson and H.L. Israel, h e r . J. Med., 55, 496 (1973).
u,
m,
w,
z,
e,
u,
Chap. 1 2 33. 34. 35.
Antifungal Agents
113
Shadomy
R.J. Holt and R.L. Newman, Develop. Med. and Child Neurol. 14 (Supp. 2 7 ) , 70 ( 1 9 7 2 ) . P.C. Bartley, Aust. N.Z.J. Med. 2, 1 8 9 ( 1 9 7 3 ) . J. Schgnbeck, A. Polak, M. Fernex and J.J. Scholer, Chemother. 18, 321 ( 1 9 7 3 ) .
36. 37 * 38.
E.R. Block, Antimicrob. Ag. Chemother., 2 , 1 4 1 ( 1 9 7 3 ) . I.L. Garriques, M.A. Sande, J.P. Utz, G.L. Mandell, J.F. Warner, R.F. McGehee and S . Shadomy, abst., 13th ICAAC, 239 ( 1 9 7 3 ) . G. Medoff, M. Comfort and G.S. Kobayashi, Proc. SOC. Exp. Biol. Med.,
138, 571 (1971). 39.
E. Titsworth and E. Grunberg, Antimicrob. Ag. Chemother. 4 , 306
40.
E.R. Block and J.E. Bennett, Proc. SOC. Exp. Biol. Med, 142, 476
41. 42. 43. 44. 45.
Birner and J.M. Nicolls, Antimicrob. Ag. Chemother. 3 , 1 0 5 ( 1 9 7 3 ) . J.M. Nicolls, J. Sirner and P. Forsell, 3 , 110 ( 1 9 7 3 ) . J.A. Bailey, J. Gen. Microbiol., 75, 1 1 9 ( 1 9 7 3 ) ; J.F. Lampard, Ann. Appl. Biol., 73, 3 1 ( 1 9 7 3 ) . D. Kotiuszko, Acta Microbiol. Pol, Ser. A., Microbiol. Gen., A, 201
46.
V.A. Tsyganov, A.F. Morgunova, Yu. E. Konev, S.N. Solovev and M.A. Malyshkina, Antibiotiki, 17,1 0 6 7 ( 1 9 7 2 ) . L.J. Hanka and A. Dietz, Antimicrob. Ag. Chemother., 2, 425 ( 1 9 7 3 ) . Yu. E. Konev. V.A. Tsygonov, E.D. Etingov, N.M. Zavalnaya and A.I. Filippova, Antibiotiki, 18,354 ( 1 9 7 3 ) . G. Moktat, N. Rezvani, M. Emami and L. Lalezari, J. Pharm. Sci62,
(1973).
(1973). J.
u.,
(1972).
47. 48. 49.
485 ( 1 9 7 3 ) . 50. 51.
S . Casadio, A. Donetti and G. Coppi, J. Pharm. Sci., 62, 7 7 3 ( 1 9 7 3 ) . W.P. Cullen, R.T. La Tonde, C.J. Wang and C.F. Wong, 62,
w.,
826 ( 1 9 7 3 ) . 52. 53. 54. 55. 56. 57. 58. 59. 60.
L.D. Boeck, G.M. Clem, M.M. Wilson and J.E. Westhead, Antimicrob. Ag. Chemother. 3 , 4 9 ( 1 9 7 3 ) . H. Gershon, M-WTMcNeil, R. Parmegiani, P.K. Godfrey and J.M. Baricko, ibid., 4, 435 ( 1 9 7 3 ) . G. Medoff, M. Comfort and G.S. Kobayashi, Proc. Soc. Exp. Biol. and Med. 1 3 8 , 5 7 1 ( 1 9 7 1 ) . M. K u G o , T. Kamiya, H. Endo and S . Komiyama, Antimicrob. Ag. Chemother., 2, 5 8 0 ( 1 9 7 3 ) . G.S. Kobayashi, G. Medoff, D. Schlessinger, C.N. Kwan and W.E. Musser, Science, 177,7 0 9 ( 1 9 7 2 ) . J.M.T. Hamilton-Miller, J. Med. Microbiol., 2, 425 ( 1 9 7 2 ) . J.M.T. Hamilton-Miller, J. Gen. Microbiol., 2, 201 (1972). J.M.T. Hamilton-Miller, Chemotherapy, 18,1 5 4 ( 1 9 7 3 ) . J.W. Love, R.G. Garrison and D.R. Johnson, Mycopathol. Myco. Appl.,
4 8 , 289 ( 1 9 7 2 ) . 61. 62.
M. Grindle, Mol. Gen. Genet., 120, 283 ( 1 9 7 3 ) . Chuen-Chin Hsuchen and D.S. Finegold, Antimicrob. Ag. Chemother.,
4 , 309 ( 1 9 7 3 ) . 63. 64.
u.,
Chuen-Chin Hsuchen and D.S. Finegold, 4 , 316 ( 1 9 7 3 ) . G. Medoff, C.N. Kwan, D. Schlessinger and G.S. Kobayashi,
u., 3
114
Sect. 111 441, (1973).
65. 66. 67. 68.
-
Chemotherapeutic Agents
Warren, Ed.
e,
S . Shadomy, C.B. Kirchoff and G. Wagner, 2, 9 ( 1 9 7 3 ) . A. Polak and H.J. Scholer, Pathol. Microbiol., 3 9 , 1 4 8 ( 1 9 7 3 ) . E.R. Block, A.E. Jennings and J.E. Bennett, Antzicrob. Ag. Chemother. , 2, 649 ( 1 9 7 3 ) . C.A. Michels, J. Blamire, B. Goldfinger and J. Marmur, 2,
s,
562 ( 1 9 7 3 ) .
69. 70. 71.
S-C. Kuo, F.R. Cano, J.O. Lampen, M y 3, 716 ( 1 9 7 3 ) . F.R. Cano, S-C. Kuo and J.O. Lampen, ?bid., 3, 723 ( 1 9 7 3 ) . F. Schroder, J.F. Holland and L.L. Bieber, Biochem., 12, 4785
72.
S . Ahmad, M.A. 3, 436 ( 1 9 7 3 ) .
(1973).
Wahid and A.Q.S. Bukhari, Antimicrob. Ag. Chemother.
73. 74.
J.M.T. Hamilton-Miller, J. Pharm. Pharmacol., 25, 4 0 1 ( 1 9 7 3 ) . E.R. Block and J.E. Bennett, Antimicrob. Ag. Chemother., 5 , 6 4 8
75.
J.A. Linquist, S . Rabinovich and I.M. Smith,
(1973).
w.,4 , 5 8
(1973).
115 Chapter 13.
Antiparasitic Agents
W. C. Campbell and H. Mrozik, Merck Institute for Therapeutic Research, Rahway, New Jersey 07065 Agents - The -Antiprotozoal comprehensively reviewed.l
chemotherapy of protozoan diseases was The Abstracts of the Ninth International Congress on Tropical Medicine and Malaria2 contain many articles on protozoan and helminthic diseases. Many aspects of parasite biochemistry and drug action were presented at a recent meeting and have been published. 3
-
The current state of malaria chemoAntimalarial Drugs. General therapy, with special emphasis on drug resistance, was reviewed by the WHO scientific group.4 This survey contains a summary of the structures of 24 antimalarial drugs from 8 chemical classes in general use, of 16 experimental compounds receiving clinical trials, and of 9 experimental compounds so far used only in animal models. A general review on antimalarial drugs and their,actions, acridine antimalarials, and diagnosis and treatment of malaria became available.
-
The phenanthrene methanol WR33063 cured a high perClinical Data centage of human volunteers infected with several Plasmodium falciparum strains, including chloroquine-resistant ones, without evidence of phototoxicity or other serious side effects, although occasionally a treatment failure occurred, even at the largest doses used in therapy; such failures might be due to drug resistance.8 In another study, WR33063 did not protect volunteers from chloroquineand pyrimethamine-resistant P. falciparum infection when given prophylactically.9 The quinolize methanol WR30090 likewise demonstrated effective antimalarial action, with low toxicity, against multi-drug-resistant P. falciparum and P. vivax in human volunteers.l0 Contrary toexpectation, the Tnduced photosensitivity was judged by these investigators to be rare, of minor degree, and easily reversed by drug withdrawal. Prophylactically, this drug provided suppressive cures to 75% of volunteers exposed to P. falciparum, but not to P. vivax.9 Although these two drugs exemElify a major advance in tge cure of P. falciparum infections, they are only suppressive against P. vivax zhere the drug of choice still remains primaquine. l1 Amodiaquin appears curative against P. falciparum infections resistant to the structurally closely reiated chloroquine. This surprising observation was supported by new experiments showing no apparent difference in binding of 14C-amodiaquin by erythrocytes of monkeys infected with chloroquine-susceptible and chloroquine-resistant P. falciparum, whereas erythrocytes of chloroquineresistant infections have a deficiency of 14C-chloroquine binding. l2 The triple combination of chloroquine, sulfamethoxydiazine, and pyrimethamine (ratio 30:lO:l) showed a high degree of potentiation of the drug effects upon P. berghei infections in mice.13
-
116
Sect. I11
-
-
Chemotherapeutic Agents
Warren, Ed.
Drua Modifications The success of suinoline methanols and Dhenanthrene methanols in the cure of p. falciparum malaria caused the recent synthesis of many analogs intended not only to increase the activity but also to reduce the phototoxicity. The more active quinoline methanols are the adamantyl derivative I (curative in mice against P. berghei at 100-320 mg/kg), 14 the phenoxy derivative 40-80 mg/kg),15 and the 7,8-benzo derivative I11 I1 (curative A low phototoxicity was observed only (curative at 100 mg/kg).16 for I. The bis-trifluoromethyl phenanthrene methanol IV (curative at 40 mg/kg)rand the naphthylthiophene V (curative at 100-320 mg/kg)18 are interesting new versions of this lead. An analysis of 107 phenanthrene methanols for the contribution of polar and partition effects of substituents according to Hansch and of a smaller number according to the Free-Wilson Additivity Scheme was reported.19 Two highly active pyridine methanols were VI (curative at 2040 mg/kg)20 and VII (curative at 20-80 mg/kg).21 The quinacrine analog VIII (curative at 80 mg/kg) is more active and less toxic than quinacrine itself.22 DHDS (X), a suspected metabolite of diaminodiphenylsulfone (DDS, IX), had no antimalarial activity but caused marked methemoglobinemia, a side effect also of DDS chemotherapy. The tetraacetyl derivative TAHDS (XI), however, had antimalarial activity (curative at 160 mg/kg) superior to DDS (TAHDS) without causing formation of methemoglobin. It is not known if TAHDS is metabolized to DDS or another active metabolite or is active in its own right, but it is clear that DHDS is not responsible for the antimalarial action of either DDS or TAHDS.23 The acylaminonitrodiphenylsulfone XI1 had curative action on the order of DDS.24 The pyrimidinylaminobenzimidazole XI11 was selected from a group of 38 compounds with curative action against 5. berghei; it is well tolerated in mice. This benzimidazole was synthesized as one of the hypothetical active metabolites of the antimalarial guanidinopyrimidine (XIV), which is toxic and less active than XIII.25 The synthetic effort in the coenzyme Q antimetabolite area produced two new quinolinequinones with antimalarial activity: XV (curative at 320 mg/kg)26 and XVI (prophylactic in chicks against P. gallinaFor inhibition of coenzyme Q enzGes by 6 ceum at 120 mg/kg).27 hydroxy-7-alkyl quinolinequinone analogs of CoQ and for antimalarial activity, an alkyl chain length of 15 carbon atoms appears optima1.28 An optimal range for the enzrgy of the lowest empty molecular orbital as calculated by the Huckel technique as well as an appropriate partition coefficient, was determined for many of the antimalarial naphthoquinones. 29 A reexamination of 295 of these compounds showed that variation of the alkyl substituent according to Topliss' scheme (methyl, isopropyl, cyclopentyl, and cyclohexyl) would lead to some of the most active analogs after synthesis of only these four compounds.30 A compound related to hetol and twice as active (curative of P. berghei in mice after a single S.C. dose of 320 mg/kg) is XVII.3f 6
zt
-
Chap. 13
Antiparasitic Agents
Campbell, Mrozik
R6m
117
HOCHCH 2NH E t
2
R8
1-11 I R2 = 1 - a d a m a n t y l , -cH (OH ) c H 2 N ( N -Bu ) 2 ,
v
F3c%
I11
HOqHCH2NH ( 4 - h e p t yl )
V
IX, X, XI, XII,
DDS, X i = X 2 = N H 2 DHDS, Xi=X2=NHOH TAHDS, X1=X2=N( O A c ) A c X1=N02,
c1
a 3 H
XIV 0
-
Coccidiosis Two v e r y c o m p r e h e n s i v e and up-to-date reviews of cocc i d i o s i s appeared. 32, 33 The c u r r e n t c o m m e r c i a l c o c c i d i o s t a t s were c l a s s i f i e d i n t o 1 2 groups according t o t h e i r m o d e of action.34 It
118
Sect. I11 - Chemotherapeutic Agents
Warren, Ed.
was p o i n t e d o u t t h a t c o c c i d i a r e s i s t a n t t o one drug of a group a r e a l s o r e s i s t a n t t o t h e o t h e r d r u g s of t h e s a m e group and t h a t a s w i t c h i n medication must be t o a d i f f e r e n t group t o overcome t h i s resistance. T i s s u e c u l t u r e s of E i m e r i a t e n e l l a have been used t o detect a n t i c o c c i d i a l a c t i v i t y , 3 5 mode of drug a c t i o n , 3 6 growth f a c t o r antagonism,37 and e f f i c a c y of drug combinations,38 and t h i s e x c i t i n g new t e c h n i q u e w a s reviewed.39 The now f a m i l i a r a s s a y of chicken embryos i n f e c t e d w i t h E. t e n e l l a c o n t i n u e s t o g i v e i n t e r e s t i n g i n s i g h t s i n t o t h e a c t i v T t y and mode of a c t i o n of c o c c i d i o s t a t s .40, 41 The c o n t i n u i n g appearance of d r u g - t o l e r a n t c o c c i d i a s t r a i n s and t h e concomitant problems f o r p r o p h y l a c t i c chemotherapy were r e f l e c t e d i n t h e h i g h a c t i v i t y aimed a t d i s c o v e r y of new a n t i c o c c i d i a l s , a s w e l l as i n numerous s t u d i e s of t h e s e n s i t i v i t y of newly i s o l a t e d f i e l d s t r a i n s t o e x i s t i n g drugs. While m e t i c l o r p i n d o l w a s e f f i c a c i o u s a g a i n s t i n f e c t i o n s by E. t e n e l l a s t r a i n s t o l e r a n t t o other a n t i c o c c i d i a l agents, a metiTlorpindo1-tolerant s t r a i n w a s not c r o s s - r e s i s t a n t t o 14 other c o c c i d i ~ s t a t s . ~ An~ o b s e r v a t i o n t h a t a m e t i c l o r p i n d o l - t o l e r a n t s t r a i n of E. a c e r v u l i n a showed g r e a t e r t h a n expected s e n s i t i v i t y t o decoq;finate43 l e d t o a s y n e r g i s t i c drug combination of a 4-hydroxyquinoline c o c c i d i o s t a t p l u s m e t i c l o r p i n d o l , which w a s e f f e c t i v e a t lower l e v e l s t h a n t h o s e used f o r e i t h e r c o c c i d i o s t a t alone. Although b o t h classes of d r u g s a r e similar i n s t r u c t u r e and b o t h i n h i b i t t h e growth of s p o r o z o i t e s a t t h e same s t a g e of t h e l i f e c y c l e , no c r o s s - r e s i s t a n c e i s p r e s e n t between them and i t w a s concluded t h a t t h e y p r o b a b l y a c t upon c l o s e l y r e l a t e d b u t n o n i d e n t i c a l m e t a b o l i c pathways.44 An E. t e n e l l a s t r a i n r e s i s t a n t t o Novastat t r e a t m e n t w a s developed aEd shown t o b e c r o s s - r e s i s t a n t t o t r e a t m e n t w i t h nihydrazone, n i t r o f u r a z o n e , T r i t h i a d o l , U n i s t a t and z o a l e n e , b u t not t o 9 o t h e r COCcidi0stats.~5
-
-
The s t r u c t u r e of t h e ionophore a n t i b i o t i c A204A w a s d e s cribed.46 The conformation of X537A, a p o l y e t h e r a n t i b i o t i c , i n complexes w i t h metal s a l t s and amines i n d i f f e r e n t s o l v e n t s w a s d i s c ~ s s e d ; ~t h7 e g e n e r i c name l a s a l o c i d h a s been adopted f o r i t , and t h e o p t i m a l d o s e a g a i n s t mixed E i m e r i a i n f e c t i o n s i n c h i c k e n s w a s determined t o b e 0.0075% i n f e e d . 4 8 The a c t i v i t y of monensin w a s s t u d i e d i n b r o i l e r s 4 9 and cal~es.~B o r o i l e r s r a i s e d on a monensin-medicated d i e t developed o n l y a low immunity t o Eimeria i n f e c t i o n s . 5 1 The b i o s y n t h e s i s of monensin w a s d i s c u s s e d . 3 2 Robenedine a t 30 g/ton of f e e d p r o t e c t e d chickens from c o c c i d i o s i s caused by 8 E i m e r i a s p e c i e s . 5 3 A t e t r a hydro analog of robenedine i s claimed i n t h e p a t e n t l i t e r a t u r e f o r u s e of c o n t r o l of malaria and c o c c i d i o s i s . 5 4 Residue and metabolism of robenedine i n r a t s was s t u d i e d . 5 5 I t w a s shown t h a t b u q u i n o l a t e does n o t i n t e r f e r e w i t h t h e h o s t ’ s development of immunity a g a i n s t c o c c i d i a l species, 56 a l t h o u g h such i n t e r f e r e n c e c o u l d have been
Chap. 13
Antiparasitic Agents
Campbell, Mrozik
119 -
expected because of the drug's mode of action upon the sporozoites in the very early part of the life cycle.34 3,5-Dinitrobenzamides substituted in the 2-position with an amino or ethoxy group appeared to be active in experimental chicken c o c c i d i ~ s i s . ~The ~ 6-hydroxy2H-pyran-3(6H)-one derivative XVIII was active against E. tenella iKfections i ; chicks at 0.05% in the feed.58 The recenF patent literature mentions 4-nitro-5-cyanoimidazole derivatives further substituted at the 1- and 2-positions,59 cinnamylideneaminobenzylideneaminoguanidines,60 6-aminopurines and their N1-oxides with further substitution at the 9-position,61 aminoethylcarbazole,62 indazolylphenylureas,63 3-amino-2,4-diarylthiophene-l, l-dioxides,b4 and a new antibiotic, salinomycin (C39H6501Na) as potential coccidiostats.65
8
-
Trypanosomiasis A review series on animal H trypanosomiasi s is being published. 66 A O a m C H 3 monograph on Chagas's disease67 contains a description of the disease, its diagnosis, XVIII and the authors' extensive experience with nifurtimox therapy of acute and chronic cases in the clinic. With a 90- to 120-day treatment using nifurtimox, a high cure N -0 0 2 N q c 3 = L H < N 4 N H 2 rate of Chagasls disease could be obtained. XIX The 2-nitroimidazole derivative Ro-7-1051 showed high efficacy in laboratory studies against T. cruzi.68 The nitrovinylfuran derivative SQ 18,506 (XIX) had curaTive action in mice infected with T. brucei and T. rhodesiense at a dosage of 200 mg/kg every 12 hours for 3 days.69 Some tetracyclic diamidines were synthesized but did not approach the high trypanocidal activity of diminazene. 70 Amoebiasis - Inhibition of Entamoeba histolytica protein synthesis was correlated with the amoebicidal activity of a number of alkaloids and antibiotics. 71 Toxoplasmosis This subject was r e ~ i e w e d . ~ The ~ , ~cat ~ was implicated as the definitive host of Toxoplasma gondii with small mammals and birds as essential intermediFte hosts.74 The therapeutic effect of several sulfa drugs and DDS, alone and in conbination with pyrimethamine or trimethoprim, in T. gondii-infected mice was reported. The effect can be reversed by F-aminobenzoic acid, and trimethoprim alone has no activity.75 T. gondii infections in mice have been cured by clindamycin and Nrdemethyl-4l -pentyl ~ l i n d a m y c i nand ~ ~ by sulfameter plus pyrimethamine or spiramycin. 77
-
-
-
Antiprotozoal Nitrofurans and Nitroimidazoles Ipronidazole at 0.00625% in feed had antihistomonal activity equivalent to that of dimetridazole and ronidazole at their recommended levels and was more effective than carbazone, nitarsone or 2-acetylamino-5-nitrothiazole in preventing mortality in turkeys.78 2-(4-Carbostyryl)-5nitro-1-vinylimidazole, a urinary metabolite of 2-(4-methylstyryl)5-nitro-l-vinylimidazole, was curative in calves against T. vivax
--
120
Sect. 111
-
Chemotherapeutic Agents
Warren, Ed.
upon multiple dosing; this compound and several carboxylic acid derivatives also showed activity against T. vaginalis, E. histol tica, T. rhodesiense, T. cruzi, T. gambieke and T. concolense. ---d c
_
- - -
-
-
-
-
Antiprotozoal Antibiotics Antibiotics with antiprotozoal activity that have been mentioned in the recent literature are G-41880 (Entamoeba), dactylarin81 (Leishmania and Entamoeba) , and viomycin82(Trichomonas )
.
-
Drugs Active Against Nematodes, General Three major reviews of this subject appeared.83-83 Methodological contributions were made to the screening of anthelmintics86,87 and to their evaluation in ruminants.88,89 The role played by fumarate reductase inhibition in the mode of action of anthelmintics was further studied. Enzyme isolated from thiabendazole-sensitive Haemonchus was inhibited by thiabendazole, cambendazole, levamisole, morantel tartrate and disophenol, but not by mebendazole. Enzyme from a thiabendazole-resistant strain was similarly affected, except that the inhibitory effect of cambendazole was reduced and that of thiabendazole was not demon~trable.~ In~ lambs, after 4 generations of Haemonchus strain BPL-2 had been exposed successively to single doses of cambendazole at 5, 5 , 10 and 20 mg/kg, this parasite was less susceptible to cambendazole than it had been initially.91 Studies on the effect of mebendazole on Trichinella larvae in vitro or in rats suggested that the mechanism of anthelmintic action is similar to that observed in certain other helminths, i.e., mebendazole reduced the parasite's uptake of exogenous glucose thus apparently depleting the endogenous energy resource (glycogen).92 Studies with Ascaris also showed a primary inhibition of glucose uptake or transport, with secondary reduction in the uptake of other nutrients.93 The physiological distribution of dichlorvos, when administered to pigs in polyvinyl chloride pellets, was studied. Radioisotope measurements indicated that certain components of the polyvinyl chloride were incorporated into the tissues, but no dichlorvos or its metabolites were found.g4 Dinitro henol inhibited the uptake of arginine by Ascaris in vitro.!9
--
-
-
0
N
xx
CH3 e C C l = N N H
H XXI
s o
I I Ii NHCNHCOEt
=I1
NHCNHCOEt II II
s
0
0
The structure-activity relationships of piperazine derivatives were studied by measuring their relative potentiation of the acetylcholine response in nervemuscle preparations from Ascaris.
MrNkEt eN,)d XXIII
Chap. 13
Antiparasitic Agents
Campbell, Mrozik
121
The results suggested that diethylcarbamazine inhibits cholinesteraseSg6 Bephenium hydroxynaphthoate gave variable and often unsatisfactory results against Necator in man, even though a high dosage was used. It was suggested that the local strain of Necator may be partially resistant to the drug.g7 Diphetarsone (a pentavalent arsenical) was reported to be highly efficacious and well tolerated when used for the treatment of Trichuris infections in man.98
-
The latest of the benzimidazole anthelmintics, oxibendaNew Drugs zole ( X X ) , was reported to have a broad spectrum of activity and a good margin of safety. The drug is insoluble in water and only slightly soluble in most organic solvents. In sheep, single oxibendazole doses of 5-10 mg/kg were highly effective against immature and adult worms of the genera Haemonchus, Ostertagia, Trichostrongylus, Strongyloides, Nematodirus, Cooperia, Bunostomum, Oesophagostomum and Chabertia. In cattle, doses of 5-20 mg/kg were 80-100% effective in removing worms of the genera Haemonchus, Ostertagia, Trichostrongylus, Cooperia and Oesophagostomum. In swine exposed to Ascaris infection, the feeding of diets containing 0.05-0.10% oxibendazole provided protection against clinical signs of disease. Radioisotope studies in sheep showed that at 53 mg/kg, the peak plasma concentration was reached in 6 hours. Approximately 34% of the drug was recovered in the urine within 24 hours after dosing, and an additional 6% was recovered within 216 hours. In toxicologic studies, sheep tolerated a dose of 300 mg/kg; and a dose of 30 mg/kg, given at various intervals during gestation in sheep or rats, did not adversely affect fetal d e v e l ~ p m e n t . ~ ~ A new anthelmintic, p-toluoyl chloride phenylhydrazone (XXI), at 40 mg/kg p.0. was at least 97% effective against Haemonchus, Ostertagia, Trichostrongylus, Cooperia and Oesophagostomum in was 87% effective against Nematodirus and inactive against Trichuris. loo P
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Thiophanate (XXII), diethyl 4,4'-0-phenylene-bis-( 3-thioallophanate), at 50 mg/kg, was at least 97%'-effective against Haemonchus, Ostertagia and Trichostrongylus in sheep and cattle. At 100 mg/kg it was highly active against Nematodirus and Cooperia, but variable against Dictyocaulus in sheep.lul Synergistic action against Dirofilaria was claimed for a combination of 3,3'-[[[4-[(4,6-diamino-l, 3,5-triazin-2-yl)amino]phenyl]arsinidine]bis(th~o)]b~s-D-val~ne (F151) and p-[ 5-[5-(4-methyl-1-piperazinyl)-2-benzimidazolyl]-2benzimidazoly1)-phenol (HOE33258). lo2 In rodents infected with Litomosoides, 3-ethyl -8-methyl-l ,3,8-triazabicyclo[4,4,O]decan-2one (XXIII) caused a transient reduction in microfilaremia but did not kill the adult worms.lo3
-
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Drugs Active Against Trematodes Major reviews of anti-trematode drugs appeared.u',lu4 A new Technical Report from W.H.O. briefly reviewed the anti-schistosome drugs that are currently in use105 (a much more comprehensive account of these drugs appeared in
Sect. I11
122 -
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Chemotherapeutic Agents
Warren, Ed.
197 2 ) .lo6
A method for screening compounds for fasciolicidal activity was described and was used to study the efficacy of many known f a s c i o l i ~ i d e s . ~A~way ~ of automatically measuring the movement of Schistosoma in response to drugs in vitro was reported and was proposed as a method for screening compounds for anti-schistosome activity.lo8 Transfer of immature flukes from the parenchyma to the bile ducts of goats gave results which suggested that the drug susceptibility of Fasciola is more dependent on degree of maturation than on location.109 The effect of bithionol on the glycolytic and oxidative metabolism of Paragonimus was studied.110 In a series of novel salicylanilides, fasciolicidal action required electron-withdrawing substituents on both the salicylic acid and aniline rings, together with a lipophilic group such as tert-butyl in the 3-position. However, not all such compounds were .___ fasciolicidal; compounds with a plasma half-life of less than 36 hours in sheep were inactive against fluke; those with a half-life of 48-84 hours were active; and those with a half-life of 5-6 days were very active. The last group included rafoxanide and several di -substituted 3-ter t -butyl -5-nit 1-0-6 -methylsalicylanilides '11
.
-
Fasciolicidal action was also reported for another series of salicylanilides in which an electronegative substituent was introduced in the 3-position. The choice of substituents in the aniline ring was restricted to halogens, methyl groups and tri-halogenated methyls (which improve lipid solubility). The most effective compounds were 3-nitro-2,6-dihydroxybenzoic acid anilides. The inhibition of succinate dehydrogenase from liver flukes has been corthe distribution of the compound between lipid and aqueous phase and not electronic or steric factors within the series is responsible for the activity. 113,114
in vitro by 25 substituted 2,6-dihydroxybenzanilides -related with the Hansch parameter*, suggesting that
-
The evolution of diamphenethide (XXIV) from 4-tert-butoxyacetanilide was recounted. Efficacy and safety of diamphenethide appear to depend on deacylation (probably to the diamino analog) by liver deacylases. Diamphenethide is not active against liver flukes in vitro unless incubated in the presence of enzymatically functional liver cells. Further, the diamino analog is rapidly effective in vitro in the absence of such cells. It was suggested that the production of this metabolite in the liver parenchyma, and its rapid destruction in the liver and dilution in the bloodstream, account for the high activity observed against immature, tissuedwelling flukes and the low activity against mature, duct-dwelling flukes.115 The unique relationship between efficacy of diamphenethide and age of F. -hepatica in shee was described and related to pathogenesis and Fontrol measures. 1
Le__
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&
Chap. 13
O ,
Antiparasitic Agents
Campbell, Mrozik
123
xxv
0
72-MZ/
cH2-cH2, 0
XXIv
The structure-activity relationships of fasciolicidal thiobisphenol derivatives were described.l17
-
Drugs Active Against Schistosomes The level of serotonin in Schistosoma mansoni was increased when hycanthone (or a benzothiopyranoindazole) was given to the host mouse. '18 Hycanthone caused morphological transformation of virus-infected rat cells in culture'" and caused mutations in Salmonella. 120 However, no mutations were observed in thousands of offspring of male mice treated with a high dosage of hycanthone.121 Successful clinical use of hycanthone continued.122 The effect of the drug on liver enzymes of infected and noninfected hosts was studied.123 Oxamniquine ( X X V ) was highly active against S. mansoni in man and was well tolerated except for pain at the siteof injection.124 The antischistosome activity of nitrofuran compounds was further investigated. Comparison of 3-( 5-nitro-2-fur yl ) -substituted derivatives of propionic, acrylic and propiolic acids showed that a vinyl group is necessary for activity.125 In derivatives of 3-(5nitro-2-furyl) acrylic acid, the 5-nitro group was required for activity.126 In a group of (5-nitro-2-furyl)-vinyl heterocycles containing a weakly basic N in a specific position, both the vinyl group and the N were essential f o r antischistosome activity.127 Tubericidin (a purine nucleoside) was tested against Schistosoma in monkeys. Erythrocytes, withdrawn from the monkeys, were allowed to absorb the drug and were then returned to the bloodstream of the donors. This procedure yielded efficacy against female schistosomes but not against males. Tubericidin is active against both sexes in vitro, and the differential in vivo is thought to be due to the greater ingestion of erythrocytes by females. '28
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In vitro, metrifonate (trichlorfon) was slightly more effective in inhibiting the cholinesterase of S. mansoni than of S. haematobium, whereas in man, the drug is Fore effective against the latter species. 129 Arylvinylsulfones were active against 2. mansoni in laboratory animals and monkeys. The most active compound (p-tert-butylvinylsulfone) -caused dermatitis.130
L_
Drugs Active Against Cestodes, General
- Major
reviews of this
124
Sect. I11
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Chemotherapeutic Agents
Warren, Ed.
subject appeared.83, At high dosage, cambendazole was highly active against Thysanosoma in sheep.131 Taenia or Echinococcus eggs incubated in vitro in bunamidine hydrochloride solutions lost their infectivity for laboratory a n i m a l ~ . A~ ~ study ~ of the effect of drugs on the metacestodes of Echinococcus in vitro revealed that many were active, especially halogenated salicylanilides, halogenated bisphenols, bunamidine, and cyanine dyes. 133 In rodents, oxyclozanide at 4 mg/kg was effective against 13-day-old Hymenolepis microstoma,134 Paromomycin was apparently effective against Diphyllobothrium in man. 135
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New Drugs Axenomycins, a new group of macrolide antibiotics, were reported active against tapeworms in mice,dogs and sheep. The antibiotics were isolated from Streptomyces lisandri in three fractions one of which (axenomycin D) was more active than the others.l36 A series of 2,6-dihydroxybenzoic acid anilides related to resorantel, were highly active against tapeworms when halogens or methyl groups were substituted in the anilide ring. The compounds apparently disrupt the energy metabolism of the tapeworm by inhibiting glucolysis and decreasing the level of ATP. 112 p-Toluoyl chloride phenylhydrazone at 50 mg/kg was 100% effective against Moniezia in sheep.100 Of 23 dithiodibenzanilides tested against Hymenolepis in vitro or in mice, none was as active as niclosamide.137
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References
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E. A. S t e c k , "The Chemotherapy of P r o t o z o a n Diseases," V o l I - I V ,
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c i n a l C h e m i s t r y , Walter Reed Army I n s t i t u t e of R e s e a r c h . " N i n t h I n t e r n a t i o n a l Congress on T r o p i c a l Medicine and M a l a r i a , " Athens 14-21,
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ibid.
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Chap. 13 25.
Antiparasitic Agents
Campbell, Mrozik
L. M. Werbel, A. C u r r y , E. F. E l s l a g e r and C. H e s s , J. H e t e r o c y c l . Chem.
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2, 2, 2,
s,
-
men,
z,
s,
11,
e,
2,
163.
126 78. 79. 80.
81. 82. 83. 84. 85. 86.
87. 88. 89. 90. 91. 92. 93. 54. 95. 96. 97. 98. 99. 100.
101. 102. 103.
104. 105. 106. 107. 108. 109. 110. 111. 112. 113. 114. 115. 116. 117. 118. 119. 120. 121. 122. 123. 124.
125. 126. 127.
Sect. 111
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Chemotherapeutic Agents
Warren, Ed.
2,
T. W.
S u l l i v a n , 0. D. G r a c e and R. J. M i t c h e l l , P o u l t r y Sc. 1287 ( 1 9 7 3 ) . i3. Jamieson and M . C. McCorven, J . Med. Chem. 1 6 , 347 ( 1 9 7 3 ) . E. H. Wageman, R . T. T e s t a , J. A. Marquez, J. A. Waitz and MTJ. W e i n s t e i n , 1 3 t h I n t e r s c i e n c e C o n f e r e n c e on A n t i m i c r o b i a l Agents and Chemotherapy, S e p t . 1973, Washington, D. C. A b s t r a c t No. 138. M. K e t t n e r , P. Nemec, J. Kovac and J. Balanova, J. A n t i b i o t . 26, 692 ( 1 9 7 3 ) . T. Kitagawa, T. Miura, S. Tanaka and H. Taniyama, J. A n t i b i o t T z , 528 ( 1 9 7 3 ) . A. D a v i s , Drug T r e a t m e n t i n I n t e s t i n a l H e l m i n t h i a s e s , W.H.O. Geneva p p . 1 2 5 (1973). J. W. McFarland, Prog. Drug R e s . 16, 1 5 7 ( 1 9 7 3 ) . R. B. Burrows, Prog. Drug R e s . E 7 1 0 8 ( 1 9 7 3 ) . T. J. Hayes and M . M i t r o v i c , Am. SOC. P a r a s i t o l . 4 8 t h Ann. Mtg., Univ. of T o r o n t o , J u n e 25-29, 1973, p. 46 ( 1 9 7 3 ) . D. K. H a s s , Exp. P a r a s i t o l . 10 ( 1 9 7 3 ) . C. J. C l a r k and J . A. T u r t o n , Exp. P a r a s i t o l . 32, 6 9 ( 1 9 7 3 ) . R. K. Reineckc, O n d e r s t e p o o r t J. V e t . Res. 3 9 , 7 5 3 (1972). R. K. P r i c h a r d , I n t . J. P a r a s i t o l . 3, 409 ( m 7 3 ) . K. C. Kates, bl. L. C o l g l a z i e r and FT D. E n z i e , J. P a r a s i t o l . 59, 1 6 9 ( 1 9 7 3 ) . S. DeNollin and H. Van den Bossche, J. P a r a s i t o l . 59, 970 (19=). H. Van den Bossche and S. DeNollin, I n t . J. P a r a s i t o l . 401 ( 1 9 7 3 ) . J. C. P o t t e r , J. E. L o e f f l e r , R. D. C o l l i n s , R . Young and A. C. Page, J. Agr. Food Chem. 21, 1 6 3 ( 1 9 7 3 ) . A. V. P a v l o c T r . G e l ' m i n t h o l . Lab., Akad. Nauk SSSR, 115 (1973). P. N. N a t a r a j a n , T. S . Yeoh and V. Zaman, Acta Pharhn. Suec., 10, 1 2 5 ( 1 9 7 3 ) . W. A. C h i n e r y , J. T. A n i m , G. O f o r i - A t t a h and D. R. W. H a d d o c q Ann. Trop. Med. P a r a s i t . 67, 75 ( 1 9 7 3 ) . C. J. R u b z g e , P. 8. O'Dorvd a n d 5 . J. P o w e l l , S. Afr. Med. J. 47, 991 ( 1 9 7 3 ) . V. J. T h e o d o r j d e s , J. Chang, C. J. D i C u o l l o , G. M. G r a s s , R. C T P a r i s h a n d G. C. S c o t t , B r . V e t . J. 129, x c v i i ( 1 9 7 3 ) . S. D. F o l z and D. L x e c t o r , Am. SOC. P a r a s i t o l . , 4 8 t h Ann. Mtg., Univ. of T o r o n t o , J u n e 25-29, 1573, p. 46 ( 1 9 7 3 ) . D. A. E i c h l e r , B r . V e t . J., 533 ( 1 9 7 3 ) . E. F r i e d h e i m , 9 t h I n t e r n a t . Cong. Trop. bled. M a l a r i a , Athens, Grefce, 12-21 O c t . , 1 9 7 3 , Vol. 11, Abst. of Communications, p. 118 ( 1 9 7 3 ) . P. E. Thompson, J. R. Z e i g l e r and J. W. McCal1, Antimicrob. Agents Chemother. 693 (1973). 241 ( 1 9 7 3 ) . P. J. I s l i p , Prog. Drug. Res. W. H. 0. Techn. Hept. S e r . No. 515 pp. 47 ( 1 9 7 3 ) . S . Archer and A . Y a r i n s k y , Prog. Drug R e s . 1 6 , 11 ( 1 9 7 2 ) . T. J. Hayes, J. B a i l e r and M. M i t r o v i c , E x p G i e n t i a , 899 ( 1 9 7 3 ) . A. W. S e n f t and 0. R . H i l l m a n , Am. J. Trop. bled. Hyg. 22, 735 ( 1 9 7 3 ) . iv (1973r D. L. Hughes and E. H a r n e s s , P a r a s i t o l o g y F. Hanajima, E x p . P a r a s i t o l . 34, 1 ( 1 9 7 3 ) . R. M. L e e , P a r a s i t o l o g y 07, x E i ( 1 9 7 3 ) . 1745 ( 1 9 7 3 ) . H . R u s c h i g , J. Konig, D.Tuwel and H. Loewe, A r m . F o r s c h . E. Druckrey and H. Metzger, J . Med. Chem. 1 6 , 536 ( 1 9 7 3 ) . D. Duewel and H. Metzger, J. Med. Chen. E r a 3 3 ( 1 9 7 3 ) . M. H a r f e n i s t , P e s t i c i d e S c i . 871 ( 1 9 7 3 ) . 883 ( 1 9 7 3 ) . D. T. Rowlands. P e s t i c i d e S c i . L. I. Denisova and V. A. O r l o v a , Khim. Farn. Zh. 7, 33 ( 1 9 7 3 ) . T. C. T. Chou. J. L. B e n n e t t , C . P e r t and E. BuedTng, J. Pharmacol. Exp. T h e r . 1 8 6 , 408 ( 1 9 7 3 ) . F. M. H e t r i c k and W. L. Kos, J. Pharmacol. Exp. T h e r . 186, 425 ( 1 9 7 3 ) . P. E. Hartman, H. Berger and Z. Hartman, J. Pharmacol. E x p . Ther. 390 ( 1 9 7 3 ) . W. L. R u s s e l l , 9 t h I n t e r n a t . Cong. Trop. bled. Malaria, Athens, Greece, 14-21 O c t . , 1 9 7 3 , V o l . 11, Abst. of Communications, p. 1 0 2 ( 1 9 7 3 ) . R. M. 5. B e l l , J. Daly, E. Kanengoni and J. J. J o n e s , T r a n s . Roy. SOC. Trop. Med. Hyg. 67, 685 ( 1 9 7 3 ) . J. S c K s t e r , G. Lammler, R. Rudolph, and H. Zahner, Z. Tropenmed. P a r a s i t . 24, 487 ( 1 9 7 3 ) . J . R. Coura, C. A. Argento, N. F i g u e i r e d o , B . Wanke, and G. C. Q u i r e z , 9 t h I n t e r n a t . Cong. Trop. Med. M a l a r i a , Athens, Greece, 14-21 O c t . , 1973, Vol. 11, Abst. of Communications. p. 1 0 3 ( 1 9 7 3 ) . P. B. H u l b e r t , E. Bueding, and C. H . Robinson, J. Med. Chem. 16, 72 ( 1 9 7 3 ) . C. H. Robinson, S. Spengel and E. Bueding, J. Ned. Chem. 16, % ( 1 9 7 3 ) . D. W. Henry, V. H. Brown, M. Cory, J. G. Johansson and E. Bueding, J. Med. Chem. 1 6 , 1287 ( 1 9 7 3 ) .
W. J. Ross, W.
2,
3,
2,
129,
2,
17,
2,
67,
2,
2,
4,
-
186,
-
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Chap. 13
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127
Campbell, Mrozik
't
128. J. J. J a f f e , H. M. Dorernus, H. A. Dunsford, W . S. Kammerer and E. Meymarian, Am. SOC. Trop. Med. Hyg. 22, 62 ( 1 9 7 3 ) . x x i i (1973). 129. B. E. Sanderson, P a r a s i t o G g y 130. H. Horstrnann, R. Gonnerl and H. H. S c h l o r , 9 t h I n t e r n a t . Cong. Trop. Med. M a l a r i a , Athens, G r e e c e , 1 1 - 2 1 O c t . , 1973, Vol. 11, Abst. of Communications, p. 90 (1Y73). 131. R . W. A l l e n , Am. J. V e t . R e s . 34, 6 1 ( 1 9 7 3 ) . 132. J. F. W i l l i a m s , C. W. C o l l i , Rrh'. Leid and R. ClacArthur, J. P a r a s i t o l . 1141 ( 1 9 7 3 ) . 133. T. Sakamoto, J a p . J. Vet. Res. 73 ( 1 9 7 3 ) . 134. C. A. Hopkins, P. M. Grant and H. S t a l l o r d , P a r a s i t o l o g y 355 ( 1 9 7 3 ) . 135. H . 8. Tanowitz and M. N i t t n e r , J. Trop. Med. Hyg. 76, 151 ( 1 9 7 3 ) . 136. C. D e l l a Bruna, M. L. R i c c i a r d i , A. S a n f i l i p o , A n t s i c r o b . Agents Chemother. 3, 708 ( 1 9 7 3 ) . 137. E. Delacoux, R. C. Moreau, R . C a v i e r , M. J. Notteghem, I. Leguen, Chim. T h e r . 8 , 303 ( 1 9 7 3 ) .
67,
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Chapter 14.
A n t i v i r a l Agents
Andrew R. S c h w a r t z , M.D., Hahnemann Medical C o l l e g e and H o s p i t a l P h i l a d e l p h i a , Pa. Introduction -
T h i s y e a r , as i n t h e p a s t , r e v i e w s of a n t i v i r a l chemot h e r a p y have appeared i n many forms, e s p e c i a l l y f o r t h o s e i n t e r e s t e d i n More e x t e n s i v e d i s c u s s i o n s of s e l e c t e d a r e a s c l i n i c a l app l i c a b i l i t y of i n t e r e s t 4 and a broad overview of t h e t y p e s of v i r a l a g e n t s i n v o l v i n g t h e r e s p i r a t o r y t r a c t 5 have a l s o a p p e a r e d . Emphasis w i l l be g i v e n t o t h e c u r r e n t s t a t u s of a s c o r b i c a c i d as a c l i n i c a l l y a v a i l a b l e t h e r a p e u t i c s u b s t a n c e i n r e l a t i o n t o v i r a l d i s e a s e s . The c l i n i c a l s t a t u s of r i f a m y c i n SV d e r i v a t i v e s , p y r i m i d i n e n u c l e o s i d e s , and i n t e r f e r o n w i l l a l s o b e commented upon. An e x c i t i n g new c l a s s of b i d - b a s i c - s u b s t i t u t e d p o l y c y c l i c a r o m a t i c compounds w a s t h e s u b j e c t of a d d i t i o n a l e v a l u a t i o n d u r i n g the p a s t y e a r . 6
A s c o r b i g Acid - Following D r . P a u l i n g ' s i n i t i a l a n e c d o t a l s u p p o r t f o r t h e e f f i c a c y of v i t a m i n C a g a i n s t c o l d s , 7 o t h e r s have drawn c o n f l i c t i n g conThe c o n t r o v e r s y h a s p r e c i p i t a t e d a d d i t i o n a l c l i n i c a l c l u s i o n s .8-12 s t u d y . An a p p a r e n t d e a r t h of r e s p i r a t o r y t r a c t i n f e c t i o n s d u r i n g t h e s c o r b u t i c s t a t e h a s a l s o been r e p o r t e d , l 3 , 1 4 b u t i n v e s t i g a t i o n s of t h e d i r e c t a n t i v i r a l e f f e c t s o f a s c o r b i c a c i d h a v e been few. No d i r e c t v i r a l i n h i b i t o r y a c t i v i t y h a s b e e n d e m o n s t r a t e d L.r G r o a g a i n s t r h i n o v i r u s e s , p o l i o t y p e 1, ECHO 11, CoxsackieA 21, i n f l u e n z a B , p a r a i n f l u e n z a 3 , res p i r a t o r y s y n c y t i a l v i r u s , a d e n o v i r u s 5 , o r h e r p e s s i m p l e x v i r u s .I5 In t h e s c o r b u t i c state, b a c t e r i a l phagocytosis is a l s o impaired, and t h e r e i s i n c r e a s e d s u s c e p t i b i l i t y t o t h e l e t h a l e f f e c t s of e n d o t o x i n c h a l l e n g e . 1 6 I t h a s a l s o been n o t e d t h a t 0 . 0 1 M a s c o r b i c a c i d i n h i b i t s t h e H202-myeloperoxidase-halide r e a c t i o n of n e u t r o p h i l s . I 7 T h i s mechanism, presumably, i s r e s p o n s i b l e f o r t h e i n t r a l e u k o c y t i c k i l l i n g of bact e r i a , y e t i n t h e model s y s t e m s s t u d i e d t h e r e was no r e d u c t i o n ( o r augm e n t a t i o n ) of l e u k o c y t e b a c t e r i c i d a l a c t i v i t y e v e n though t h e H2021nvelop e r o x i d a s e - h a l i d e s y s t e m was i n h i b i t e d , T i s s u e , l e u k o c y t e , and plasma l e v e l s of a s c o r b i c a c i d are q u i t e v a r i a b l e and t e n d t o f a l l i n a "stress" s i t u a t i o n (even m y o c a r d i a l i n f a r c t i o n ) , w i t h a c o r r e s p o n d i n g f a l l i n u r i n a r y e x c r e t i o n of a s c o r b i c The amount of v i t a m i n C w i t h i n a c i d i n t h e unsupplemented s t a t e . 1 8 - 2 4 l e u k o c y t e s on t h e 2nd o r 3 r d day a f t e r t h e o n s e t of a c o l d i s o f t e n a t s c o r b u t i c l e v e l s , l 9 a l t h o u g h t h e mechanisms of a s c o r b i c a c i d u p t a k e by l e u k o c y t e s are n o t i m p a r i e d by t h e stress of i n f e c t i o n . 2 3 Supplementary a s c o r b i c a c i d g i v e n t o a d u l t s r e s u l t s i n t r a n s i e n t i n c r e a s e s i n plasma l e v e l s and i n c r e a s e d e x c r e t i o n i n t h e u r i n e , w i t h a d e g r e e of a u g m e n t a t i o n r e l a t i n g t o d o s e , b u t n o t u n i f o r m l y , i n t h e s e subs t r a t e s . Both l e u k o c y t e a s c o r b i c a c i d (LAA) and serum a s c o r b i c a c i d (SAA) are s i g n i f i c a n t l y e l e v a t e d o v e r unsupplemented l e v e l s a t 1 g / d a y , r i s i n g p r o p o r t i o n a t e l y t o maximum u p t a k e s , a t a b o u t 6 g / d a y . I t h a s been
Chap. 14
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129 -
observed t h a t w h i l e s u f f e r i n g from a c o l d , t h o s e r e c e i v i n g s u p p l e m e n t a l vitamin C i n d o s e s of 1 g o r g r e a t e r do n o t e x h i b i t t h e e x p e c t e d d e c l i n e i n LAA t o s c o r b u t i c levels on t h e 2nd day.19 The o p t i m a l d o s e f o r m a i n t a i n i n g t h e LAA above s c o r b u t i c levels h a s n o t y e t been c l e a r l y e s t a b l i s h e d . S e v e r a l s t u d i e s of t h e c l i n i c a l e f f i c a c y of a s c o r b i c a c i d have been r e p o r t e d .15,21-23 925-33 Three c l e a r l y f a i l e d t o r e v e a l any p r o p h y l a c t i c b e n e f i t of 3 g/day i n o u t p a t i e n t - a c q u i r e d c o l d s 2 6 o r a g a i n s t induced i n f l u e n z a B and r h f n o v i r u s e s l 5 and induced r h i n o v i r u s 44 i n f e c t i o n 2 5 i n ad u l t human v o l u n t e e r s . Some a u t h o r s 3 1 , 3 2 were unimpressed w i t h t h e d i f f e r e n c e s i n t h e i n c i d e n c e and s e v e r i t y of t h e c o l d s i n e a r l i e r s t u d i e s , b u t t h i s i n t e r p r e t a t i o n h a s b e e n d e b a t e d . 7 On t h e o t h e r hand, a s t u d y by R i t z e 1 3 3 and more r e c e n t o u t p a t i e n t t r i a l s i n s c h o o l c h i l d r e n i n Dublin, 21-23 on a Navajo I n d i a n r e s e r v a t i o n , 2 8 among u n i v e r s i t y s t a f f and s t u d e n t s i n G l a s g 0 w , ~ 7 and among a d u l t s i n Toronto29 r e v e a l e d t h a t v i t a m i n C s i g n i f i c a n t l y r e d u c e s t h e number of e p i s o d e s o f r e s p i r a t o r y t r a c t i n f e c t i o n s (and o t h e r a i l m e n t s ! ) and t h a t t h e c a t a r r h a l m a n i f e s t a t i o n s of a c o l d a r e more s u s c e p t i b l e t o a m e l i o r a t i o n t h a n t h e t o x i c m a n i f e s t a t i o n s . S i g n i f i c a n t l y less t i m e was l o s t from s c h o o l o r employment among t h o s e rec e i v i n g s u p p l e m e n t a l a s c o r b i c a c i d , I t i s of i n t e r e s t t h a t a t t h e 200 mg p e r day d o s e , s c h o o l g i r l s were s i g n i f i c a n t l y b e n e f i t e d b u t n o t s c h o o l b o y s . I n f a c t , some m a n i f e s t a t i o n s were more severe among t h e l a t t e r , 2 3 s u g g e s t i n g s e x - r e l a t e d d i f f e r e n c e s i n r e s p o n s e t o , and p e r h a p s i n r e q u i r e ments f o r , v i t a m i n C . There h a s been no d e m o n s t r a b l e e f f e c t of a s c o r b i c a c i d ( a t 3 g / d a y ) on t h e magnitude of v i r a l m u l t i p l i c a t i o n o r s h e d d i n g from t h e n o s e , t h e r a t e of development o r magnitude of l o c a l o r c i r c u l a t i n g a n t i b o d y , o r t h e d e g r e e of i n f e c t i o n - i n d u c e d r e d u c t i o n i n t h e flow of t h e n a s a l m u c o c i l i a r y b l a n k e t .25 ,34 I n t e r f e r o n p r o d u c t i o n a p p e a r s t o b e n e i t h e r enhanced n o r s u p p r e s s e d by t h e p r e s e n c e of a d d i t i o n a l a s c o r b i c a c i d o r by t h e s c o r butic state, O v e r a l l , t h e r e does a p p e a r t o b e some b e n e f i t a s s o c i a t e d w i t h supp l e m e n t a l a s c o r b i c a c i d i n p r e v e n t i n g c o l d s , b u t t h e mechanism and dosage requirements r e q u i r e f u r t h e r e l u c i d a t i o n . Rifamycin A n t i b i o t i c s - Derived from t h e f e r m e n t a t i o n of Streptomyces m e d i t e r r a n e i , t h i s f a m i l y of ansa-macrolide d r u g s p o s s e s s e s a v e r y b r o a d s p e c t r u m of a n t i m i c r o b i a l a c t i v i t y a g a i n s t g r a m - p o s i t i v e and gram-negative b a c t e r i a , m y c o b a c t e r i a , chlamydia, and s e v e r a l v i r u s e s . The a c t i v i t y o f r i f a m p i n , t h e o n l y c l i n i c a l c a n d i d a t e of t h i s g r o u p , h a s r e c e n t l y been reviewed by L e s t e r . 3 5 I t s e f f i c a c y is presumably due t o t h e i n h i b i t i o n of b a c t e r i a l d e r i v e d DNA-dependent RNA polymerase and t h e i n h i b i t i o n of v i r a l RNA-dependent DNA polymerase ( r e v e r s e t r a n s c r i p t a s e )
.
V i r a l - d i r e c t e d RNA s y n t h e s i s by pox v i r u s e s i s a f f e c t e d by h i g h conc e n t r a t i o n s of r i f a m p i n (100 p g / m l ) ; v i r a l - d i r e c t e d DNA s y n t h e s i s is p a r t i a l l y a f f e c t e d , and l a t e v i r a l p r o t e i n s y n t h e s i s is b l o c k e d . The t a r g e t seems t o b e t h e l a t e s y n t h e s i z e d p a r t i c u l a t e RNA polymerase t h a t is t o b e i n c o r p o r a t e d i n t o t h e i n t a c t v i r u s , Riva and S i l v e s t r i 3 6 review t h e n o t e worthy i n h i b i t i o n of r e v e r s e t r a n s c r i p t a s e a c t i v i t y of r i f a m p i n i n DNA vi-
130
Sect. I11
- Chemotherapeutic Agents
Warren, Ed.
r u s e s and RNA tumor v i r u s e s . Rif ampin ( t h e 3-iminomethyl d e r i v a t i v e of r i f a m y c i n SV) is i n a c t i v e a g a i n s t t h e reverse t r a n s c r i p t a s e of murine sarcoma v i r u s (MSV), b u t i f t h e 4 - s i d e c h a i n on t h e p i p e r a z i n e m o i e t y of r i fampin ( s e e accompanying s t r u c t u r e ) i s l e n g t h e n e d ( i . e . , from m e t h y l t o o c t y l ) i t becomes an e f f e c t i v e i n h i b i t o r . Lengthening of t h e 4 - s i d e c h a i n by a d d i n g a r o m a t i c s u b s t i t u e n t s may a l s o y i e l d d e r i v a t i v e s which i n h i b i t MSV polymerase a c t i v i t y .
I CH,
'0
3-(4-methypiperazinyliminomethyl)r i f a m y c i n SV ( r i f a m p i n )
The 3-hydrazonomethyl d e r i v a t i v e s of r i f a m y c i n SV w i t h b u l k y sPde c h a i n s ( c y c l o h e p t y l and c y c l o o c t y l g r o u p s ) a r e e x c e l l e n t i n h i b i t o r s of MSV p o l y m e r a s e , In 3-formyl r i f a m y c i n SV oxime d e r i v a t i v e s , good c o r r e l a t i o n i s s e e n between t h e l e n g t h of t h e a l k y l - o - s u b s t i t u e n t and RNA polymerase i n h i b i t o r y a c t i v i t y . Rifamycin SV-3-substituted d e r i v a t i v e s o t h e r t h a n 3-iminomethyl, 3-formylhydrazo, o r 3-formyl oxime, a r e e i t h e r i n a c t i v e o r on$minimally active a g a i n s t RNA p o l y m e r a s e s . Very low conc e n t r a t i o n s of some of t h e s e d e r i v a t i v e s may i n h i b i t reverse t r a n s c r i p t a s e a c t i v i t y , p r o b a b l y by combining w i t h t h e enzyme d i r e c t l y r a t h e r t h a n by a f f e c t i n g t h e t e m p l a t e t These compounds do n o t m a t e r i a l l y a f f e c t DNAdependent DNA p o l y m e r a s e s . Yang, e t a l . 3 7 r e p o r t e d i n h i b i t o r y a c t i v i t y of some r i f a m y c i n SV d e r i v a t i v e s a g a i n s t normal and leukemic human lymphocyte-derived and muri n e (Rauscher) leukemia v i r u s - d e r i v e d DNA polymerases , The 3 - p i p e r a z i n o iminomethyl d e r i v a t i v e s and 3 - ( 2 , 4 - d i n i t r o p h e n y l hydrazonomethy1)rif amycin Sv were more i n h i b i t o r y a g a i n s t v i r a l polymerase t h a n a g a i n s t normal lymphocyte-derived DNA polymerase, and a l s o p r e f e r e n t i a l l y i n h i b i t e d t h e leukemic lymphocyte-derived DNA polymerase, b u t n o t t o t h e same e x t e n t as t h e v i r a l polymerase. T h i s s u g g e s t s a p o s s i b l e a n t i v i r a l o r a n t i l e u k e m i c a c t i v i t y f o r these preparations.
G?aconcluded t h a t l a r g e and hydrophobic s u b s t i t u e n t s Tischler, a t t h e 3 - p o s i t i o n are a s s o c i a t e d w i t h h i g h p o t e n c y i n s t u d i e s of r e v e r s e
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t r a n s c r i p t a s e ( i n h i b i t i o n by r i f a m y c i n d e r i v a t i v e s ) . Gurgo, e t a l . 3 9 r e v i e w e d t h e s t r u c t u r a l r e l a t i o n s h i p s of 180 rifamyDNA polymerase and a l s o concluded t h a t s u i t a b l e m o d i f i c a t i o n s of r i f a m y c i n SV d e r i v a t i v e s i n t h e 3 - p o s i t i o n can i n h i b i t v i r u s - d e r i v e d RNA-dependent DNA polymerase a t low c o n c e n t r a t i o n s (5-20 u g / m l ) . The a n s a r i n g s t r u c t u r e is n e c e s s a r y f o r e f f e c t i v e i n h i b i t i o n of b o t h b a c t e r i a l DNA-dependent RNA polymerase and MSV RNA-dependent DNA polymerase a c t i v i t y . The b a c t e r i a l DNA-dependent RNA polymerase may be i n h i b i t e d by r i f a m y c i n SV d e r i v a t i v e s w i t h o u t a 3 - s i d e c h a i n , b u t f o r s u c c e s s f u l i n h i b i t i o n of t h e MSV RNAdependent DNA polymerase a l a r g e 3 - s u b s t i t u e n t is e s s e n t i a l . Compounds s u c h as s t r e p t o v a r i c i n and topolymycin, which c o n t a i n no 3 - s u b s t i t u e n t s b u t have t h e a n s a r i n g , i n h i b i t t h e b a c t e r i a l RNA p o l y m e r a s e , S t r e p t o v a r i c i n s A, C , and D do n o t a p p r e c i a b l y i n h i b i t t h e MSV polymerase o r do s o o n l y w i t h weak a c t i v i t y , Geldanomycin, an ansomycin w i t h o u t a 3-subs t i t u e n t , does n o t i n h i b i t MSV polymerase a t 100 ug/ml. F u r t h e r s t u d y of t h e a n t i v i r a l and a n t i n e o p l a s t i c p r o p e r t i e s of t h e s e d e r i v a t i v e s i s des i r a b l e . 40 - a
tin SV and r i f a m y c i n B d e r i v a t i v e s a g a i n s t MSV RNA-dependent
I n t e r f e r o i t and I n t e r f e r o n I n d u c e r s
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I n t e r f e r o n and i n t e r f e r o n i n d u c e r s have been r e c e n t l y -d.hl--T The e f f e c t i v e n e s s of l o c a l s e c r e t o r y i n t e r f e r o n i n induced i n f e c t i o n a p p e a r s t o be l i m i t e d and p r o b a b l y unrel a t e d t o i t s a n t i v i r a l a c t i v i t y .44 The l o c a l a p p l i c a t i o n of i n t e r f e r o n t o t h e n a s a l mucous membranes p o v i d e d p r o t e c t i o n a g a i n s t s u b s e q u e n t c h a l l e n g e w i t h a r h i n o v i r u s and p a r a i n f l u e n z a I v i r u s i n monkeys45 and ag a i n s t r h i n o v i r u s 4 c h a l l e n g e i n man.46 Large d o s e s and a p r o l o n g e d c o u r s e of p o s t - c h a l l e n g e a p p l i c a t i o n s were n e c e s s a r y t o a c h i e v e t h i s e f f e c t . T o p i c a l u s e of i n t e r f e r o n i n d u c e r s i n HSV k e r a t i t i s h a s b e e n reviewed .47 N o n - i n t e r f e r o n a n t i g e n i c i m p u r i t i e s i n v i r u s - s t i m u l a t e d c e l l d e r i v e d i n t e r f e r o n p r e p a r a t i o n s have been o b s e r v e d , 4 8 b u t t h e i r s i g n i f icance i s u n c e r t a i n . Induced i n t e r f e r o n may n o t b e t h e s o l e f a c t o r i n v o l v e d i n t h e augmented h o s t r e s p o n s e t o v i r a l i n f e c t i o n a s s o c i a t e d w i t h p o l y n u c l e o t i d e a d m i n i s t r a t i o n . 4 9 The improlred e f f i c a c y of i n t e r f e r o n i n c'ucers p l u s s p e c i f i c ganima g l o b u l i n t h e r a p y i n HSV k e r a t i t i s and v a c c i n i a i n f e c t i o n s (even i n t h e immune-suppressed h o s t 5 O y 5 1 ) , and of i n t e r f e r o n i n d u c e r s p l u s vaccines52, h a s b e e n r e p o r t e d . The a n t i v i r a l a c t i v i t y of p o l y n u c l e o t i d e i n t e r f e r o n i n d u c e r s is dependent upon many f a c t o r s . 5 3 I n Poly I : C t h e more s t r i n g e n t c o n t i n u i t y and b a s e p a i r i n g r e q u i r e m e n t s of t h e p o l y I s t r a n d l i m i t s i g n i f i c a n t modi f i c a t i o n s t o t h e p o l y C s t r a n d . The s y n t h e s i s of g u a n i d i n e o r u r i d i n e "loops" i n t h e p o l y C s t r a n d may b e a s s o c i a t e d w i t h more r a p i d h y d r o l y s i s , l e s s e n e d t x i c i t y , and p r e s e r v a t i o n of t h e a n t i v i r a l i n t e r f e r o n - i n d u c i n g a c t i v i t y . 58 A series of b i s - b a s i c s u b s t i t u t e d a r o m a t i c compounds h a v i n g d i r e c t a n t i v i r a l and i n t e r f e r o n - i n d u c i n g e f f e c t i v e n e s s h a s been d e s c r i b e d . 6 9 4 2 The b i s a l k a m i n e esters of f l u o r e n o n e , f l u o r e n o l and f l u o r e n e had dimini s h i n g a n t i v i r a l p o t e n c y , r e s p e c t i v e l y . B i s @-dibutylaminopropy1)-9-oxof luorene-2,7-dicarboxylate d i h y d r o c h l o r i d e p r o l o n g e d t h e s u r v i v a l of mice
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a g a i n s t EMC, v a c c i n i a , and S e m l i k i F o r e s t v i r u s i n f e c t i o n s . S t r u c t u r e a c t i v i t y r e l a t i o n s h i p s h a v e been e l u c i d a t e d and t h e f i r s t member of t h i s c l a s s of drugs--tilorone--has been e x t e n s i v e l y s t u d i e d . Another low m o l e c u l a r w e i g h t i n t e r f e r o n i n d u c e r , BL-20803 , h a s a l s o been r e p o r t e d t o b e s u c c e s s f u l i n i n h i b i t i n g v a c c i n i a 1 i n f e c t i o n .55 P y r i m i d i n e N u c l e o s i d e s - An improved a n i m a l model f o r t h e s t u d y of idoxu r i d i n e (IUDR) and t h e e f f i c a c y of t o p i c a l a n t i v i r a l t h e r a p y i n t h e i m mune h o s t h a s been d e s c r i b e d . 5 6 T o p i c a l a p p l i c a t i o n of Poly I : C enhances t h e e f f i c a c y of IUDR t h e r a p y f o r HSV k e r a t i t i s and d e l a y s t h e emergence of r e s i s t a n t v i r u s .57 T r i f l u o r o t h y m i d i n e (F3T) h a s been found t o b e less t o x i c t o c o r n e a l e p i t h e l i u m , i n a n i m a l s and man t h a n are IUDR and c y t o s i n e a r a b i n o s i d e The i n v i t r o and i n v i v o spectrum of anti-DNA v i r u s a c t i v i t y and (Ara-C). t h e marked s o l u b i l i t y i n aqueous s o l u t i o n r e p r e s e n t d e s i r a b l e p r o p e r t i e s . I n a c l i n i c a l t r i a l comparing F3T and IUDR i n HSV k e r a t i t i s i n man,58 t h e r e w a s more r a p i d h e a l i n g i n t h e F3T group t h a n i n t h e IUDR g r o u p , w i t h a 7.5% v e r s u s a 39% f a i l u r e r a t e , r e s p e c t i v e l y , The p o t e n t i a l f o r v i s c e r a l a b n o r m a l i t i e s i n t h e newborn a n i m a l , esp e c i a l l y i n v o l v i n g t h e r e t i n a , makes t h e u s e of s y s t e m i c IUDR a s e r i o u s u n d e r t a k i n g f o r t h e t h e r a p y of c o n g e n i t a l HSV o r CMV i n f e c t i o n . 5 9 The v a l u e of IUDR t h e r a p y i n s y s t e m i c HSV i n f e c t i o n is s t i l l c o n t r o v e r s i a l , and a new a p p r o a c h t o i t s u s e h a s been o u t l i n e d 6 0 t o minimize i t s t o x i c side e f f e c t s . A controlled c l i n i c a l t r i a l is s t i l l required t o establish e f f i c a c y i n HSV e n c e p h a l i t i s . Study i n a n i m a l models61762 b s emphasized t h e need f o r d e f i n e d p r o t o c o l s of d r u g a d m i n i s t r a t i o n . I n an a s s e s s m e n t of t h e i n v i t r o s u s c e p t i b i l i t y of HSV t o a n t i v i r a l d r u g s , u t i l i z i n g a p l a q u e r e d u c t i o n a s s a y , 6 3 a r a b b i t kidney c e l l c u l t u r e s y s t e m produced lower M I C ' S t h a n d i d a h a m s t e r kidney c e l l s y s t e m , Of t h e two t y p e s of HSV t e s t e d ( t y p e s I and 11, a t o t a l of 2 1 s t r a i n s ) , b o t h exh i b i t e d a n i n v i t r o spectrum f o r s u s c e p t i b i l i t y t o IUDR, which may acc o u n t f o r some of t h e d i f f e r e n c e s p r e v i o u s l y r e p o r t e d . HSV-I w a s most c o n s i s t e n t l y i n h i b i t e d by IUDR and Ara-C, w h i l e Ara-C w a s most e f f e c t i v e a g a i n s t HSV-I1 Adenine a r a b i n o s i d e (Ara-A) and hypoxanthine-arabinoside were less e f f e c t i v e a g a i n s t b o t h HSV t y p e s .
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2 '-Deoxyuridine (UDR) enhances t h e a c t i v i t y of 5 - f l u o r o u r a c i l (FU) and 5-fluoro-2' -deoxyuridine (FLJDR) a g a i n s t L-1210 leukemia and adenocarcinoma-755 i n mice, The r e s u l t a n t s t a b i l i z a t i o n of t h e FUDR formed & v i v o from FU i n t h e p r e s e n c e of added UDR64 might p r o v i d e an i n t e r e s t i n R approach t o augmenting t h e a n t i v i r a l e f f i c a c y of IUDR as w e l l .
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Ara-C Ara-C h a s been u t i l i z e d i n t h e t h e r a p y of MSV, CMV, and v a r i c e l l a z o s t e r (VZ) i n f e c t i o n s w i t h i n c o n c l u s i v e c l i n i c a l r e s u l t s .65-71 The o n l y c o n t r o l l e d c l i n i c a l t r i a l r e p o r t e d f a i l e d t o reveal any b e n e f i t from t h e u s e of A r a - C i n d i s s e m i n a t e d z o s t e r and s u g g e s t e d t h a t t h e d i s e a s e may b e prolonged by i t s use.72 A r a - C may a d v e r s e l y a f f e c t t h e d e v e l o p i n g n e r v o u s
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system,73 and i t s u s e i n c o n g e n i t a l CMV and HSV i n f e c t i o n should be cont r o l l e d i n view of t h e u n c e r t a i n outcome of b o t h t h e d i s e a s e and t h e t r e a t m e n t . 7 4 HSV i n h i b i t i o n i s a p p a r e n t i n v i v o i n r a t s , even w i t h delayed t h e r a p y , b u t h i g h doses appear l e s s T f T E 3 i v e than lower ones ,75 perhaps because of i n c r e a s e d immunosuppression, as i n t h e c a s e of dissemin a t e d z o s t e r . The r e p o r t of a r e m i s s i o n a s s o c i a t e d w i t h Ara-C therapy76 i n a c a s e of p r o g r e s s i v e m u l t i f o c a l leukoencephalopathy (PML) i s extremely e x c i t i n g . PML may be caused by a papova v i r u s , and t h u s f u r t h e r t r i a l s a r e indicated. The f r e q u e n t l y observed temporary i n h i b i t i o n of v i r a l m u l t i p l i c a t i o n i n -v i t r o , 7 7 and shedding d u r i n g Ara-C t h e r a p y , as w e l l as k i n e t i c s t u d i e s _ s u g g e s t t h a t A r a - C does n o t i n h i b i t t h e development of a l r e a d y e x i s t i n g v i r a l genomes i n t o mature v i r i o n s and t h a t c e l l u l a r damage may occur bef o r e t h e s t a g e of v i r a l DNA s y n t h e s i s t h a t Ara-C i n h i b i t s . S t r u c t u r a l m o d i f i c a t i o n of Ara-C may r e n d e r i t more r e s i s t a n t t o deamination -i n v i v o . The u s e of a p o o r l y s o l u b l e p r o d r u g , such as 1-parabinofuranosylcytosine-5'-adamantoate, o r o t h e r 5 ' - a c y l a t e s , r e s u l t s i n a p r o l o n g a t i o n of a c t i v e blood l e v e l s .78 C y c l o c y t i d i n e y i e l d s Ara-C by in v i v o h y d r o l y s i s and is less t o x i c and more e f f e c t i v e a g a i n s t t h e L-1210 mouse leukemia t h a n is Ara-C. S u b s t i t u t i o n a t C-6 of t h e p y r i m i d i n e r i n g improves r e s i s t a n c e t o deamination i n v i t r o , b u t competing d e g r a d a t i o n r e a c t i o n s i n v i v o r e s u l t i n no i n c r e a s e d s t a b i l i t y ,
L .
- _ _ L
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C y c l i c Ara-CMP s y n t h e s i s h a s been d e s c r i b e d . 7 9 I n v i t r o a n t i v i r a l a c t i v i t y a g a i n s t HSV I and 11, v a c c i n i a and myxoviruses w a s comparable t o I n -9v i v o HSV I k e r a t i t i s responded w e l l t o t o p i c a l t h e r a p y , t h a t of A r a - C . and i n t r a c e r e b r a l i n j e c t i o n r e s u l t e d i n i n h i b i t i o n of HSV and v a c c i n i a v i r u s e s . The i n c r e a s e d c e l l p e n e t r a t i o n of t h i s compound should make i t s f u r t h e r s t u d y of i n t e r e s t .
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Adenine A r a b i n o s i d e (Ara-A) This compound h a s been reviewed.80 The in i n h i b i t o r y e f f e c t a g a i n s t DNA v i r u s e s a p e a r s t o b e dose r e l a t e d , w i t h e q u i v a l e n t s e n s i t i v i t y of HSV I and II.gl The l a c k of noteworthy t o x i c i t y a s s o c i a t e d w i t h Ara-A has l e d t o c o n t r o l l e d c l i n i c a l t r i a l s i n HSV i n f e c t i o n s i n man.82 Marked d e c r e a s e i n v i r a l shedding and e a r l i e r h e a l i n g of o r o f a c i a l HSV I was n o t e d . Less e f f e c t was n o t e d on g e n i t a l HSV I1 l e s i o n s . Encouraging r e s u l t s were r e p o r t e d i n n e o n a t a l HSV i n f e c t i o n , e s p e c i a l l y when t r e a t e d e a r l y . The v i r u s t a t i c e f f e c t of A r a - A on CMV i n f e c t i o n s i n man83 and t h e e q u i v a l e n t e f f i c a c y t o IUDR i n t h e t o p i c a l t h e r a p y of HSV k e r a t i t i s 8 4 have been r e p o r t e d , M o d i f i c a t i o n s of Ara-A s u g g e s t t h a t changes i n t h e c a r b o h y d r a t e n o i e t y a t C-3 may eliminate t h e a n t i v i r a l a c t i v i t y ,85 The w a t e r - s o l u b l e N6-(dimethylamino)methylene d e r i v a t i v e of A r a - A w a s found t o p r o t e c t ag a i n s t HSV and v a c c i n i a , probably by c o n v e r s i o n t o Ara-A i n vivo.86 A series of 5 ' - e s t e r s of Ara-A showed i n v i t r o e f f i c a c y of t h e 5 I - p a l m i t a t e ; n o a c t i v i t y a g a i n s t RNA v i r u s e s w a s
not-.
A number of 5-butylpyrimidine n u c l e o s i d e s r e v e a l e d only l i m i t e d ac-
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t i v i t Y a t h i g h c o n c e n t r a t i o n s - 8 8 Carboxamide-ribonucleosides r e v e a l e d i n v i t r o a c t i v i t y a g a i n s t HSV I , r h i n o v i r u s 13 and p a r a i n f l u e n z a 3 and & v i v o a c t i v i t y a g a i n s t i n f l u e n z a A2.89,90
A
V i r a z o l e , a p-D-ribofuranosyl d e r i v a t i v e , h a s demonstrated a broad spectrum of i n v i t r o i n h i b i t o r y a c t i v i t y a g a i n s t a d e n o v i r u s , HSV I and 11, v a c c i n i a , myxoma v i r u s , p a r a i n f l u e n z a , r h i n o v i r u s , Coxsackie v i r u s and i n f l u e n z a A and B,91 It i s more p o t e n t t h a n o t h e r a n t i - i n f l u e n z a 1 compounds i n v i t r o g 2 and i s e f f e c t i v e a g a i n s t HSV k e r a t i t i s and l o c a l i z e d v a c c i n i a , i n v i v o . The a n t i F r i e n d leukemia v i r u s , i n f l u e n z a A and p a r a i n f l u e n z a I -v i r a l e f f e c t may b e due t o t h e i n h i b i t i o n of guanosine-5'-phosphate synthesis i n the infected ce11.9~ Benzimidazole D e r i v a t i v e s - The a c t i v i t y of hydroxy b e n z y l benzimidazole (HBB) a g a i n s t p i c o r n a v i r u s e s h a s been a s t i m u l u s t o continued s t u d y of i t s d e r i v a t i v e s . 2- (alpha-Amino b e n z y l ) benzimidazole (ABB) and 1-phenyl ABB were s i g n i f i c a n t l y e f f e c t i v e i n r e d u c i n g c y t o p a t h i c e f f e c t of ECHO-12 v i r u s a t n o n t o x i c c o n c e n t r a t i o n s .94 A l k y l a t i o n o r a c e t y l a t i o n of t h e amino f u n c t i o n r e s u l t e d i n l o s s of a n t i v i r a l a c t i v i t y . The p r e s e n c e of a 5- or 1-methyl group i n t h e N-methylamino series i n c r e a s e d t h e a c t i v i t y a g a i n s t ECHO-12. The a d d i t i o n of a phenyl s u b s t i t u e n t i n t h e 1 - p o s i t i o n of t h e alpha-methoxy and alpha-amino d e r i v a t i v e s of HBB a l s o i n c r e a s e d t h e a c t i v i t y a g a i n s t t h e ECHO v i r u s e s . The a c t i v i t y of a series of 1 - a l k y l d e r i v a t i v e s of HBB w a s dependent Unsatuupon t h e l e n g t h of t h e s i d e c h a i n , b e i n g maximal a t C-3 o r C-4.95 r a t i o n i n t h i s s i d e chain did not g r e a t l y influence a c t i v i t y , Bisbenzimidazoles have demonstrable a c t i v i t y a g a i n s t r h i n o v i r u s e s g 6 and a r e n a v i r u s e s .97 H e t e r o c y c l i c analogues of HBB have n o t shown s p e c i f i c a c t i v i t y a g a i n s t w i l d s t r a i n s of p o l i o v i r u s . 9 8 Adamantine D e r i v a t i v e s - These compounds c o n t i n u e t o b e of i n t e r e s t , Adamantine s p i r o compounds have di.splayed a broad range of i n v i t r o a c t i v i t y a g a i n s t myxoviruses, paramyxoviruses, and some p i c o r n a v i r u s e s . Of a s e ries of N - s u b s t i t u t e d adamantine s p i r o compounds, N-methyladamantanes p i r o - 3 ' - p y r r o l i d i n e had t h e b r o a d e s t spectrum of a c t i v i t y Six- and seven-membered r i n g analogues were found t o b e mere c y t o t o x i c . 100 C l i n i c a l t r i a l s 1 0 1 r e v e a l e d p r o p h y l a c t i c e f f i c a c y a g a i n s t i n f l u e n z a A/HK 68 b u t n o t a g a i n s t r h i n o v i r u s e s 2 and 9 . A z i r i d i n e s and annulene were compared t o 1-amino adamantine and found t o p o s s e s s less 5 v i t r o a n t i - i n f l u e n z a 1 a c t i v i t y . 1 0 2 F u r t h e r c l i n i c a l t r i a l s w i t h c y c l o - ~ c t y l a m i n e r~e~v ~ ealed prophylactic efficacy assoc i a t e d w i t h t o p i c a l u s e i n v o l u n t e e r s challenged w i t h A2 i n f l u e n z a , I s o q u i n o l i n e s - These compounds have shown i n v i t r o a c t i v i t y a g a i n s t r h i n o v i r u s e s , b u t have had l i t t l e o r no s i g n i f i c a n t p r o p h y l a c t i c e f f i c a c y i n human t r i a l s . 104,105 Isoprinosine
-
This drug h a s demonstrated a v a r i a b l e spectrum of a c t i v i t y
Chap. 1 4
Schwar t z
A n t i v i r a l Agents
135
i n v i t r o and i n viv0.106,107 a g a i n s t s e v e r a l DNA and RNA v i r u s e s -A recent c l i n i c a l t r i a l s u g g e s t s e f f i c a c i n t h e e a r l y t r e a t m e n t of p r i m a r y h e r p e s p r o g e n i t a l i s i n f e c t i o n s i n man. 108 _.-
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485 ( 1 9 7 2 ) . McCracken, J r . , J.P. Luby, J . P e d i a t r . 80, 488 ( 1 9 7 3 ) . S.A. P l o t k i n , J. P e d i a t r . 8 0 , 493 (1972). W.A. F a r r i s , M.E. B l a w , A r x , N e u r o l . 27, 99 (1972). V.N; Mankad, A.A. Gershon, P.A. B r u n e l z J . I n f . D i s . __ 1 2 8 , 442 (1973). D.A. S t e v e n s , G.W. J o r d a n , T.F. Waddell, T.C. Merigan, N. Eng. J . Med. 282, 873 (1973). S . Ashwal, I. F i s h , M. F i n g g o l d , P.A. B r u n e l l , A b s t . 1 3 t h I n t e r s c i . Conf. A n t i m i c r o b . Ag. and Chemother. S e p t . 19-21, 1973, Washington, D.C., p a p e r 247. H.S. P a n i t c h , J . R . B a r i n g e r , Arch. Neurol. 28, 37 (1973) H.E. R e n i s , A n t i m i c r o b , Ag. and Chemother. 4 , 439 (1973). W.K. Bauer, AP.P. T u r e l , Jr., K.P. Johnson, JAMA 226, 174 ( 1 9 7 3 ) . L. V a c z i , E , Gonczol, Acta V i r o l . 1 7 , 189 (1973). R.E. N o t a r i , J. Pharm. S c i . 62, 8 6 5 ( 1 9 7 3 ) . L.N. Simon, R.K. Robbins, J . M e d . Chem. 15, 1215 (1972). L.T. Ch'ien, F.M. S c h a b e l , J r . , C.A. A l f z d , J r . , i n S e l e c t i v e Inh i b i t o r s of Viral F u n c t i o n s , Wm. Carter, Ed., CRC P r e s s , C l e v e l a n d , (1973). B . J . S l o a n , F.A. Miller, I . W . McLean, J r . , Antimicrob. Ag. and Chemot h e r . 3 , 74 (1973). L.T. CK'ien, N . J . Cannon, L . J . Charamella, W.E. Dismukes, R.J. W h i t l e y , R.A. Buchanan, L.A. A l f o r d , Jr., J . I n f . D i s . 128,658(1973). N . J . Cannon, L.T. C h ' i e n , W.E. Dismukes, R.A. Buchanan, C.A. A l f o r d , C l i n . R e s . 2 1 , 594 (1973). D . P a v a n - L a c s t o n , C.H. Dohlman, Amer. J. Ophthal. 74, 8 1 (1972). K, Miyai, R.K. Robbins, R.L. Tolman, J. Med. Chem. 15, - 1092 ( 1 9 7 2 ) . S . H a n e s s i a n , J. Med. Chem. 1 6 , 290 (1973). H.E. R e n i s , D.T. Gish, B.A. c u r t , E,E. Eidson, W . J . Wechter, J . Med. Chem. l6, 754, (1973). E.H. Hamamura, K. S a t o , J . G . M o f f a t t , J . Med. Chem. 15, 1 0 6 1 (1972). J.T. Witkowski, R.K. Robbins, G.P. Khase, R.W. Sidwe'iT, J . Med. Chem. 1 6 , 935 (1973). Y , Togo, Antimicrob. Ag, and Chemother. 4 , 6 4 1 ( 1 9 7 3 ) . J.T. Witkowski, R.K. Robbins, R.W. S i d w e i l , L.N. Simon, J. Med. Chem. 15, 1150 (1972). Y. Togo, Antimcirob. Ag. and Chemother. 4 , 6 4 1 (1973). D.G. S t r e e t e r , J . T . Witkowski, G.P. Khare, P r o c . Nat. Acad. S c f . USA 70, 1174 (1973)e F. G a u l t i e r i , G. Brody, A.H. F i e l d s t e e l , W.A. S k i n n e r , J . Med. Chem. 1 5 , 420 ( 1 9 7 2 ) . D.G. O ' S u l l i v a n , A.K. Wallis, J. Med. Chem. 15, 103 (1972). W.R. Roderick, C.W. Nerdeen, J r . , A.M. Von E G h , R.N. A p p e l l , J . Med. Chem. 1 5 , 655 (1972) J.P. S t e l l a 7 K . D . Yankaskas, C. J. P f a u , A b s t . 1 3 t h I n t e r s c i . Conf. on Antimicrob. Ag. and Chemother., S e p t . 19-21, 1973,Washington,D.C., p a p e r 147. H. Berner, H. Reinshagen, J. Med. Chem. 1 6 , 1296 (1973). G.H.
138 99.
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a
Chapter 15. Antineoplastic Agents A . Bloch
Roswell Park Memorial Institute, Buffalo, New York 14203
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Introduction A review of the progress made during 1973,with respect to the development of new antineoplastic agents, clearly reflects the profound difficulties encountered in the development of such drugs. Little concrete information i s , generally, available concerning biochemical differences between tumor and normal tissues which could be exploited for selective therapy. Thus, with the exception of some of the hormones, the currently used antitumor agents a r e essentially inhibitors of cell growth, normal as well a s tumor, and it is for this reason that some host toxicity is an almost invariable component of tumor chemotherapy. However, subtle biochemical differences between tumor and normal tissues are, in some instance , sufficient to allow for selective chemotherapeutic intervention, and remarkable progress has been made in the treatment of various neoplasms 1
.
The question facing the medicinal chemist is not whether new antitumor agents should be developed-on that point the cancer chemotherapists a r e in nearly unanimous agreement the question is what type of agents carry the greatest promise for success. Here the opinions diverge, depending on one's chemical interests, the type of tumor one wishes to treat, and the analogies one draws from past clinical or experimental experience gained with a given class of compounds. For, the development of antineoplastic agents falls, at best, under the heading of rational empiricism. Thus , the systematic introduction of small structural changes into metabolite molecules led to such useful antimetabolites a s methotrexate, 6 mercaptopurine, 5-fluorouracil and 1-P-D_-arabinofuranosyl cytosine. The routine screen for antibiotics provided such valuable agents as actinomycin D and daunomycin, and serendipity paired with perspicacity led to the use of alkylating agents such a s the nitrogen mustards, mitotic inhibitors such a s vincristine, and hormones including the corticosteroids and sex hormones. A l l further cancer drug development has relied, essentially, on these approaches. When one type of compound is found active, innumerable structural modifications a r e then carried out,aimed at decreasing toxicity and improving selectivity. New insights COP cerning novel structures with antitumor activity a r e encountered only infrequently. The antineoplastic agents reported during 1973 follow this pattern, and in reporting on some of the agents, an attempt is made to correlate structure with antitumor activity. From such correlations, rational advances in cancer chemotherapy may, eventually, emerge.
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Vitamin Analogs
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The folate analog methotrexate is of great value in the treat-
140 -
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ment of acute leukemia, choriocarcinoma and some other solid tumors. In some instances this toxIts utility is limited by its pronounced toxici 3 denoting the administration icity can be ameliorated by leucovorin "rescue", of folinic acid to a patient treated at some prior interval with high doses of methotrexate
%;
.
The observation that esterification of nucleoside analogs can improve their Estertherapeutic properties, has now been applied to methotrexate a s well 4 y ification of the carboxyl groups of the glutaN N C02R moyl moiety of methotrexate with primary o r secondary alcohols of various chain lengths Ri)2CCH2CH2C?NHCO~NMeCH2~N I (C1- C ) provided derivatives (Iwhich ) NH:! 8 R=Me, E t , B ~ ( C , H ' ) ~ M ~( C . H 2 ) 5 Me. ( C H 2 ) 7 ~e were, however, only a s active a s the parent compound in increasing the life span of mice r with both intracerebrally and i.p. implanted leukemia L-1210. A s early a s 15 minutes after i.p. administration, 80-90% of the inhibitory activity in the serum was due to free methotrexate. It is of interest, that these lipophilic esters did not apparently enhance the transport of methotrexate through the blood-brain barrier, as evidenced by the lack of increased activity against the intracerebrally implanted tumor. Of course, the observed rapid hydrolysis of the esters may have abolished the potential therapeutic advantage. The increased transport of methotrexate through the barrier is desired,to achieve concentrations high enough to be effective against leukemic cells sequestered in the brain.
.
-
1x,;16""'
Among newly synthesized active inhibitors of dihydrofolate reductase is the water-soluble 1-[3-chloro-4- (m-dimethylcarbamoylbenzyloxy)phenyl]-4 ,6-diamino1,2-dihydro-2, 2-dimethyl-s-triazine ethanesulfonate6 , which is markedly effective against Walker ascites carcinosarcoma 256 in vivo. In a series of 2,4-diamino-6,7-bis (aralkyl)pteridines, the 3,4-dimethylbenzyl derivative was the most potent inhibitor of the growth of S. faecium in vitro , but was inactive against leukemia L-1210 o r P 1534 in the m o u s n r o c e e d i n g on the assumption that a decrease in the electron density at position 10 (N1o) of folate may affect its one carbon transfer function, the synthesis of neohomofolic acid (N-[p - [ [2-amino-3,4dihydro-4-0~0-6-pteridinyl) methyl] aminolphenylacetyl] glutamic acid was carried out Although toxic to animals, the compound showed little inhibitory activity towards dihydrofolate reductase, and was inactive against leukemia L-12 10 in mice. Correlations of the structure of various 2 ,4-diaminopyrimidine derivatives with their activity a s dihydrofolate reductase o r tumor inhibitors suggest that the extent of activity is related to the steric bulk and the hydrophobicity of the 5- substituent 9 9 lo. With an adamantyl group at position 5 , the introduction of an alkyl C ) at position 6 also increased activityx1. chain (C 1 3
'.
-
Among a series of 6-halogen-substituted vitamin B (pyridoxol, pyridoxal and 6
Chap. 15
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141
--
pyridoxamine) analogs, only the fluoro derivatives inhibited the in vitro growth of two tumor cell lines in the presence of low pyridoxal concentrations, but they were less active than 4-deoxypyridoxine 12. In c0ntrast~4-deformyl-4-vinylpyridoxal was shown, in vivo , to be a more potent vitamin B antagonist than is 4-deOW6 pyridoxine 1 3 7
-
Amino acid analogs do not, currently, play a significant part in cancer chemotherapy. The therapeutic use of DON (diazo-oxo-norleucine) and azaserine has essentially been abandoned. However, because of the clinical utility of L-asparaginase14, the preparation of analogs which interfere with the synthesis o r utilization of asparagine appeared indicated. Among such agents, _L -2-amino-5-bromo4-oxopentanoic acid and its 5-chloro-analog inhibited the synthesis of L-asparagine by P 815 Y tumor cells, as well a s the growth of asparagine dependent G5178 Y lymphoblasts in culture 15. A vivid example of the value of perspicacity i n drug development is provided
by the evaluation of mimosine (II) as an antitumor agent. Because sheep eating the browse tree, Leucaena glauca, shedtheir wool and expe0pNCH2CH(NH2)C02H rience weight loss and reversible infertility a pattern of toxicity seen with several of the currently used antitumor MO drugs -the active principle, mimosine, was considered a poII tential antineoplastic agent 16. When provided in the drinking water (0.4-0.8 g / 100 ml), a dose dependent reduction in the growth of Walker 256 carcinosarcoma and a concomitant prolongation of life-span was achieved. Similarly, by taking into account that prolactin is essential for the development of spontaneous and carcinogen-induced mammary tumors in rats 17,the lowering of prolactin levels by _L -dopa [3-(3,4-dihydroxyphenyl-L-alanine] was envisioned to decrease tumor growth, which was indeed achieved in rats bearing carcinogeninduced mammary carcinomas18. Screening for new agents has furnished (as,5S)a-amino-3-chloro-4,5-dihydro-5-isoxazoleacetic acid (111) from KrFtoniyces sviceus , which has significant act'vity against leukemia L-12 10 in vivo, with min19 imal toxicity Of interest to the researcher using tissue is the observation that pyrrolidone carboxylic acid, cultures CI ;$CH(NH~)CO~H formed spontaneously upon incubation of glutamine,at 37", in phosphate o r bicarbonate medium, inhibited the growth of a 20 line of hamster fibrosarcoma requiring glutamine for growth. 111
-
'Y
.
The utility of pyrimidine and purine analogs in cancer chemotherapy (e.g. 6-mercaptopurine, 5- fluorouracil, arabinosyl cytosine) serves a s a continued stimulus for the preparation of new compounds of this type. Among these, 3-deazauridine [ 1-(&_D-ribofuranosyl)-4-hydroxy-2 -pyridone] was markedly active against leukemia L-1210 in vivo 21. On the other hand, the 5-fluoro derivative of this nucleoside was i n a c t i v e 2 . Another pyrimidine nucleoside modified at the 3-position, 3- (2-deoxy- P-_D -erythro -pentofuranosyl)- 2,3-dihydro-l,3-6H-
142
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oxazine-2,6-dione ) was found moderately active against leukemia L-12 10 cells -in vitro‘, whereas 1,2-dihydro-1-(2-deoxy-P-D- rihofuranosyl)-2oxopyrazine-4-oxide was inactive against two in vitro tumor cell lines, but was a highly potent inhibitor (50% at 4 x 10-11 M) of bacterial HOCHD cells24. This fact is mentioned because bacterial infections a r e a major cause of mortality of cancer patients 25926, and the identificaOH tion of a potentially useful antibacterial agent is acceptable compenrv sation for the lack of antitumor activity. This finding also indicates that the evaluation of agents designed for potential antitumor action should not necessarily cease, once they a r e found to lack this potential.
-
Among nucleosides isolated from natural sources o r synthesized during previous years, pyrazomycin [3 (5)-ribofuranosyl-4-hydroxypyrazole-5 (3) carboxamide] has demonstrated pronounced activity against various experimental tumors in vivo 27. Also reported active28 is a- (but not P) glucopyranosylnordoPan New potential anticancer agents are also found among the puHN’!$-~ C H ~ C H ~ C rine ~ J ~ analogs. Several l-deazapurines (imidazo [4,5-b]pyridine derivatives) were found active against leukemia L-12 10 in v i t r ~ the ~ ~most ; effective of these, ethyl 7- { [bis (diphen3.1) methyl] amino 1 -3H-imidazo [ 4 ,5-blpyridine-5-carbamate provided a 59% increase in life span at a sin le dose of 4 0 CHIOH mg/kg (toxic at 200 mg/kg) 8-Aza-inosine prolonged the life span of the tumor bearing mice by 55%. Among a large V series of 6-ureidopurine~~ l, phenylpropylureidopurine and phenylbutylureidopurine showed some inhibition of a cell line derived from human leukemia, and some unsaturated 6-methylthiopurine nucleosides (e g 9-(2,3-dideoxy-P-g- glyceropent-2 -enopyranosyl)-6-methylthiopurine were markedly cytotoxic to HeLa cell&“2 R.eplacement of the furanose ring with cyclopentane furnished various carbocyclic analogs of purines. The 8-azaadenosine analog [ ( )-trans -3-V-amino-3H- 9 triazolo-[4,5-d]pyrimidin-3-yl)-trans -5- (hydroxymethyl)cis-l,2 -cyclopentane. cyclodiol) was effective against leukemia P 388 but not against L - 1 2 1 0 ~ ~The pentyl analog of 8-azaguanosine and various carbocyclic analogs of 6-substituted purine ribonucleosides were similarly inactive against L-1210 , although some were cytotoxic to human epidermoid carcinoma # 2 cells34, 35. Of interest is the observation that formaldehyde: R.NA (5:l and 10: 1molar ratios) o r formaldehyde: polyadenylic acid (20: 1 molar ratio) significantly prolonged the survival of mice bearing leukemia P-38836.
or);
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--
.
..
*
.
Cyclic Nucleotides R.eports on the inhibition of in vitro and in vivo tumor cell growth by cyclic AMP o r its dibutyryl derivative, and loss of tumorigenicity of certain cell lines treated with these agents have accumulated extensively during recent years. To be added to this list a r e observations on the inhibition of the in vitro growth of human r h a b d o m y o ~ a r c o m ,B16 a ~ ~ melanoma3* ,and murine
--
Chap. 15
Antineoplastic Agents
14 3 -
Bloch
39 2’ P 815 Y cells by N6, 0 -dibutyryl cyclic AMP. The in vivo growth of two hormone-dependent mammary tumors was also arrested by this cyclic nucleotide ester40, and when administered, in vivo, together with aminophylline, cyclic AMP decreased the growth of Ehrlich carcinoma ~ i g n i f i c a n t l y ~The ~ . apparent ability of cyclic AMP to suppress tumorigenicity was demonstrated with human cancer cells (KB) which, after growth in a medium containing this cyclic nucleotide, lost the ability to form solid tumors in hamster cheek pouches42.
Carbohydrates. Relatively few pentose o r hexose derivatives have been prepared as potential antitumor agents during the reporting period. In a series of deoxyfluoro-D-glucopyranoses , only 6-deoxy-6-fluoro-_D-glucose inhibited (up to 90%) R-l lymphoma growth in vivo 43. Polysaccharides, on the other hand, are receiving wider attention. These polymers appear to stimulate the reticuloendothelial tissues, thereby activating the immune defenses of the host. For instance, mannan from Saccharomyces cerevisae injected into mice 1-2 weeks prior to sarcoma 180, prevented the growth of these tumors44. Similarly, zymosan, from 45 yeast cell walls, enhanced the survival of rats bearing a benzpyrene induced tumor But stimulation of the R.ES may not be the only parameter of polysaccharide action, since the chitin derivative (l+ 4)-2 -amino-2-deoxy-B-D-gluco11yranuronan showed marked in vitro -- growth inhibition of leukemia L - E ~ O & ~ ~ I S ~ ~ - .
.
Lipids, too, have not fared prominently as potential antitumor agents, probably due, in part, to the difficulties involved in their isolation and characterization. But such materials do possess potential antitumor activity, a s own by the observation that certain lipid fr tions from group A streptococci and from try88 panosomes (cruzi and lewisi) exert an inhibitory effect on the growth of Ehrlich ascites and sarcoma 180 cells in mice. Indications a r e that it is the free fatty acids and monoglyceride components of these fractions which awount for the observed activity. A glycopeptide fraction, isolated from bovine liver, 4yas also reported markedly effective against the Ehrlich ascites tumor in vivo
ZP
.
Hormone derivatives with reported antitumor activity include 2 or, 3 a-epithio5 a-androstan-17 P 01 50, which suppressed the growth of estrogen-dependent mammary tumors in rats, and enhanced the growth of androgen-dependent mammary carcinoma in mice. Similarly, in a large series of estrone and estradiol analogs prepared, some compounds such a s dl-8-iso-D-homoestrone o r dl-8 -isoor , 17 0-est diol displayed significant activity against mammary tu-D-homo-3 5 3 Androgen derivatives have also been prepared, and 9mars of rat and mouse or-fluoro- 11P-hydroxybenzo [d, el testosterone 17-acetate was reported mark5dly inhibitory to 7 , 12 dimethylbenzanthracene induced mammary tumors in rats’ .
-
.
Enzymes. The clinical utility of L-asparaginase has acted a s a stimulus for the evaluation of other enzymes for possible antitumor activity. A phenylalanine ammonia-lyase from yeast rapidly decreased the plasma phenylalanine and tyrosine
144
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- Chemotherapeutic
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levels in mice, and significantly increased the life span of mice with lymphoblastic leukemia53. Similarly, a L-methioninase from Clostridia effectively decreased the plasma methionine level of rats, and inhibited the growth of Walker carcinosarcoma 25654. A bacterial L-leucine dehydrogenase and a glutaminase increased the survival time of mice wi6Ehrlich ascites carcinoma by 200-300%55, and a pumpkin ascorbate oxidase interfered with the growth of Sarcoma-180 in mice56. Modification of E coli L-asparaginase with maleic anhydride, ethyl acetimidate o r methyl P-dimethylaminopropionimidate gave enzyme derivatives with altered isoelectric points, but not significantly changed catalytic activity. The biological half-life increased with increasing isoelectric points, and this relationship was associated with increased therapeutic effectiveness in leukemic mice 57.
-.
Diphtheria toxin , a protein secreted by lysogenic strains of Corynebacterium diphtheriae , has been shown to be an effective inhibitor of the growth of Ehrlich carcinoma, both ascitic and solid, in mice 58. The effect is thought to be mediated primarily through inhibition of protein synthesis. Similarly, the parasporal crystal ( 6 -endotoxin) of B. thuringiansis has been reported to exert otent anti-Yoshida ascites carcinoma activity, with a high therapeutic index 5 8 Shigella endotoxin, too, caused the regression of some established S.C. o r intradermal tumors in mice, when administered systemically o r directly into the tumor 60
.
.
.
Materials from plant and animal tissues Although empirical in approach, the isolation and evaluation of materials from natural sources has been extraordinarily successful in the development of antimicrobial agents, and has contributed significantly towards obtaining agents of great value in cancer chemotherapy e.g. the antibiotics vincristine, actinomycin D, daunomycin and others). Thus, this source of antineoplastic agents deserves closest attention, since it also reveals information about chemical structures with antitumor activity. In this very restricted review it is obviously impossible to cite all the reports of natural products with antitumor action which appeared during the past year, and only a few examples a r e rovided. Thus, extracts frQm Western Samoan plants (Barringtonia samoensis)!l from lichens (Cetraria S P , ) ~ from ~ , the evening primrose Oenothera caespitosa f3from Steganotaenia araliacea (which yielded two lignan lactoand from leaves of Ptemcarpus indicus and vidalianus 65 had antitumor activity in vitro and/or in vivo The glycosidic lignan derivative 4'-demethylepipodophyllotoxin 9- (4,6-0-2-then lidine-P-D_.-gIucopyranosidea6 (XI), and its ethylidine H a d o g !7 proved h i m y effective against leukemia L-12 10 in mice, and appear to be of value in clinical therapy68. of o\._o - --Lo various derivatives of daunomycin examined:g only 13-dihydrodaunomycin-HCl showed significant antitumor activity O w o 0 -in vivo. Similarly, actinomycin D lactam, [ l ' ,1'-bis(&-threo'if a, P-diaminobutyric acid 1 actinomycin D, had a lower thera0 cn 0 ~ ~ ~ peutic index against Ridgeway osteogenic sarcoma then had
.
--
qo7 HC
HLO
VI
Chap. 15
Antineoplastic Agents
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Bloch
the parent compound7o. Of interest is the observation that ergot alkaloids a r e effective in suppressing the growth of pituitary tumors in rats, relieving thereby the adrenal hypertrophy caused by tumor ACTH71. Tissue extracts with reported antitumor activi include an ethanolic fraction from the tentacles of R.eteterebella queenslandia ,' a tropical sea annelid used by early Hawaiians for cancer therapy73 , and thymic extracts from calf and horse embryosrir4.
-
Platinum compounds and other metal derivatives: The promising antitumor activity of cis-dichloro-diammineplatinum 76 has stimulated the search for related compounds with improved therapeutic properties. A comparison of B p : ety of Pt (II) complexes a s active inhibitors of sarcoma 180 in mice showed 7 that, in general, only neutral complexes exhibited activity, whereas charged species were inactive. C i s - reactive ligands appear essential for antitumor activity, the trans -isomers being relatively inactive. Among newly prepared derivatives, dichloro (4,5-dimethyl-o-phenylenediamine-N,N') platinum (VII increased the survival time of L-1210 bearing mice by 74% 79) Of poMe NH~-. 4 , tential interest is the report8' that sodium selenite, a t 1 I PC!* mg/kg i .p., retarded the growth of various experimental tuMe mors in rats and mice. Also of interest to the medicinal VII chemist may be a discussion on the potential utility of boron8 1 and silicon82 compounds in cancer therapy.
n,,, -
.
Thiosemicarbazones. Because of the pronounced antitumor activity of various Q (N)-heterocyclic carboxaldehyde thiosemicarbazones, some 4 '-diethyleneoxy derivatives were prepared, in an effort to improve the therapeutic index. Among these, 4 -hydroxyisoquinoline-1-carboxaldehyde 4 '-diethyleneoxythiosemicarbazoneand 5-hydroxypyridine-2 -carboxaldehyde 4 '-diethyleneoxythiosemicarbazonewere less toxic but also less active than the corresponding unsubstituted thiosemicarbazones against sarcoma 180 in mice 83. Similarly, the #-phenyl- and ally1 thiosemicarbazone derivatives of 6-formylpurine were less active than the parent compound, o r were inactive against 6-mercaptopurine resistant leukemia L-12 10 in mice 84. Alkylating Agents are continuing to receive attention as antineoplastic agents. In view of the favorable therapeutic indices of some aromatic carbamate mustards in the Walker 256 system, several new compounds were prepared, wherein the 2phenyl _N- phenylcarbamate moiety was linked to bis (1-aziriding1)- and bis (2,2dimethyl- 1-aziridiny1)phosphinate. No improvement of the chemotherapeutic in~ . the other hand, replacement of one dex was achieved by this m ~ d i f i c a t i o n ~On ethyleneimine group in thio-TEPA with a methyl substituted oxypiperidine moiety 86 Ma (VIII), furnished a compound which reportedly was less toxic, and exerted a greater antitumor effect E N than equivalent doses of thio-TEPA Steroidal alkylating Lh
N!.H-Qi* . M4
VIII
146
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Chemotherapeutic Agents
Warren, Ed. 88
agents, such as "estradiol mustard" 87 (M), and some homoaza-steroid mustards showed significant inhibition of the growth of 0 2 C C H z ~ N I C k Z C H 2 C I 1 2 various experimental tumors. A comparison of the effects of "estradiol mustard" with those of its estrogen and alkylating moieties on a transplanted mammary carcinoma in the rat showed that the mustard produced a considerably small0 I C I C H p i H p 1 2 N ~ C H p CO ~ er antitumor effect and toxicity than did an equimolar amount of its two constituents- however its M uterotropic action was less profounde7. A number of naphthoquinone derivatives with side chains potentially capal& of alkylation after reduction,as for instance 2-chloromethyl-l,4-naphthoquinone, significantly increased the life span of mice bearing adenocarcinoma 755 and Sarcoma 1808'. Similarly, some azobenzene derivatives containing an alkylating group chemically unreactive because of the deactivating influence of the azo linkage, were converted by liver azoreductase to toxic metabolites, suggesting their potentially selective use against liver tumorsg0. Polymers as carriers of alkylating agents have also been prepared, and poly (4-vinylpyridine) containing 10-40% sarcolysine was reported to have a similar o r greater effect, as compared to free sarcolysine, against various experimental tumorsgl. Various compounds containing the m A anesulfonate moiety have shown activity against a number of experimental tumor systems 92. New members of this class of compounds include methanesulfonic acid esters of amino glycols, among which 3,3'- (morpho1inopropylimino)di-1-propanol dimethanesulfonate (X) and the corresponding dibutylaminopropylimino ester were reported active against various experimental tumorsg3. Also effective were a p-toluenesulfonate derivative of N, N-bis (3-methylsul0 ~ ~ ( C H ~ ! , l . r [ ~ n 2 ) , 0 ,z5.2HCI He) fobloxypropyl)amine94, and some bifunctional alkylating
JJp i
u
agents containing methanesulfonate as one componentg5. The methanesulfonic acid esters of aminoglycols have. reX portedly demonstrated encouraging results in the treatment of human malignant lymphoma 96, with minimal toxicity being encountered. Nitrosoureas such as 1,3-bis (2-chloroethy1)- 1-nitrosourea (BCNU) and 1-(2chloroethyl)-3-cyclohexyl- 1-nitrosourea (CCNU) have shown significant activity in a wide spectrum of animal and human neoplasms 97-99. Of particular value is their ability to penetrate the blood-brain barrier. These compounds appear to act by alkylation of nucleic acids loo and/or by cyclohexylcarbamoylation of the lysine residues of proteins lol. 3- (Tetraacetyl glucopyranos-2 -yl) - 1-(2-chloroethy1)- 1nitrosourea (GCNB) (XI), which is structurally related to streptozotocin, is markedly active against leukemia L-1210, without the marked CH Z O A ~ bone marrow toxicity exhibited by BCNU o r CCNUlo2. Marked inhibition of L-1210 growth has also been reportAcO ed for 1,1'-ethylenebis (1-nitrosourea) and for the hexaNHCON1NOlCHp cH2CI methylene derivative
k0*. XI
Chap. 15
A n t i n e o p l a s t i c Agents
Bloch
147 -
Triazene derivatives also continue to receive attention a s potential antineoplastic agents, based on the e n c o % i w t i t u m o r activity of 5- (3,3-dimethyl- 1-triazeno) imidazole-4-carboxamide in man. The compounds 4-carbethoxy-5 (3,3dimethyl- 1-triazino)-2-phenylimidazole , and the corresponding 2-methylimidazole derivative (XII) showed activity against various experimental rodents tumors comparable to t ?;43,3-dimethyl- 1-triazino)imidazole-4 RJJyZEt N=N,,~.~ carboxamide
?@!
H
R3Ph.w
In relating the structures of the newly synthesized compounds to their reported antitumor activity, one needs to keep in mind that the evaluation, even of members of the same class of compounds, was carried out in widely differing tumor systems, and that generalizations must, therefore, be derived with great caution. The pronounced inhibitory activity of a compound against a highly sensitive tumor, may be of only limited value in predicting its potential clinical utility. Nevertheless, the findings reported serve as indications that such a potential exists, and that a given structural modification may be usef u l in eliciting a tumor growth-inhibitory response.
What this brief review shows is, that the development of new antineoplastic agents is a slow and tedious process, but the achievements of past years clearly demonstrate, that each new contribution adds to the progress of cancer chemotherapy. References
1. C. Zubrod, Proc. N a t . Acad. S c i . , 69, 1 0 4 2 ( 1 9 7 2 ) . 2 . M. L e v i t t , M.B. Mosher, R.C. DeConti, L.R. F a r b e r , R.T. S k e e l , J . C . Marsh, M.S. M i t c h e l l , R . J . Papac, E.D. Thomas and J . R . B e r t i n o , Cancer Research, 33, 1 7 2 9 (1973). 3. N. J a f f e , S. F a r b e r , D. T r a g g i s , C. Geiser, B.S. K i m , L. Das, G. Fraue n b e r g e r , I. Djerassi and J . R . Cassady, Cancer, 31 1 3 6 7 ( 1 9 7 3 ) . 4. D.G. Johns, D. Farquhar, B.A. Chabner, M.K. Wolpert, R.H. Adamson, Exp e r i e n t i a , 29, 1104 (1973). 5. A. Rosowsky, J . Med. Chem., 16 1190 ( 1 9 7 3 ) . 6. B.R. Baker, W.T. Ashton, J . Med. Chem., 16,209 ( 1 9 7 3 ) . 7. A. Rosowsky, M. Chaykovsky, M. L i n , E . J . Modest, J. Med. Chem., 3, 869 (1973). 8. E.C. R o b e r t s , Y.F. S h e a l y , J. Med. Chem. 16,697 ( 1 9 7 3 ) . 9. E . J . Lien, G.L. Tong, Cancer Chemother. Rep., P a r t 1, 57, 2 5 1 ( 1 9 7 3 ) . 10. Y.K. Ho, S.F. Zakrzewski, L.H. Mead, Biochemistry, 12, 1 0 0 3 ( 1 9 7 3 ) . 11. J . P . Jonak, L.H. Mead, Y.K. Ho and S.F. Zakrzewski, J . Med. Chem., 16, 724 (1973). 1 2 . W. Korytnyk and S.C. S r i v a s t a v a , J . Med. Chem., 16,6 3 8 (1973). 1 3 . W. Korytnyk, G.B. Grindey and B. Lachmann, J. Med. Chem., 16,8 6 5 ( 1 9 7 3 ) . 1 4 . J . M . H i l l , J . R o b e r t s , E. Loeb, A. Kahn, A. MacLellan and R.W. H i l l , J . h e r . Med. ASSOC., 202, 882 ( 1 9 6 7 ) . 15. P.K. Chang, L . J . S c i a r i n i , R.E. Handschumacher, J. Med. Chem., 16,1 2 7 7 (1973).
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16. W.D. DeWys, T.C. Hall, Eur. J. Cancer, 2, 281 (1973). 17. J. Meites, J. Nat. Cancer Inst., 48, 1217 (1972). 18. S.K. Quadri, G.S. Kledzik, J. Meites, Proc. SOC. Exp. Biol. Med., 142, 759 (1973). 19. L.J. Hanka, D.G. Martin, G.L. Neil, Cancer Chemother. Rep., Part 1, 57, 141 (1973). 20. I.E. Goetz, C. Weinstein, and E. Roberts, In Vitro, 8, 279 (1973). 21. A. Bloch, G. Dutschman, B.L. Currie, R.K. Robins and M.J. Robins, J. Med. Chem., 16,294 (1973). 22. S. Nesow, T. Miyazaki, T. Khwaja, R.B. Meyer, Jr., C. Heidelberger, J. Med. Chem., 16,524 (1973). 23. M. Bobek, A. Bloch and S. Kuhar, Tetrahedron Letters, 36, 3493 (1973). 24. P.T. Berkowitz, T.J. Bardos and A. Bloch, J. Med. Chem., 16,183 (1973). 25. J. Klastersky, D. Daneau, A. Verherst, Europ. J. Cancer, 8, 149 (1972). 26. J.L. Ambrus, C.M. Ambrus, I.B. Mink and J. Pickren, J. Med., in press. 27. M.J. Sweeney, F.A. Davis, G.E. Gutowski, R.L. Hamil, D.H. Hoffman and G.A. Poore, Cancer Res., 33, 2619 (1973). 28. D.V. Popov, G.N. Platonova, E.P. Studentsov, L.F. Larinov, Byull. Eksp. Biol. Med., 75(5), 73 (1973). 29. C. Temple, Jr., B.H. Smith, R.D. Elliott and J . A . Montgomery, J. Med. Chem., 16,292 (1973). 30. L.L. Bennett, Jr., M.H. Vail, P. Allan, W.R. Laster, Jr., Cancer Res. 33, 465 (1973). Hong, 11, G.B. Chheda, S.P. Dutta, A. O'Grady-Curtis, G.L. Tritsch, 31. J. Med. Chem. 16,139 (1973). 32. G. Alonso, M. Fuertes, G. Garcia-Munoz, R. Madronero, M. Stud, J. Med. Chem., 16,1056 (1973). 33. Y.F. Shealy and J.D. Clayton, J. Pharm. Sci., 62, 858 (1973). 34. Y.F. Shealy and J.D. Clayton, J. Pharm. Sci., 62, 1252 (1973). 35. Y.F. Shealy and J.D. Clayton, J. Pharm. Sci., 62, 1432 (1973). 36. T. Alderson, Nature (London), New Biol. 244, 3 (1973). 37. R. Sandor, J. Natl. Cancer Inst., 51, 257 (1973). 38. J.W. Kreider, M. Rosenthal and N. Lengle, J. Natl. Cancer Inst., 50, 555 (1973). 39. D.B. Thomas, G. Medley and C.A. Lingwood, J. Cell. Biol., 57, 397 (1973). 40. Y.S. Cho-Chung and P.M. Gulling, Science (Wash., D.C.) 183,87 (1974). 41. M.J. Seller, P.F. Benson, Eur. J. Cander, 9, 525 (1973). 42. E.E. Smith and A.H. Handler, Res. Commun. Chem. Pathol. Pharmacol. 5, 863 (1973). 43. E.M. Bessell, V.D. Courtenay, A.B. Foster, M. Jones and J.H. Westwood, Eur. J. Cancer 2, 463 (1973). 44. H. Hojo, M. Uchiyama and S. Suzuki, Yakugaku Zasshi, 93,947 (1973). 45. E. Matthies, K. Pfordte and W. Ponsold, Arch. Geschwulstforsch, 5, 110 (1973). 46. D. Horton and E.K. Just, Carbohydr. Res., 2, 173 (1973). 47. S. Kigoshi, Experientia, 3,375 (1973). 48. N.N. Sukhareva-Nemakova, V.M. Urinyuk, A.B. Silaev, Aktual. Vop. Sovrem. Onkol. No. 3, 293 (1973). 49. V.I. Rykova, G.M. Ronichevskaya, L.F. Nikiforovskaya, L. Chernichenko, Dokl. Akad. Nauk SSSR, 209, 486 (1973). 50. 0 . Takatani, S. Kumaoka, K. Yamaguchi, Gann, 64,305 (1973).
c.
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51. I.B. Sorokina, T.I. Barkova, A.A. Zakharychev, R.N. Chigir, S.N. Ananchenko, I.V. Torgov, Izv. Akad. Nauk SSSR, Ser. Biol. (5), 664 (1973). 52. G. Briziarelli, P.P. Castelli, R. Vitali, R. Gardi, Experientia, 3, 618 (1973). 53. C.W. Abell, D.S. Hodgins, W.J. Stith, Cancer Res., 33, 2529 (1973). 54. W. Kreis and C. Hession, Cancer Res., 33, 1866 (1973). 55. T. Oki, M. Shirai, M. Ohshima, T. Yamamoto, K. Soda, (Fed. Eur. Biochem. SOC.) Lett., 33, 286 (1973). 56. H. Omura, Y. Nakamura, Y. Tomita, K. Yamafuji, J . Fac. Agr., Kyushu Univ., 17,187 (1973). 57. L.E. Hare and R.E. Handschumacher, Mol. Pharmacol., 2, 534 (1973). 58. S. Buzzi, I. Maistrello, Cancer Res., 33, 2349 (1973). 59. S.S.V. Prasad, H.L. Kumari, Y.I. Shethna, Curr. Sci., 42, 568 (1973). 60. I. Parr, E. Wheeler, P. Alexander, Brit. J. Cancer, 27, 370 (1973). 61. T.R. Norton, M.L. Bristol, G.W. Read, O.A. Bushnell, M. Kashiwagi, C.M. Okinaga and C.S. Oda, J. Pharm. Sci., 62, 1077 (1973). 62. F. Fujikawa, T. Hirayama, M. Watanabe, S. Nakazawa, H. Kuroda, Chemotherapy (Tokyo), 21, 11 (1973). 63. G.R. Pettit, P.M. Traxler, C.P. Pase, Lloydia, 2, 202 (1973). 64. S.M. Kupchan, R.W. Britton, M.F. Ziegler, C.J. Gilmore, R.J. Restivo and R.F. Bryan, J . Am. Chem. SOC., 95, 1335 (1973). 65. H. Endo and Y. Miyazaki, Bull. Natl. Inst. Hyg. Sci. (Tokyo), 90, 69 (1973). 66. T.L. Avery, D. Roberts, R.A. Price, Cancer Chemother. Rep., Part 1, 57, 165 (1973). 67. H. Staehelin, Eur. J. Cancer, 9 , 215 (1973). 68. M.A. Goldsmith, S.K. Carter, Eur. J . Cancer, 9, 477 (1973). 69. A. DiMarco, A.M. Casazza, T. Dasdia, F. Giuliani, L. Lenaz, A. Necco, C. Soranzo, Cancer Chemother. Rep., Part 1, 57, 269 (1973). 70.E. Atherton, R.P. Patel, Y. Sano, J. Meienhofer, J. Med. Chem., 16,355 (1973). 71. R.M. MacLeod and J.E. Lehmeyer, Cancer Res., 33, 849 (1973). 72. T.R. Norton, M. Kashiwagi, R.J. Quinn, J. Pharm. Sci., 62, 1464 (1973). 73. F.L. Tabrah, M. Kashiwagi and T.R. Norton, Science, 170, 181 (1970). 74. S.M. Milcu and I. Potop, Thymic Horm. 97 (1973). 75. D.J. Higby, H.J. Wallace, Jr., and J.F. Holland, Cancer Chemotherap. Repts., 57, 459 (1973). 76. B. Rosenberg, Naturwissenschaften, 60, 399 (1973). 77. M.J. Cleare, J.D. Hoeschele, Bioinorg. Chem., 2, 187 (1973). 78. M.J. Cleare, J.D. Hoeschele, Platinum Metals Rev., 17,2 (1973). 79. G.R. Gale, L.M. Atkins, E.M. Walker, Jr., A.B. Smith, S . J . Meischen, Proc. SOC. Exp. Biol. Med., 142, 1349 (1973). 80. G.B. Abdullaev, G.G. Gasanov, R.N. Ragimov, G.V. Teplyakova, M.A. Mekhtiev, A.I. Dzhafarov, Dokl. Akad. Nauk Azerb, SSR, 29(3), 18 (1973). 81. W. Kliegel, Pharm. Unserer Zeit, 2, 21 (1973). 82. M.G. Voronkov, Chemistry in Britain, 9, 411 (1973). 83. K.C. Agrawal, B.A. Booth, A.C. Sartorelli, J . Med. Chem., 16,715 (1973). 84. A. Giner-Sorolla, M. McCravey, J . Longley-Cook, J.H. Burchenal, J . Med. Chem., 16,984 (1973). 85. Y.Y. Hsiao, T.J. Bardos, J . Med. Chem., 16,891 (1973). 86. N.P. Konovalova, R.F. D'yachkovskaya, E.G. Kiseleva, Vop. Onkol. 3, 58
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(1973). 87. R.B. Everson, T.C. Hall and J.L. Witliff, Cancer Chemotherap. Repts., 57, 353 (1973). 88. p. Catsoulacos and L. Boutis, Cancer Chemotherap. Repts., 57, 365 (1973). 89. A.J. Lin, R.S. Pardini, L.A. Cosby, B.J. Lillis, C.W. Shansky, A.C. Sartorelli, J. Med. Chem., 16,1268 (1973). 90. A. Bukhari, T.A. Connors, A.M. Gilsenan, W.C.J. Ross, M.J. Tisdale, G.P. Warwick, D.E.V. Wilman, J. Nat. Cancer Inst., 50, 243 (1973). 91. O.V. Zubova, Y.E. Kirsh, T.S. Lebedeva, L.I. Samoilovich, A.A. Sharokhova, A.B. Silaev, V.A. Kabanov, Aktual. Vop. Sovrem. Onkol. No. 3, 260 (1973). 92. J.S. Sandberg, H.B. Wood, Jr., R.R. Engle, J.M. Venditti, A. Goldin, Cancer Chemother. Rep., Part 2, 3, 137 (1973). 93. M.M. El-Merzabani, Y. Sakurai, Chem. Pharm. Bull., 21, 1560 (1973). 94. H. Imamura, K. Ikegami, T. Okumoto, H. Hoshino, Y. Sakurai, Yakugaku Zasshi, 93,47 (1973). 95. M.J. Tisdale, L.A. Elson and W.C.J. Ross, Eur. 3. Cancer, 2, 89 (1973). 96. N. Gad-el-Mawla, F. Hammuda, M.M. El-Merzabani, M.I.R. Hamza, A. Osman, A.S. Ibrahim, Y. Sakurai and A.L. Abul-Nasr, Cancer Chemotherap. Repts., 57, 159 (1973). 97. T K . Carter, Cancer Chemotherap. Repts., Part 3, 2, 33 (1972). 98. B. Hoogstraten, J.A. Gottlieb, E. Caoili, W.G. Tucker, R.W. Talley and A. Haut, Cancer, 32, 38 (1973). 99. R.C. DeConti, S.P. Hubbard, P. Pinch, and J.R. Bertino, Cancer Chemotherap. Repts., 57, 201 (1973). 100. G.P. Wheeler and S. Chumley, J. Med. Chem., 10,259 (1967). 101. C.J. Cheng, S. Fujimura, D. Grunberger and I.B. Weinstein, Cancer Res., 32, 22 (1972). 102. T S . Schein, M.G. McMenamin and T. Anderson, Cancer Res., 33, 2005 (1973). 103. M. Nakadate, M. Anzai, I. Suzuki, M. Ishidate, Jr. and S. Odashima, Gann Jap. J. Cancer Res., 64,415 (1973). 104. Y.F. Shealy, J.A. Montgomery and W.R. Laster, Jr., Biochem. Pharmacol., 11, 674 (1962). 105. T K . Carter and M.A. Friedman, Europ. J. Cancer, 8, 85 (1972). 106. J. Heyes, E.J. Catherall, A. Cockburn, Cancer Chemother. Rep., Part 1, 57, 263 (1973). 107. R.C.S. Audette, T.A. Connors, H.G. Mandel, K. Merai, W.C.J. Ross, Biochem. Pharmacol., 22, 1855 (1973).
Chapter 16.
Immunotherapy of Cancer
A n i t a Hodson and E. F r e d e r i c k Wheelock, J e f f e r s o n Medical C o l l e g e , Thomas J e f f e r s o n U n i v e r s i t y , P h i l a d e l p h i a , Pennsylvania
Many e x c e l l e n t review a r t i c l e s on t h e immunolo y and immune t h e r a p y The of c a n c e r have appeared i n t h e l a s t few y e a r s . 1 , 2 , 3 , ~ , 5 , 6 , 7 , 8 , g , 1 0 , 1 1 major g o a l of t h i s c h a p t e r is t o p r o v i d e t h e r e a d e r w i t h i n f o r m a t i o n on immunotherapy of c a n c e r which h a s been p u b l i s h e d d u r i n g t h e p a s t y e a r . The e x i s t e n c e of an immunological r e s p o n s e by a h o s t t o h i s tumor h a s been w e l l documented.12 D e f i c i e n c i e s i n one o r more components of t h i s r e s p o n s e may e i t h e r p r e d a t e and be a p r e r e q u i s i t e f o r development of t h e tumor o r b e a consequence of p r o g r e s s i o n of t h e tumor.3,13,14,15 I m munotherapy a i m s a t r e s t o r i n g immune competence o r a m p l i f y i n g a competent b u t i n e f f e c t i v e h o s t r e s p o n s e t o t h e t ~ m o r . ~ , Experience ~ , ~ w i t h immunot h e r a p y h a s r e v e a l e d t h a t t h e h o s t w i t h t h e minimal r e s i d u a l tumor burden f o l l o w i n g s u r g e r y , radio-and/or chemotherapy r e c e i v e s t h e most b e n e f i t from immunotherapy. 1 6 A c t i v e S p e c i f i c Immunotherapy - The r e j e c t i o n of tumor c e l l s f o l l o w i n g act i v e s p e c i f i c immunotherapy i s probably mediated through s e n s i t i z e d lymphoc y t e s , l 7 macrophages18 a n d / o r antibody.19 R e j e c t i o n of t h e tumor f o l l o w s r e c o g n i t i o n of tumor s p e c i f i c t r a n s p l a n t a t i o n a n t i g e n s on n e o p l a s t i c c e l l s by an immune-competent h o s t . Tumor a n t i g e n s t h a t a r e e i t h e r weak o r hidden may n o t b e recognized by t h e h o s t and t h e r e b y e s c a p e r e c o g n i t i o n ; 2 0 some s u p p o r t f o r t h e e x i s t e n c e of t h i s phenomenon comes from Witz who h a s d e s c r i b e d t h e u b i q u i t y of IgG on tumor c e l l s found o n l y i n v i v o and n o t on tumor c e l l s grown i n v i t r o ? 2 1 Thus t h e g o a l of a c t i v e s p e c i f i c immunot h e r a p y i s t h e a m p l i f i c a t i o n of t h e a f f e r e n t arc of t h e immune system by i n c r e a s i n g t h e a n t i g e n i c s t i m u l u s t o t h e tumor b e a r i n g h o s t . Vaccines - Vaccines c o n s i s t i n g of whole autogenous o r a l l o g e n e i c c e l l s o r membrane f r a c t i o n s from tumors have been used i n a n i m a l s t o i n d u c e r e s i s t a n c e t o subsequent tumor c h a l l e n g e , and i n man, t o improve t h e immune r e s p o n s e t o an e s t a b l i s h e d tumor. A l l tumors induced by a s p e c i f i c v i r u s have common a n t i g e n s b u t c h e m i c a l l y induced tumors do n o t s h a r e common a n t i g e n s . Gross demonstrated t h e immunization of ' s t r a i n 2 ' guinea p i g s w i t h L2C v i a b l e leukemic c e l l s u s p e n s i o n s by s u p e r f i c i a l s k i n s c a r i f i c a t i o n . 22 The m o r t a l i t y of 30% due t o t h e s c a r i f i c a t i o n a l o n e w a s less t h a t i n prev i o u s t r i a l s w i t h i n t r a d e r m a l immunization. P r a g e r and co-workers have shown that r e s i s t a n c e t o 6C3HED lymphoma i n C3H mice could b e produced by immunization w i t h s o l u b l e 6C3HED membrane a n t i g e n ( s ) 23
.
Human t r i a l s w i t h o s t e o g e n i c sarcoma24 u s i n g f r o z e n o r f r e s h i r r a d i a t e d a u t o l o g o u s tumor have been t o o small t o o f f e r c o n c l u s i v e r e s u l t s b u t t h e r e s p o n s e s were as good as w i t h c o n v e n t i o n a l t r e a t m e n t s . O t h e r , l a r g e r s t u d i e s have shown enough f a v o r a b l e r e s p o n s e t o w a r r a n t f u r t h e r t r i a l s .
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The f o l l o w i n g v a c c i n e s have been among t h o s e used: aqueous pooled bronchog e n i c carcinoma extract i n a series of p a t i e n t s w i t h lung c a n c e r ; 2 5 irrad i a t e d , animal a s s a g e d and g o a t gamma g l o b u l i n coupled v a c c i n e i n malign a n t melanoma; 2g and autogenous c e l l - f r e e extracts i n complete F r e u n d ' s a d j u v a n t i n a series of p a t i e n t s w i t h v a r i e d t y p e s of tumors.27 C o r r e l a t i o n of t h e c l i n i c a l r e s p o n s e t o tumor v a c c i n e s w i t h changes i n s k i n r e a c t i o n t o r e c a l l and tumor a n t i g e n s and i n v i t r o measures of c e l l - m e d i a t e d immunity and humoral immunity are i n p r o g r e s s . I n a randomized prospect i v e t r i a l i n p a t i e n t s w i t h g l i o b l a s t o m a multiforme no a n t i t u m o r e f f e c t w a s produced f o l l o w i n g a c t i v e immunotherapy. 28 Following s u r g e r y and rad i o t h e r a p y , t h e s u r v i v a l of 27/62 p a t i e n t s r e c e i v i n g i r r a d i a t e d tumor c e l l s w a s n o t improved o v e r t h e c o n t r o l group r e c e i v i n g t h e c o n v e n t i o n a l therapy alone. Enzyme Treatment of Tumor Cells - Another mode of a c t i v e s p e c i f i c immunotherapy h a s i n v o l v e d V i b r i o c h o l e r a neuraminidase (VCN), an enzyme t h a t can remove g l y c o p r o t e i n s from t h e s u r f a c e of tumor c e l l s . Vaccines c o n s i s t i n g of neuraminidase t r e a t e d tumor c e l l s have been shown t o i n d u c e r e g r e s s i o n of p r e e x i s t i n g tumors. To d a t e , such v a c c i n e s have been l i m i t ed t o e x p e r i m e n t a l systems b u t t h e y o f f e r t h e b e s t model f o r c l i n i c a l s i t u a t i o n s where t h e r a p y must be designed f o r a tumor-bearing h o s t . Neuramini d a s e t r e a t m e n t increases t h e immunogenicity of tumor c e l l s b u t t h e mechRios and Simmons have anism of tumor r e j e c t i o n h a s n o t been e s t a b l i s h e d . shown t h a t murine methycholanthrene-induced f i b r o s a r c o m a s (MC-43), mammary carcinomas (M-2) and B16 melanoma up t o 1 c m i n s i z e can b e r e p r e s s e d w i t h t h e r e s p e c t i v e s p e c i f i c n e u r a m i n i d a s e - t r e a t e d tumor v a c c i n e and t h i s rej e c t i o n can b e enhanced w i t h BCG.29 Such combined t h e r a p y i s more e f f e c t i v e when t h e tumor burden is reduced p r i o r t o t h e immunotherapy. VCN t r e a t m e n t is immunospecific, i n c r e a s i n g t h e a n t i g e n i c i t y of o n l y t h e uns h a r e d a n t i g e n s of mammary carcinomas and n o t t h e s h a r e d mammary tumor v i r u s a n t i g e n . Other workers have extended t h i s t r e a t m e n t m o d a l i t y t o exp e r i m e n t a l leukemia w i t h c o n f l i c t i n g r e s u l t s . 309 31 A c t i v e Non-Specific Immunotherapy - A c t i v e n o n - s p e c i f i c immunotherapy i n v o l v e s a g e n e r a l s t i m u l a t i o n of t h e h o s t ' s immuno system. Yashphe h a s rec e n t l y w r i t t e n a v e r y comprehensive review of t h i s t o p i c . 3 2 BCG - Receiving much c u r r e n t a t t e n t i o n is a s t r a i n of Mycobacterium E a r l y s t u d i e s r e v e a l e d t h a t BCG b o v i s , b a c i l l u s Calmette-Guerin (BCG) s t i m u l a t e d t h e r e t i c u l o e n d o t h e l i a l system.33 A d m i n i s t r a t i o n of BCG t o AKR mice d e c r e a s e d t h e i n c i d e n c e of spontaneous leukemia. 34 A r e t r o s p e c t i v e s t u d y by Davignon r e v e a l e d t h a t BCG v a c c i n a t i o n w a s a s s o c i a t e d w i t h a dec r e a s e d i n c i d e n c e of childhood leukemia; 35 however, o t h e r s t u d i e s have ref u t e d t h i s c o n c l u s i o n . 36 Mathe r e p o r t e d t h a t r e m i s s i o n s produced i n p a t i e n t s w i t h a c u t e lymphocytic leukemia f o l l o w i n g c o n v e n t i o n a l t h e r a p y could b e s i g n i f i c a n t l y prolonged i n t h e a b s e n c e of chemotherapy by treatment w i t h P a s t e u r BCG p l u s i r r a d i a t e d leukemic cells.37 An e n l a r g e d s t u d y by t h e B r i t i s h who used a d i f f e r e n t p r e p a r a t i o n of BCG (Glaxo) and no i r r a d i a t e d leukemic c e l l s f a i l e d t o s u p p o r t t h i s f i n d i n g . 38 Powles, on t h e o t h e r hand, h a s found t h a t i n a c u t e myelogenous leukemic (AML) remiss i o n s on maintenance chemotherapy p l u s immunotherapy (BCG and i r r a d i a t e d
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AML v a c c i n e ) were prolonged compared t o chemotherapy a l o n e . j Y The v a r i a t i o n i n c l i n i c a l r e s p o n s e s may stem from t h e u s e of d i f f e r e n t s o u r c e s and p r e p a r a t i o n s ( f r e s h v e r s u s l y o p h i l i z e d ) of BCG r e s u l t i n g i n d i f f e r e n t numb e r s of l i v e organisms a t t h e t i m e of u s e . Another problem i n t h e f a i l u r e t o reproduce c l i n i c a l t r i a l s h a s been t h e tendency t o q u a n t i t a t e dosage by m i l l i g r a m of BCG p r o t e i n r a t h e r t h a n t h e number o f v i a b l e organisms and t o a c c e p t t h e number of v i a b l e organisms as s t a t e d by t h e s u p p l i e r when u s i n g b o t h t h e f r e s h o r l y o p h i l i z e d p r e p a r a t i o n s . The v a r i o u s methods of admini s t r a t i o n of BCG create v a r i a b i l i t y i n number of organisms a c t u a l l y penet r a t i n g i n t o t h e s u b j e c t . Mackaness h a s shown t h a t c e l l mediated immunity i s t h e dominant h o s t r e s p o n s e t o v i a b l e organisms whereas non-viable organisms e l i c i t a predominantly humoral r e s p o n s e .40 Experimental s t u d i e s on t h e immune r e s p o n s e t o d i f f e r e n t s t r a i n s have r e v e a l e d t h e P a s t e u r s t r a i n t o b e t h e most immunogenic.41 F i n a l l y i t i s p o s s i b l e t h a t t h e ant i t u m o r e f f e c t s of BCG may r e s u l t from a d i r e c t a n t i t u m o r e f f e c t r a t h e r t h a n mediated through t h e immune system.
Gutterman used t h e s c a r i f i c a t i o n method of immunization w i t h BCG i n p a t i e n t s w i t h S t a g e I11 and I V melanomas and produced a s i g n i f i c a n t l y lowe r r a t e of r e l a p s e w i t h t h e T i c e ( l y o p h i l i z e d ) s t r a i n t h a n t h e l i v e Past e u r s t r a i n . 4 2 I n b o t h groups, p a t i e n t s r e c e i v i n g t h e h i g h e s t dose of o r ganisms (6 x 108) developed t h e g r e a t e s t s k i n r e a c t i v i t y on t e s t i n g w i t h a b a t t e r y of a n t i g e n s . I n j e c t i o n s of BCG d i r e c t l y i n t o i n t r a d e r m a l malign a n t melanomas c o n t i n u e s t o produce s t r i k i n g l y p o s i t i v e r e s u l t s . 4 3 ~ 4 4 One s t u d y w i t h BCG i n malignant melanomas s u p p o r t s t h e h y p o t h e s i s t h a t BCG p r i m a r i l y augments t h e h o s t 5 response t o a n t i g e n s t o which t h e h o s t h a s had p r e v i o u s exposure. 45 BCG h a s a l s o been s t u d i e d i n s e v e r a l e x p e r i m e n t a l models. Maximum tumor i n h i b i t i o n and p r e v e n t i o n of pulmonary metastases i n a n i m a l s w i t h m e t h y l c h o l a n t h r e n e sarcoma followed BCG p l u s l i v e tumor v a c c i n e i n o c u l a t e d i n t r a l e s i o n a l l y . 4 6 , 4 7 Bansal and S j o g r e n have shown t h a t BCG must be g i v e n b e f o r e t h e tumor r e a c h e s a c r i t i c a l s i z e ; o t h e r w i s e BCG enhances t h e b l o c k i n g e f f e c t s on t h e e n l a r g i n g tumor.48 F i n a l l y BCG d e c r e a s e s t h e tumo r s i z e i n Moloney sarcoma v i r u s i n f e c t e d mice which had been immunosupp r e s s e d by cytoxan. 49 A n a l y s i s of t h e guinea p i g hepatoma model r e v e a l e d t h a t b o t h t h e BCG c e l l w a l l s u s p e n s i o n i n o i l and i s o l a t e d BCG c e l l w a l l components were a s e f f e c t i v e as t h e l i v e organisms.50 Another BCG prod u c t , m e t h a n o l - e x t r a c t i o n r e s i d u e (MER) h a s a l s o been e x t e n s i v e l y s t u d i e d and shown t o s t i m u l a t e a p o s i t i v e h o s t r e s p o n s e towards tumor r e j e c t i o n . 5 1 I n t r a l e s i o n a l BCG t h e r a p y i n s t r o n g l y PPD-positive i n d i v i d u a l s h a s been complicated by malaise, i n f l u e n z a - l i k e syndrome and h e p a t i c dysfunct i o n . 52,53 Noncaseating g r a n u l o m a t o s i s h e p a t i t i s developed i n t h r e e p a t i e n t s who were PPD n e g a t i v e . Treatment w i t h a n t i - t u b e r c u l o u s a g e n t s have r e v e r s e d t h e more s e v e r e c o m p l i c a t i o n s of BCG. Corynebacterium parvum - The anaerobe Corynebacterium parvum (5. parvum) h a s been s t u d i e d i n t e n s i v e l y because of i t s a b i l i t y t o s t i m u l a t e a n immune r e s p o n s e r e s u l t i n g i n tumor r e g r e s s i o n . Contrary t o BCG whose a c t i v i t y depends upon v i a b i l i t y , C. parvum i s a c t i v e when h e a t - k i l l e d o r
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as a phenol extract. In animal systems, it is most active when inoculated directly into an established tumor or administered with the tumor inoculum. Inoculation into the contralateral limbs or as intravenous doses was not effective. Maximum tumor regression occurred about 14 days after treatment, and the tumor-specific resistance that followed inoculation of 5 x lo7 organisms was permanent .54,55 The mechanism of regression appeared to involve cellular immunity. Several large clinical series with 5. parvum have been reported by In one, composed of patients with metastatic cancer, the treated group survived longer than the controls. A subsequent study involving squamous cell cancer of the lung compared chemotherapy alone with chemotherapy plus C. parvum. The mean survival (MS) was 6.1 months and 10.5 months, respectively. Although the response rate was the same in each group, the duration of response was longer in the group with the combined treatment. Of further interest is a breakdown of the combined groups into PPD status:those which were PPD positive had a MS of 13 months whereas those which were negative had a MS of 3.6 months. L. Israe1.56
Listeria monocytogenes - Listeria monocytogenes (LM) has also been effective in experimental tumor rejection.57 Inbred mice previously immunized with LM were able to impede or prevent growth of a syngeneic sarcoma when the tumor cells were inoculated together with viable LM. The mechanism of action was consistent with a delayed-type of hypersensitivity at the site of tumor implantation. Macrophage Activation - The role of macrophages in tumor rejection is being actively investigated.58 Certain compounds which exhibit antitumor activity has been shown to act via the macrophage, probably through macrophage-mediated cytotoxicity 59 T ese include endotoxin and double stranded Penicillium stoloniferum RNA," Triton mi 133gy6I and pyran.62~63 One such compound, tilorone, is in a Phase I1 human trial and has produced antitumor responses in 5/41 cases .64
.
Interferon - The role of interferon in tumor rejection is unknown. Interferon enhances phagocytosis ,65 antibody production,66 sensitized lymphocyte mediated cytotoxicity and tumor cell a n t i g e n i ~ i t yand ~ ~ thus may induce host-mediated anti-tumor effects in addition to a direct tumoricidal effect. Interferon prolonged survival in DBA/2 mice inoculated with interferon resistant strains of L1210. In these mice, lymphocyte and macrophage functions were compromised with anti-lymphocyte serum and silica, respectively, leaving the mode of action of interferon unexplained.68 Statolon, a double stranded RNA mycophage from Penicillium stoloniferum, induces interferon in DBA/2 mice and suppresses established Friend virus leukemia (FV) .69 Suppression of FV erythroleukemia is dependent upon the immunostimulatory effects of statolon, a property not shared by other interferon inducers such as Newcastle disease virus and poly 1:poly C, that have no leukemosuppressive effects.70 Macrophage Recognition Factor - Macrophage recognition factor (MRF) may be important as a mediator of immunotherapy.71 Found in the a2-globu-
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lin fraction of normal plasma, MRF enhances phagocytosis. Depleted levels of MRF in tumor patients return to normal after reduction of tumor load. Inoculation of MRF into a subcutaneous Shay chloroleukemic tumor reduced the tumor size by 86% compared to controls. Direct cytotoxicity by MRF €or tumor cells was not demonstrated. Allogeneic Cell Transfers - Another approach to immunotherapy has been the initiation of a graft versus host reaction in an immunocompetent tumor-bearing host. As the only form of therapy transfer of non-sensitized allogeneic immunocompetent lymphocytes significantly altered the course of guinea ig leukemia L2C, and produced long-term protection in 21% of cases.72,75 Optimum protection resulted when the allogeneic lymphocytes were transferred one week prior to leukemic cell challenge; prolonged survival still occurred when a 30-fold increase in the lethal dose of L2C leukemia was used. Significantly prolonged survival also occurred when the allogeneic cells were transferred after the leukemic challenge. The use of human allogeneic cell transfers in early malignancies in immunocompetent host was suggested. Skin Sensitizers - A final mode of active specific immunotherapy utilizes primary skin sensitizing agents such as 2,4-dinitrochlorobenzene (DNCB), 2,3,5-trisethyleneiminobenzoquinon and 5-mercapto-2-deoxyuridine. Reports show 5445% favorable response in premalignant and primary cutaneous neoplasia and 33% in neoplasias metastasizing to skin.74 Anti-tumor effects in neoplastic lesions distant to the site of sensitization and challenge were a l s o observed. A response was defined as the. complete disappearance of a lesion €or the period of survival (12 months) or period of observation (20 months). Anergic patients who could not respond to primary antigens were challenged with antigens which could test for retained immunological memory. A much smaller study limited to the response rate of basal cell carcinoma to sensitization and challenge by DNCB revealed that only 32% of lesions showed complete, and 29% partial r e g r e ~ s i o n . ~ ~ Large lesions had the poorest anti-tumor response and no distant effects were noted. Croton oil, which is an irritant without being a sensitizer, produced complete regression in 23% of treated basal cell carcinomas. This sheds doubt on the mechanism of response to DNCB as being due to cell-mediated immunity. The authors noted, however, that the patient most sensitive to DNCB had the best response and the patient who was most tolerant to DNCB had the poorest response. This supported a host-mediated immunological reaction to DNCB. Miscellaneous - The anti-tumor effects of nonspecific stimulants of the immune system were first described in 1906 by Coley who reported a series of 31 patients with sarcoma who improved after receiving extracts of hemolytic streptococci and Serratia marcesans. 76 In a retrospective study Ruckdeschel found that 50% of the patients with post-operative empyema were alive following surgery for lung cancer compared to a nonempyema control series where the survival was 182.77 By excluding patients with metastatic symptoms, the survival rates increased to 73% in the study group compared to 23% in the control. He concluded that the host responses to infection may influence survival in the cancer patient.
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Passive Immunotherapy - In passive immunotherapy, immune modalities are given to a tumor host which will enable it to more effectively reject its tumor. These modalities may consist of activated mononuclear cells, extracts of sensitized lymphocytes or serological factors such as specific antibodies or complement. Lymphocytes, macrophages or thymocytes may be transferred in passive immunotherapy. Lymphocytes Activated In Vivo - Lymphocytes may be presensitized either in a syngeneic or an allogeneic host. An example of syn eneic cell transfer comes from guinea pig primary lymphatic leukemia, L2C .98 All animals were protected from a lethal dose of leukemic cells if 580-750 x lo6 sensitized spleen and lymph node cells were transferred 2 days after the leukemic challenge; cell transfer 4 and 7 days after leukemic challenge was less successful. Gaugas et.al. 79 similarly showed that transfer of sensitized syngeneic spleen cells tothymectomized and/or antilymphocyte globulin-treated (ALG) mice inhibited polyoma virus and mammary tumors. Passive immunotherapy with sensitized allogeneic lymphocytes has been employed in patients with metastatic carcinoma, Pairs or groups of 3 patients, matched for ABO-Rh and similar tumors, were serially immunized with another patient’s tumor. Then, sensitized lymphocytes were serially transfused into the host with the target tumor. Krementz et.&.80 reported a series of 64 patients and observed an overall anti-tumor response of about 20%. This type of immunotherapy was most effective when the patients were immunocompetent and carried a minimal amount of residual tumor. Lymphocytes Activated In Vitro - Lymphocytes activated in vitro may also be effective in tumor inhibition. Balb/C spleen cells sensitized _ in _ vitro _ for seven days to a syngeneic plasma cell tumor, HPC 108, exhibited in vitro cytotoxicity for this target ce11.81 In this system sensitized lymphocytes admixed with tumor cells at a 20:l ratio inhibited subcutaneous growth of tumor. Autochthonous lymphocytes activated in vitro with phytohemaglutinin (PHA) and applied to subcutaneous metastatic nodules resulted in a greater
percent regression than non-activated lymphocytes but the degree of regression in those nodules which were affected was not significantly different from the control group.82 This study reemphasized the need for effector lymphocytes at the site of the tumor. Non-Activated Lymphocytes - Non-activated lymphocytes from healthy HLA-identical siblings have been harvested and transfused into the other sibling with advanced cancer with the rationale of increasing the number of immunocompetent cells for the tumor host.83 It is conceivable that these lymphocytes may have been sensitized in the healthy donor to the same carcinogenic stimulus as was present overtly in the recipient. In any case transfer of lymphocytes had no effect on the tumor. Non-specific or specific in vitro lymphocyte activation plus active immunotherapy were suggested for the future.
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Macrophages - Adoptive transfer of BCG-activated macrophages has also been shown to play a role in tumor rejecti0n.8~ Transfer of these cells together with tumor-immunized lymph node cells and tumor cells into normal syngeneic hamsters resulted in significant reduction of tumor growth. Substitutions of non-activated macrophages or of normal lymph node cells was not effective. This study was unique in that extracellular BCG itself was not present in the inoculum. Furthermore, the requirement of both lymphocytes and macrophages in the inoculum for antitumor effects indicated that cell-cell cooperation was needed for inhibition of tumor growth. Thymic Cells - There are scattered reports of the anti-tumor effects of thymic transplants in immunodeficient tumor-bearing hosts ?5,86 In contrast, ablation of the thymus has been cited as cause for decreased incidence of experimental neoplasias.87 The role of the thymus in neoplasia is implicated by a retrospective study which described an increased incidence of neoplasia in patients with myasthenia gravis prior to thymectomy. After the second post-thymectomy year the incidence of neoplasia in the thymectomized patient equaled the general population.88 Cellular extracts - Sensitized lymphocytes have served as a source of extracts such as immune RNA89390 or transfer factor.91 Immune RNA from presensitized lymphoid tissue has been given either directly to the tumor bearing host or to lymphoid cells in vitro which have then been adoptively transferred to such hosts. Such treatment has conferred immunity to tumor specific transplantation antigens; its use in conferring tumor immunity has been largely limited to experimental systems. However, immune-RNA has been shown to transfer cell-mediated skin reactivity in Hodgkin's disease and other neoplastic states.92 Since transfer factor has been strikingly effective in the restoration of cell-mediated immunity in some immunodeficiency diseases,93 its use in a variety of human neoplasias is being tested.80 Its specificity, non-antigenicity, ability to be stored and quantitated in vitro and its long lasting effects would make it a desirable form of immunotherapy. Serotherapy - The administration of tumor-specific antisera to a tumor-bearing host is an old approach to tumor therapy and early reports were disappointing.94 In contrast, the intraperitoneal administration of heterologous immune antisera to animals with experimental peritoneal ovarian carcinomas95 produced 100% survivors when administered concurrent with a lethal dose of tumor cells. Survivors were reduced to 30% when administration of the antisera was delayed until the fourth to the eighth day. The intraperitoneal administration of antisera may have avoided the dilution or non-specific organ binding of heterologous antibody given intravenously. With the recognition that certain antibodies can protect tumor cells against the host's immune response thereby resulting in tumor enhancement, therapy with unblocking antibodies has been attempted. Bansal and Sjogren96 studied a rat polyoma tumor system and found unblocking activity
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in sera from rats in which tumors were excised and bled 5-7 days later or from animals non-specifically immunized with BCG 14 days prior to grafting with the polyoma tumor and again bled 5-7 days after grafting. Blocking effects were abrogated by such sera and growth of tumors in vivo was either arrested or regressions observed. DiLorenze et .&?vitiated the 'blocking effects' in a mouse mammary carcinoma with low dose chemotherapy, i.e. cytosine arabinoside. The use of antibodies as carriers of cytotoxic drug, radioisotopes, toxins, and enzymes offers hope of achieving greater specificity by chemotherapeutic means. 98999
.&.
Drake et loo,lol found that endogenous complement may be a limiting factor in serotherapy and showed that exogenous complement plus heterologous antisera was needed to achieve in vivo tumor cytolysis. A C i inhibitor produced by tumor cells in culture and found on human leukemia and carcinoma cells has been described and may serve to block host effector mechanisms; the overall scope of this phenomena is being studied.102 Imunotherapy today is in its infancy, but recent results are encouraging and prospects for the future are bright. The wide range of approaches to immunotherapy and the multiple agents which can be employed in each approach will form the basis for much research in the coming years. Several considerations should serve as important guidelines for effective immunotherapy: 1) early diagnosis is essential since maximum beneficial effects are produced in patients with the smallest tumor load; 2) immunotherapeutic agents must be selected with great caution since they may enhance production of factors that block the host's normal immune response to tumor cells; 3) immunotherapy demonstrated to be effective against one tumor may work to the detriment of the host bearing another tumor. Ideally immunotherapy should be directed at amplification of the specific host function that is crucial to the defense against a specific tumor. References 1. D.L. Morton,EC.Holmes, F.R. Eilber and W.C. Wood, Ann. Intern. Med., 74, 587 (1971). 2. B. Cinader, Med. Clin. N. her., 56, 801 (1972). 3 . R.A. Good, Proc. Nat. Acad. Sci. USA, 69, 1026 (1972). 4. C.A. Currie, B. J. Cancer, 2, 141 (19n). 5. D.L. Morton,Cancer, 30, 1647 (1972). 6. T.A. Waldmann, W. Strober and R.M. Blaese, Ann. Intern. Med., 77, 605 (1972). 7. C.F. McKhann in J.S. Najarian and R.L. Simmons (editors), Transplantation, Lea & Febiger, Philadelphia, 1972, p. 297. 8. G. Klein, Transplant. Proc., 5, 31 (1973). 9 . C.W. Parker,Pharmacol. Rev., 25, 325 (1973). 10. H.F. Oettgen and K.E. Hellstrum in J.F. Holland and E.Frei (editors),
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Cancer Medicine, Lea & Febiger, Philadelphia, 1973, p. 951. 11. E.M. Hersh, J.V. Gutterman, G. Mavligit, Immunotherapy of Cancer in Man, Charles C. Thomas, Springfield, 1973. 1 2 . R.T. Prehn and J.M. Main, J. Nat. Cancer Inst., 769 (1957). 13. I. Penn and T.E. Starzl in Seventh National Cancer Conference Proceedings, J.B. Lippincott Co., Philadelphia, 1973, p. 425. 14. F.R. Eilber and D.L. Morton, Cancer, 25, 362 (1970). 15. C.M. Southam, Cancer Res., 28, 1433 (1968). 16. P. Alexander, Pr0c.R. SOC. Med., 1044 (1971). 17. C. O'Toole, P. Perlmann, H. Wigzell, B. Unsgaard and C.G. Zetterlund, 1085 (1973). Lancet 18. R. Evans and P. Alexander, Nature 236, 168 (1972). 19. M.G. Lewis, R.L. Ikonopisiv, R.C. g r n , T.M. Phillips, G.H. Fairley, D.C. Bodenham and P. Alexander, Brit. Med. J., 3:547 (1969). 20. N.A. Mitchison, Transplant. Proc., 2, 92 (1970): 21. I.P. Witz in Current Topics in Microbiology and Immunology, 61, 1 5 1 (1973). 22. L. Gross, Proc. Nat. Acad. Sci. USA, 70, 3432 (1973). 23. M.D. Prager, A.C. Hollinshead, R.J. Rzble and I. Derr, J. Natl. Cancer Inst, 51, 1603 (1973). 199 (1973). 24. C.M. Southam, Front. Radiation Ther. Onc., 25. G. Alth, H. Denck, M.Fischer, K. Karrer, 0. Kokron, E. Eorizek, M.
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42. J . V . G u t t e r m a n , G . M a v l i g i t , C . McBride, E . F r e i , 111, E . J . F r e i r e i c h , E.M. H e r s h , L a n c e t , i , 1208 ( 1 9 7 3 ) . 43. L . Nathanson, C a n c e r C h e m o t h e r . Rep., 56, 659 ( 1 9 7 3 ) . 44. M . J . M a s t r a n g e l o , Y.H. K i m , R.S. B o r n s t e i n , D . O . Chee, H.L. S u l i t , J . W . Y a r b r o and R . T . P r e h n , J . Natl. Cancer I n s t . , 5 2 , 1 9 ( 1 9 7 4 ) . 45. L. C h e s s , G . N . Bock, P.C. Ungaro, D . H . Buchhalz and%.R. Mardiney, Jr., J . Natl. Cancer I n s t . , 51, 57 ( 1 9 7 3 ) . 46. R.W. Baldwin and M.V. Pimm, B r . J . C a n c e r , 48 ( 1 9 7 3 ) . 47. R.W. Baldwin and M.V. Pimm, B r . J . C a n c e r , 28, 281 (1973). 162 ( 1 9 7 3 ) . 48. S.C. B a n s a l and H . O . S j o g r e n , I n t . J . C a n c e r , 49. T . J . Meyer, E.E. R i b i , I. Azuma and B . Zbar, J . Natl. Cancer I n s t . , 52, 103 (1974). 50. X P . Houchens, A . I . G o l d b e r g , M.R. G a s t o n , M . Kende and A. G o l d i n , Cancer R e s . , 33, 685 ( 1 9 7 3 ) . 51. D.W. Weiss, N a t i o n a l Cancer I n s t . Monogr. 35, U.S. Government P r i n t i n g O f f i c e , 1972. 52. J . S . Hunt, M . J . S i l v e r s t e i n , F.C. S p a r k s , C.M. H a s k e l l , Y.H. P i l c h , D.L. Morton, L a n c e t , 2, 820 (1973). 53. F.C. S p a r k s , M . J . S i l v e r s t e i n , J . S . H u n t , C.M. H a s k e l l , Y.H. P i l c h and 827 (1973). D.L. Morton, N . E n g l . J . Med., 9,
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162
Section IV - Metabolic Diseases and Endocrine Function Editor: Walter T. Moreland, Pfizer, Inc., Groton, Connecticut Chapter 17. Prostaglandins and Related Compounds Richard A. Mueller and Lloyd E. Flanders, G. D. Searle and Co., Chicago, I l l .
This review will include advances in chemical synthesis that have occurred since the last article in this series1 and selected aspects of prostaglandin biosynthesis. Specialized up-to-date reviews of most areas in prostaglandin biological research have appeared; CAMP interactions,3,4, prostaglandin antagonists,6 effects on the res iratory s y ~ t e m , ~ ,in~ , ~ blood,lO ,11 in cardiova~cular-renal,1~ occular,193 20,21 fertility and reproduction,22923324 gastro-intestinal,25 9 26,27 nervous system,28 29 radio-immunoassay,30 and veterinary applications.31 Chemical reviews12 l3 ,I4 and general books and monographs have been published.32 9 33,34 Synthesis of natural prostaglandins and their analogs. Corey has disclosed three direct syntheses of PGA2. The first involves conversion of the key intermediate 1 to 2 [R = Si(CH ) C(CH ) ] and subsequent treatment by pre2 viously reported procedures to3yield $Ga2 as the primary product.35 0
-3
1 -
The second sequence involves the preparation of 2 from a bicyclo[2,2,1]heptene intermediate.36 Addition of the carboxyl sidechain in the usual fashion followed by Collins oxidation of the resulting 9-hydroxy-PGA2
& & (yO q .
\FQ
-
~THP
OR
Prostaglandins, Related Compounds Mueller, Flanders
Chap. 17
163
formed gave PGA2. The third is a biosynthetically derived process which involves Lewis acid cyclization of 3 to give c - 2 (R = H ) and its 15epimer.37 Corey has also disclosed38 a total synthesis of PGC2, 5 (R = R' = H ) involving reduction of 2 (R = THP) to the lactol which gave 5 when treated with Fe3(CO)1?. Addition of the carbuxyl sidechain and Collins oxidation, which also disrupted the iron carbonyl complex, gave the desired product. 8-Methyl-PGC2, 5 (R = H, R' = CH3), synthesized from 5, had about 3% of the activity of PGE2 in "the smooth muscle test". The epimeric methyl esters had about 60% of the potency of PGA2 as inhibitors of gastric secretion in the rat.39 Crabbe has also reported40 the synthesis of 1 (R = THP, R ' = H) via 7 and 8. A synthesis of optically pure L was published, which
OH
OH
(2'"" '0
10 -
OCH~
'~1-c EC -CHW
-
/
OR
CH3
dH
11
12 -
is based on a highly stereoselective hydroboration with (+)-di-3-pianylborane.41 Epoxidation of 1 with peracetic acid gave a 36% yield of the epoxylactone ?. This represents an efficient route to the optically pure intermediates 2 and 10,which have been converted to the natural prostaglandins by Corey and Fried respectively. Fried and his group disclosed42 the details of their method for obtaining 100% regioselectivity in the ring opening of epoxide 10 by displacement with the organoaluminum reagent 2 [R = OC(CH3)3] This process, which has been shown to lead to the natural prostaglandins,l, was used to pre are 13,14-dehydro analogs of PGE2, PGF2, (g), and their enantiomorphs.45 Compound 12 is 2 times more potent subcutaneously and 5 times more active orally than the natural product in a hamster anti-fertility test, while having about 113 of its activity in vitro on the gerbil colon, thus showing some separation of pharmacological activity. The enantiomer of 12, although less potent than the natural isomer, shows an even broader separation of smooth muscle and anti-fertility activities and effectively inhibits prostaglandin 15-dehydrogenase, the enzyme primarily responsible for inactivation of the natural compounds. The most active dehydrogenase inhibitor in the series, 13,has a Ki of 7 pm. The lactone 14 (R1,Rz = 0) or closely related intermediates have been the focal point of work from a number of laboratories. A Woodward synthesis of PGF2a was based on the conversion of 15,which was made starting
164
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with c&-cyclohexanetriol, via the amine 16 to give 14 (R1 = H, R2 = OCH3).44 The key reactions were diazotization followed by rearrangement of the resulting carbonium ion. Corey has disclosed45 a similar sequence starting with the cyclohexene analog of 2, whose resolution was reported this year.46 An additional variant started with norbornadiene and proceeds through
14 -
13 -
15 -
0 II
17 -
16 -
18 -
intermediate 17.47Kelly has reportedh8 on an improved synthesis of PGF2, which starts with the endo-aldehyde 18 and utilizes intermediate 19 (R1, R2 = 0). The resolution of a key cyclobutanone intermediate was discussed in a separate paper.49 An alternate synthesis of 19 was reported by another dl-PGF2, via 19 (R1 = H, group.50 Turner has disclosed the synthesis of R2 = OH), starting with dicyclopentadiene.51
R
5H
19
-
bR '
20
W
0
21-a (X=CH=CH)
b
(X'CH2CH2) A synthesis of g-PGD2 by oxidation of the 15-THP derivative of PGF2a was described;52 PGE2 and 11-keto PGE2 were also obtained. In a second communication,53 16(R) and 16(S)-methyl PGE's and PGF's of both the 1 and 2 series were disclosed. Another laboratory has also published the synthesisg4 and biological activity123 of alkyl prostaglandins. 16 (R)-Methyl-PGE2 is reported to be 100 to 200 times more active than PGE2 as an inhibitor of gastric secretion in the rat. The 15-epi compound is also reported to have "strong activity". Two additional papers54~55disclose the synthesis, but not the biological activity, of 2-carboxy- and 15 16-dimethyl prostaglandins in the E, F, and A series. Gandolfi has reported$6 a synthesis of 14-chloroPGE2 and 14-chloro PGFza. Base catalyzed elimination of chloride yielded
Chap. 17
Prostaglandins, Related Compounds Mueller, Flanders
165
the corresponding 13,14-dehydro derivatives whose biology is reported above. The 1,4-addition of organometallic reagents to cyclopentenone derivatives continued to be pursued. dl-11-Deoxy-PGE2 and its 15 epimer were made57 by the addition of 20 (R T L i , R' = CH(CH3)0C2H5) to 21a and the 1,4-alanate addition, reported last year,l was further d e v e l G d to yield PGEl using 21b (R = THP, R' = OTHP) as substrate.58 A second paper reports59 the tri-n-butylphosphine copper (I) iodide complex catalyzed addition of the Grignard reagent 20 (R = MgBr, R1 = trityl) to the same substrate. The synthesis60 of 20 (R = I, R1 = H) by microbial reduction of the corresponding ketone in 10% yield with an optical purity of 80% was reported. The reduc-
22-a (X'CH2CH2)
- (X=CH=CH) b
23 -
H
OCH3 24 -
tion of the 11-ketone in 3 was also accom lished by fermentation, thus expanding the synthesis reported last year.! Compound 21b (R = OH, R 1 = H) has been microbiologically hydroxylated to 21b (R = OH, R1 = OH) .61 Details of the bacterial and chemical resolution of 7-(2-trans-styryl-3-hydroxy-5oxocyclopentenyl)-n-heptanoic acid have been published.62, 63 Another group used 3 as starting material for the synthesis of 8-epi-PGF by a multi1.. step pathway.64 Alvarez and Wren reported65 the Michael addition of nitro(R = R1 = H) as the basis of a synthesis of 11-deoxy PGE1, methane to PGFla, and PGFIB. 15-Keto-tetrahydro-PGA1 has been resolved67 using fermentation methods and both enantiomers have served as substrates for the synthesis o f 23. Minimal anti-fertility action was observed, whereas both enantiomers had oral and parenteral anti-secretorylanti-ulcer effects, with (+)-ent-z being the more potent.68,69 9-Deoxy-PGE1 has been synthesized by another group.66
=
PGA2 from the coral, p. homomalla, continued to provide starting material for the synthesis of the other natural prostaglandins and their analogs. Crabb; reported70 on the intramolecular photochemical addition of the 5,6 and 10,11 double bonds of PGA2 in non-protic solvents which afforded three isomers of 24. A full paper has appeared7I on the photochemical addition of enes and dienes to PGA2 disclosedl earlier. Also r e p 0 r t e d ~ ~ ~were 7 3 the synthesis of 13,14-dihydro-14-methyl-PGB2 methyl ester, 11-methyl-PGE2, 11deoxy-PGE2, PGF2a, their 15-methyl analogs, and 9,11-dimethyl-ll-deo~y-PGF2~ by the reaction of PGA2 with lithium dimethyl copper and of the difluoromethylene adduct of 1 and the resulting PGE2 and PGF2 derivatives. The reaction of PGA2 with diazomethane was investigated in a separate study.74 Photochemical decomposition of the pyrazoline derived addition to the 10,lldouble bond gave both 10,11B-cyclopropyl-PGA2 and 11-methyl PGA2. PGA2 was
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Moreland, Ed.
converted into PGE2 via epoxidation of the l0,ll-double bond and aluminum amalgam reduction by researchers at Upjohn.75 The total yield was 47%, 40% without chromatography. In a second full paper, catalytic reduction of various prostaglandins was discussed and complete experimental details were given.76 The hydrolysis of PGA2 esters using enzymes endogenous to the coral was described75 and a group at Syntex reported77 details applicable to large (10 kg) batches. Corey has also disclosed78 the conversion of PGA2 into PGE2 by epoxidation of the 10,ll-double bond and subsequent reduction. The key step is the use of the bulky tri-2-xylylsilyl derivative of the 15-hydroxyl to give an a/B epoxide ratio of 94/6. He has also published79 details for converting PGF2a into PGE2 using n-chlorosuccinimide/ dimethyl sulfide in 90% yield and the preparation of aldehyde 14 (R1,R2 = 0) in 93% yield from the corresponding alcohol using chlorine/metGl phenyl sulfide. In other publications, Finch,go Miyano,91 Sih,92 and Taube93 have disclosed details or improvements of their previously reportedl,2 syntheses o f prostaglandins. PGAl was tentatively identified as a constituent of yellow onion80 and a metal ion assisted (especially FeC13) thin layer chromatographic method for separating and identifying PGA2 and PGB2 was reported.81 Reports on the PMR spectrum of prostaglandin derivatives using shift reagents82 and 1% nmr spectra83 have ap eared. The X-ray structure of PGA1 has been completed.84 Five papers85-88 were published on the stability and/or kinetics of decomposition of the natural prostaglandins or their salts. Prostaglandin synthetase mechanism studies & inhibition by non-steroidal anti-inflammatory compounds. Although many details of prostaglandin biosynthesis have yet to be worked out, the early work of Bergstrom, Samuelsson and their co-workers from Sweden and that of Van Dorp and his group in the Netherlands forms much of the basis for a possible reaction pathway shown in Scheme 1.192932934 The synthesis of the so-called natural prostaglandins proceeds in several steps and the enzyme catalyzing these reactions are referred to collectively as prostaglandin synthetase (PGS'ase). Many mammalian tissues as well as some cell cultures, invertebrates and fish gills are capable of converting substrate to prostaglandin^.^^,^^ It is implied that PGS'ase is not one enzyme, but rather an enzyme complex. The specific enzymes catalyzing the formation of the PGE's, PGF's, PGD's and PGA's have not been identified to date. Isomerase activity is postulated in the formation of PGE's and PGD's and a reductase activity for formation of PGF's.103 The enzymatic formation of PGAz in kidney has been claimedg7 but it is still uncertain whether the material found was formed enzymatically or by non-enzymatic dehydration of PGE2. Corey -et al.loo have demonstrated that g. homomalla can synthesize PGAl and PGA2; little or no PGE synthesis was detected.
Chap. 17
Prostaglandins, Related Compounds Mueller, Flanders 167
PGDl
-
OH
Scheme
I;
Both the Swedish and Dutch workers have now confirmed the existence of the endo eroxides 27 and 28. Hamberg and Samuelssonlo2 and Nugteren and Hazelhoff03 have isolated 28 from ram seminal vesicle PGS'ase incubations with eicosatetraenoic acid and shown enzymatic as well as non-enzymatic conversion of 28 to PGE2, PGFzcland PGD2. Nugteren and Hazelhof were also able to isolate 27. Compounds known to act as cofactors in the biosynthesis of prostaglandins in vitro were shown by the Dutch workers to affect non-enzymatic breakdown of both 27 and 28. Thus, glutathione (GSH), a "stimulator'' of PGE2 biosynthesis in vitro reduced the 15-hydroperoxy group (measured by reduction of 15-hydroperoxy PGEl to PGE1) .95,103 In combination with ferrihaem, GSH addition catalyzed the reduction of 28 to PGF 2"' The ratio of prostaglandins formed from 28 differs quite markzly in different tissues; for example, rat lung homogenate produced predominantly PGD2 while ram seminal vesicle tissue gave predominantly PGE2. That the endoperoxide does exist in detectable concentrations during biosynthesis for a fairly long time period - T$ = 2-5 min - has raised the question of its role as an active prostaglandin. Samuelsson has shown 28 to be more active than some of the other natural prostaglandins in various biological tests.lO4 These data and that of others has led Samuelsson to propose that 27 and/or 28 may be the physiologically active agent in some tissues or cell types while in others, the PGE's or PGF's may be the physiologically active prostaglandins.
-in vitro
Whatever the lhatural" prostaglandin may be, it is clear that inhibition of the initial dioxygenase step will sto production of prostaglandin by PGS'ase. It was shown in 1970 by Downinglg5 that 5, 8, 11,14-eicosatetrynoic acid inhibited the formation of PGEl from 8, 11,14-eicosatrienoic acid and the following year Vane and Willis published their data showing that aspirin inhibited prostaglandin bios nthesis in human platelets, guinea What has followed these pig lung and dog spleen homogenates.1063187,108 initial papers can be described as a flurry of activity directed toward the following objectives:
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1. to explain the anti-inflammatory, antipyretic and analgesic activity of aspirin on the basis of i.ts observed inhibition of PGS'ase. 2. to establish that most, if not all, non-steroidal anti-inflammatory and antipyretic drugs are capable of inhibiting PGS'ase in vivo and thus, like aspirin, owe their efficacy to this activity. 3. to utilize inhibitors of PGS'ase to explore the role of prostaglandins in normal physiological processes (as opposed to their pharmacological effects). 4 . to develop structure-activity relationships for inhibition of PGS'ase. Vane proposed that antipyretic, analgesic and anti-inflammatory drugs owe their activity to their ability to inhibit PGS'ase.lo6 Flower showed a remarkable correlation between potency of PGS'ase inhibitors and their antiinflammatory activity (carrageenin adema test in rat hind paw) Evidence for the specificity of the enzyme system is that analogs of aspirin lacking anti-inflammatory activity and an enantiomer of naproxen with lowered antipyretic and anti-inflammatory potency also showed much less inhibitory actinn against PGS'ase. Clinical studies involving the measurement of prostaglandins before and after drug therapy are just beginning to appear. Hamberg has shown112 that the appearance of urinary metabolites of PGEl and PGE2 in man is strongly suppressed after therapeutic doses of indomethacin or aspirin. This offered early clinical support for the Vane hypothesis.
Table I Inhibition of PGS'ase by Non-Steroidal Anti-Inflammatory Agents Compound Meclofenamic Acid Niflumic Acid Indomethacin Mef enamic Acid Phenylbutazone Aspirin Paracetamol Paracetamol Indomethacin Naproxen Ibuprofen 2,7-dihydroxynaphthalene
Dexamethasone Hydrocortisone
Tissue Source of PGS'asea Dog Spleen-M Dog Spleen-M Dog Spleen-M Dog Spleen-M Dog Spleen-M Dog Spleen-M Dog Spleen-M Rabbit Brain-H Rabbit Brain-H Bovine S. V.-M Bovine S. V.-M Bovine S. V.-M Dog Spleen-M Dog Spleen-M
ID5o* (w/ml) 0.03 0.03 0.06 0.17 2.23 6.61 100
14 1.3 0.37 2.0 2.0 -
aS.V. - seminal vesicle; M = Microsomes; H = homogenate. *Dose at which compound produced 50% inhibition of PGE2 synthesis i% inhibition at 100 pg/ml.
Ref. 111 111 111 111 111 111 111 120 120 121 121 122 111 111
Chap. 17
Prostaglandins, Related Compounds Mueller, Flanders
169
Research efforts on the inhibitory mechanisms of these drugs against PGS’ase is at an early stage; however, Smith and Lands have demonstrated that indomethacin and aspirin irreversibly inhibited the dioxygenase activity.ll3 This allows one to interpret the actions of these drugs on prostaglandins physiologically, irrespective of whether the active prostaglandin in vivo is the endoperoxide or one o r more of the end products of the biosynthesis. Table I contains a list of dru s with confinned activity of PGS’ase inhibitors. Shen114 and Gryglewskillf have independently proposed structure activity relationships to account for the inhibitory action of most, if not all, of the compounds mentioned in Table I. Other structures reported as PGS’ase inhibitors are corticosteroids,ll6 some psychotropic drugs,ll7 antiestrogens,118 fatty acid hydroperoxides,103 and various acetylenic fatty acids.119 REFERENCES
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2
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Chap. 17 97. 98. 99. 100. 101. 102. 103. 104. 105. 106. 107. 108. 109.
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5,
3
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231.
133,
s,
238.
-
2,
3
2.
186.
Chapter 18.
D i s o r d e r s of L i p i d Metabolism
Gerald F. Holland and Joseph N . P e r e i r a , P f i z e r , I n c . , Groton, Conn. 0 6 3 4 0 I n t r o d u c t i o n - Previous reviews i n t h i s series have been l i m i t e d t o l i p i d a b n o r m a l i t i e s p r e d i s p o s i n g t o c a r d i o v a s c u l a r d i s e a s e which accounts f o r more t h a n f i f t y p e r c e n t of a l l d e a t h s i n t h e United S t a t e s . Although prog r e s s i n a t h e r o s c l e r o s i s r e s e a r c h c o n t i n u e s t o be t h e main f o c u s of t h i s review, i m p o r t a n t advances i n o t h e r a s p e c t s of l i p i d metabolism have prompted us t o expand our scope and c o n s i d e r s e v e r a l p o t e n t i a l new approaches i n o t h e r t h e r a p e u t i c areas. Prominent among t h e s e a r e enhancement of c h o l e s t e r o l g a l l s t o n e d i s s o l u t i o n by i n c r e a s i n g t h e r e l a t i v e conc e n t r a t i o n of b i l i a r y b i l e a c i d s and c o r r e c t i o n of t h e i n a p p r o p r i a t e m o b i l i z a t i o n of f a t t y a c i d s d u r i n g t h e p e r i o d immediately f o l l o w i n g a myocardial infarction. Pathogenesis of A t h e r o s c l e r o s i s - Two new hypotheses on p l a q u e development have been proposed. Ross and Glomset s u g g e s t t h a t t h e l e s i o n i s i n i t i a t e d by t h e accumulation of smooth muscle c e l l s at t h e damaged i n t i m a l s i t e between t h e endothelium and t h e i n t e r n a l e l a s t i c lamina a t a r t e r i a l branch p o i n t s where t h e e n d o t h e l i a l s t r u c t u r e i s thought t o be under g r e a t e s t hemodynamic stress .I With i n c r e a s i n g a g e , numbers of cells i n c r e a s e , t h e i n t i m a t h i c k e n s , and extra- and i n t r a - c e l l u l a r l i p i d s and c o n n e c t i v e t i s s u e a r e d e p o s i t e d . The m u l t i p l i c a t i o n of i n t e r n a l smooth muscle c e l l s i s thought t o be under t h e i n f l u e n c e of plasma l i p o p r o t e i n s (LP). Once plaque formation has been i n i t i a t e d , t h e l e s i o n may remain f i x e d , r e g r e s s o r become a n i d u s f o r d e p o s i t e d c h o l e s t e r o l . The second p r o p o s a l , l i k e t h e f i r s t , s u g g e s t s t h a t t h e l e s i o n is a p r o l i f e r a t i o n of smooth muscle c e l l s i n t h e a r t e r i a l wa11.2 However, i t maintains t h a t t h e smooth muscle c e l l s i n plaques o r i g i n a t e from a s i n g l e mutant c e l l produced e i t h e r by chemical mutagens o r v i r u s e s . A d d i t i o n a l s t u d i e s are needed t o determine whether such t r a n s f o r m i n g f a c t o r s p l a y a c a u s a l r o l e i n a t h e r o s c l e r o s i s . Attempts t o e l u c i d a t e t h e mechanism of l i p i d accumulation i n p l a q u e s continue. C h o l e s t e r o l i n atheromata is known t o be d e r i v e d from plasma c h o l e s t e r o l and n o t from s y n t h e s i s i n s i t u , b u t t h e p r o c e s s e s of accumulat i o n and combination w i t h o t h e r s u b s t a n c e s remain poor.ly understood. Smith and S l a t e r 3 3 4 examined postmortem a o r t i c LP-bound and unbound c h o l e s t e r o l i n p a t i e n t s whose blood c h o l e s t e r o l levels were measured w i t h i n a week of d e a t h and found a c o r r e l a t i o n between t h e amount of l o w d e n s i t y l i p o p r o t e i n s (LDL) i n normal intima and plasma c h o l e s t e r o l l e v e l s . An i n c r e a s e of 100 mg p e r c e n t i n serum c h o l e s t e r o l gave a 50% i n c r e a s e i n i n t i m a l LPbound c h o l e s t e r o l , b u t d i d n o t appear t o i n c r e a s e t h e unbound c h o l e s t e r o l . The f i n d i n g t h a t i n normal a o r t i c i n t i m a t h e r e is i n t a c t LP which appears t o be i n e q u i l i b r i u m w i t h p l a s m a LP h a s prompted Adams5 t o s u g g e s t t h a t t h e a r t e r i a l ( i n t i m a l ) LP c h o l e s t e r o l p o o l should be more a c c e s s i b l e t o metabolic and exchange p r o c e s s e s t h a n t h e unbound c h o l e s t e r o l p o o l . Smith and ~ l a t e r 6conclude t h a t t h e p e r i f i b r o u s l i p i d i n normal i n t i m a , t h e pool of amorphous atheroma l i p i d under r a i s e d p l a q u e s and t h e l i p i d w i t h i n f a t f i l l e d cells i n f a t t y s t r e a k s are no l o n g e r i n t h e form of i n t a c t LP i n e q u i l i b r i u m w i t h plasma LP, b u t r e p r e s e n t unbound c h o l e s t e r o l . I f t h i s
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i s t r u e , r e d u c i n g i n t i m a l LP c h o l e s t e r o l l e v e l s should reduce t h e rate of d e p o s i t i o n of unbound c h o l e s t e r o l b u t i s n o t l i k e l y t o remove unbound chol e s t e r o l from t h i s p o o l . A s i g n i f i c a n t c o r r e l a t i o n between a o r t i c and serum c h o l e s t e r o l l e v e l s i n a t h e r o s c l e r o t i c swine demonstrates t h a t serum c h o l e s t e r o l l e v e l s are indeed good measures of a t h e r o g e n e s i s . 7 The work of Smith and S l a t e r s u g g e s t s t h a t t h e e p i d e m i o l o g i c a l r e s u l t s e s t a b l i s h i n g high serum c h o l e s t e r o l l e v e l s and e l e v a t e d blood p r e s s u r e as a d d i t i v e r i s k f a c t o r s p r e d i s p o s i n g t o coronary h e a r t d i s e a s e nay b e e x p l a i n a b l e by t h e simple f a c t t h a t h y p e r t e n s i o n i n c r e a s e s t h e t o t a l volume of serum e n t e r i n g t h e i n t i m a , and hence i n c r e a s e s t h e d e p o s i t i o n of LP c h o l e s t e r o l . The f a c t o r s which c o n c e n t r a t e and keep i n t a c t LP w i t h i n t h e i n t i m a are n o t known. However, t h e s e f i n d i n g s p r o v i d e evidence of an e q u i l i b r i u m between plasma LP c h o l e s t e r o l and a o r t i c LP c h o l e s t e r o l which u n d e r s c o r e s t h e v a l u e of reducing plasma c h o l e s t e r o l l e v e l s . F i b r i n o g e n , a n o t h e r plasma compon e n t , has a l s o been shown t o e n t e r t h e i n t i m a . 8 A d d i t i o n a l s t u d i e s w i l l be r e q u i r e d t o determine whether f i b r i n o g e n i s converted t o f i b r i n which could c o n t r i b u t e t o t h e formation of t h e a t h e r o s c l e r o t i c l e s i o n .
There has been an i n c r e a s e d i n t e r e s t i n t h e r o l e t h a t plasma and c e l l u l a r enzymes p l a y i n c h o l e s t e r o l e s t e r i f i c a t i o n and h y d r o l y s i s , and t h e p a t h o l o g i c a l consequences t h e r e o f . For example, i t appears t h a t t h e accumulation of c h o l e s t e r o l esters i n t h e arterial w a l l , e i t h e r by f i l t r a t i o n o r s y n t h e s i s , i s a c h a r a c t e r i s t i c f e a t u r e of a t h e r o g e n e s i s . E i t h e r a f t e r o r d u r i n g t h e passage of f r e e c h o l e s t e r o l i n t o t h e a r t e r i a l w a l l , 9 m e t a b o l i c changes occur t h e r e , which promote t h e s y n t h e s i s of f a t t y a c i d s . The f a t t y a c i d s combine w i t h c h o l e s t e r o l t o form c h o l e s t e r o l esters which are unable t o d i f f u s e o u t , and remain f i x e d . The enzyme t h a t c a t a l y z e s t h e formation of c h o l e s t e r o l esters i n plasma i s 1 e c i t h i n : c h o l e s t e r o l a c y l t r a n s f e r a s e (LCAT) .lo O r i g i n a l l y thought t o be a c h o l e s t e r o l esterase of l i t t l e importance i n plasma LP metabolism, i t h a s now been shown t o c a t a l y z e t h e t r a n s f e r of f a t t y a c i d s from l e c i t h i n t o t h e c h o l e s t e r o l of plasma LP. LCAT i s mainly e x t r a c e l l u l a r and i s s e c r e t e d i n t o plasma by t h e l i v e r . Evidence i n man s u g g e s t s t h a t c h o l e s t e r o l e n t e r s t h e plasma i n t h e f r e e form and is e s t e r i f i e d w i t h i n t h e plasma by t h e a c t i o n of LCAT f o r which f h e p r e f e r r e d s u b s t r a t e s are h i g h d e n s i t y l i p o p r o t e i n (HDL) c h o l e s t e r o l and l e c i t h i n . LCAT i n d i r e c t l y r e d u c e s t h e u n e s t e r i f i e d c h o l e s t e r o l and l e c i t h i n of plasma LP o t h e r t h a n HDL, by v i r t u e of t h e non-enzymatic e q u i l i b r a t i o n of t h e s e l i p i d s among o t h e r LP. The p h y s i o l o g i c a l r o l e of t h e plasma LCAT r e a c t i o n is n o t f u l l y understood, b u t i t is t h o u g h t t o b e of major importance f o r c h o l e s t e r o l homeostasis. It h a s been s u g g e s t e d t h a t LCAT, by i n c r e a s i n g t h e l e v e l of plasma HDL c h o l e s t e r o l esters a t t h e expense of HDL f r e e c h o l e s t e r o l , promotes t h e u p t a k e and t r a n s p o r t of f r e e c h o l e s t e r o l from p e r i p h e r a l t i s s u e s , such as t h e a r t e r i a l w a l l , t o t h e l i v e r . 1 1 The f a c t t h a t c h o l e s t e r o l e s t e r i f i c a t i o n i n blood i s impaired i n p a t i e n t s w i t h a c u t e myocardial i n f a r c t i o n and coronary a r t e r y d i s e a s e t e n d s t o s u p p o r t t h i s sugges t i o n . 1 2 A d d i t i o n a l s u p p o r t € o r t h i s h y p o t h e s i s 1 s found i n t h e r e p o r t t h a t t h e t u r n o v e r of HDL and LDL c h o l e s t e r o l esters i s reduced i n t y p e I1 s u b j e c t s . 1 3 I f one assumes t h a t d e c r e a s e d c h o l e s t e r o l e s t e r t u r n o v e r i s mainly due t o d e c r e a s e d c h o l e s t e r o l e s t e r i f i c a t i o n , t h e n t h e i n c r e a s e d r i s k of a t h e r o s c l e r o s i s observed i n such s u b j e c t s may be due t o reduced LCAT a c t i v i t y . On t h e o t h e r hand, the s p e c u l a t i o n of Glomset
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and Norum t h a t t h e f u n c t i o n of LCAT might b e t o i n s u r e t h a t c e l l membranes r e c e i v e an adequate s u p p l y of f r e e c h o l e s t e r o l from plasma a p p e a r s t o b e less t e n a b l e . 1 0 The p o s s i b i l i t y t h a t t h e primary f u n c t i o n of LCAT i s t o m a i n t a i n LP s t r u c t u r e and s u r f a c e area h a s a l s o been suggested.14 C h o l e s t e r o l e s t e r i f i c a t i o n (and h y d r o l y s i s ) t a k e s p l a c e a t a number of o t h e r s i t e s , e.g. l i v e r , p a n c r e a s , small i n t e s t i n e , s k i n , a o r t a , l 5 , 1 6 o v a r i e s ( c o r p o r a l u t e a and i n t e r s t i t i a l t i s s u e l 7 ) and a d i p o s e t i s s u e . 1 8 L i v e r and a r t e r i a l t i s s u e are of s i g n i f i c a n c e i n a t h e r o g e n e s i s s i n c e b o t h are involved i n t h e uptake and d e g r a d a t i o n of plasma c h o l e s t e r o l esters. I n man, a r e v e r s i b l e h e p a t i c a c i d c h o l e s t e r o l esterase whose main f u n c t i o n is probably ester h y d r o l y s i s h a s been d e s c r i b e d . 1 9 S i n c e c h o l e s t e r o l esters i n plasma LP are good s u b s t r a t e s f o r t h i s enzyme, i t h a s been proposed t h a t i t f a c i l i t a t e s t h e h y d r o l y s i s of LP c h o l e s t e r o l esters p r i o r t o b i l i a r y s e c r e t i o n . I n normal and a t h e r o s c l e r o t i c a r t e r i a l t i s s u e , b o t h t h e s y n t h e s i s and h y d r o l y s i s of c h o l e s t e r o l esters have been shown t o o c c u r . Two pathways f o r a r t e r i a l c h o l e s t e r o l e s t e r i f i c a t i o n have been claimed, d i rect e s t e r i f i c a t i o n w i t h acyl-CoA and t r a n s e s t e r i f i c a t i o n v i a a t i s s u e LCAT. The remarkable i n c r e a s e i n t h e a c y l CoA-dependent e s t e r i f i c a t i o n d u r i n g a t h e r o g e n e s i s s u g g e s t s t h a t t h e former r e a c t i o n i s of s u b s t a n t i a l l y g r e a t e r s i g n i f i c a n c e .20 Both t h e enhanced rate (28-40 f o l d ) of c h o l e s t e r o l e s t e r i f i c a t i o n i n t h e a t h e r o s c l e r o t i c i n t i m a and t h e g r e a t e r p e r m e a b i l i t y of LP c h o l e s t e r o l ester i n t o a t h e r o s c l e r o t i c l e s i o n s s u g g e s t t h a t t h e s e p r o c e s s e s may c a u s e t h e observed accumulation of c h o l e s t e r o l esters .20 s 2 l I n a d d i t i o n , an impaired rate of c h o l e s t e r o l ester h y d r o l y s i s may c o n t r i b u t e t o c h o l e s t e r o l ester accumulation.22 There a p p e a r s t o b e l i t t l e doubt t h a t c h o l e s t e r o l es t e r i f i c a t i o n and t h e h y d r o l y s i s of c h o l e s t e r o l esters are u b i q u i t o u s and i m p o r t a n t p r o c e s s e s i n c h o l e s t e r o l economy. However , u n t i l t h e r e is f u r t h e r c l a r i f i c a t i o n of t h e s i g n i f i c a n c e of t h e s e r e a c t i o n s i n t i s s u e and plasma, t h e y appear t o o f f e r a l i m i t e d b a s i s f o r a therapeut i c approach t o a t h e r o s c l e r o s i s . E a r l y D e t e c t i o n and Treatment of Hyperlipidemia - The r e c o g n i t i o n t h a t c e r t a i n forms of h y p e r l i p i d e m i a are f a m i l i a l i n n a t u r e h a s s t i m u l a t e d invest i g a t i o n s i n t o t h e u n d e r l y i n g p a t t e r n s of i n h e r i t a n c e . Only i n t h e case of F r e d r i c k s o n t y p e I d i s o r d e r , which arises from an autosomal r e c e s s i v e 23 t r a i t of l i p o p r o t e i n l i p a s e d e f i c i e n c y , h a s t h e p a t t e r n been e s t a b l i s h e d . Three f a m i l i a l l i p i d d i s o r d e r s , d e s i g n a t e d f a m i l i a l h y p e r c h o l e s t e r o l e m i a , f a m i l i a l hy e r t r i g l y c e r i d e m i a and f a m i l i a l combined h y p e r l i p i d e m i a were i d e n t i f i e d . 34-26 I n f a m i l i a l h y p e r t r i g l y c e r i d e m i a and f a m i l i a l combined h y p e r l i p i d e m i a reduced e x p r e s s i o n of t h e g e n e t i c d e f e c t (incomplete p e n e t r a n c e ) w a s observed i n r e l a t i v e s below age 25. The a u t h o r s conclude t h a t t h e t h r e e major f a m i l i a l l i p i d d i s o r d e r s r e p r e s e n t dominant e x p r e s s i o n of t h r e e d i f f e r e n t autosomal genes. S e v e r a l ~ t u d i e s ~ ’ have - ~ ~ been undertaken t o determine whether l i p i d a n a l y s e s of u m b i l i c a l c o r d blood are p r e d i c t i v e of h y p e r l i p o p r o t e i n e m i a i n later l i f e . These s t u d i e s s u g g e s t t h a t d i a g n o s i s of t h e t y p e I1 d i s o r d e r can be made on t h e b a s i s of h i g h c o r d blood serum c h o l e s t e r o l concentrat i o n s . On t h e o t h e r hand, Darmady e t a1.33 concluded t h a t c h o l e s t e r o l v a l u e s i n cord blood are n o t a r e l i a b l e b a s i s f o r e a r l y d e t e c t i o n of t y p e
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IIa h y p e r c h o l e s t e r o l e m i a . The w e i g h t of t h e s e v e r a l s t u d i e s i n d i c a t e s t h a t d i a g n o s i s a t b i r t h on t h e b a s i s of plasma c h o l e s t e r o l c o n c e n t r a t i o n s i s , at b e s t , d i f f i c u l t and can b e improved markedly by a knowledge of LDL l e v e l s and t h e f a m i l y h i s t o r y w i t h r e g a r d t o e s s e n t i a l h y p e r c h o l e s t e r o l e m i a . The a b i l i t y t o d e t e c t h y p e r l i p i d e m i a i n childhood h a s l e d t o N a t i o n a l Heart and Lung I n s t i t u t e - s u p p o r t e d d e t e c t i o n programs which are p r e s e n t l y b e i n g c a r r i e d o u t a t t h r e e d e s i g n a t e d r e s e a r c h c e n t e r s and w i l l b e expanded t o i n c l u d e twelve c e n t e r s a t a l a t e r d a t e .
D e t e c t a b i l i t y of t y p e I1 h y p e r l i p o p r o t e i n e m i a i n e a r l y childhood s u g g e s t s t h e p o s s i b i l i t y of e a r l y t h e r a p e u t i c i n t e r v e n t i o n . Toward t h i s end, s e v e r a l s t u d i e s have examined t h e e f f e c t s of d i e t a r y m a n i p u l a t i o n on i n f a n t plasma c h o l e s t e r o l l e v e l s . I n t y p e I1 h y p e r l i p o p r o t e i n e m i c i n f a n t s ,34 a p r o g r e s s i v e h y p e r c h o l e s t e r o l e m i a develops on cow's milk o r b r e a s t milk d i e t s . On t h e o t h e r hand, formulas r i c h i n c o r n o i l and low i n c h o l e s t e r o l d e p r e s s plasma c h o l e s t e r o l t o l e v e l s s e e n i n normal i n f a n t s . T h i s r e s p o n s i v e n e s s t o d i e t a r y management p e r s i s t s through t h e f i r s t y e a r of l i f e b u t i n o l d e r c h i l d r e n w i t h t h e t y p e I1 d e f e c t , a much reduced I n a d d i t i o n t o d i e t a r y m a n i p u l a t i o n , West r e s p o n s i v e n e s s i s observed.34-36 and Lloyd37 have a d m i n i s t e r e d c h o l e s t y r a m i n e t o heterozygous t y p e 11 c h i l dren and produced a 36 p e r c e n t r e d u c t i o n i n c h o l e s t e r o l l e v e l s . D i e t a r y m o d i f i c a t i o n had no a d d i t i o n a l e f f e c t on serum c h o l e s t e r o l v a l u e s . 38 I n a l i m i t e d number of homozygous t y p e I1 s u b j e c t s , n i c o t i n i c acid and c h o l e s t y r a m i n e , a l o n e o r i n combination , produced l i m i t e d e f f e c t s on plasma c h o l e s t e r o l . However, t h e f a c t t h a t t h e combination reduced t h e s i z e of atheromatous l e s i o n s s u g g e s t s t h a t t h e mass o f t o t a l body exchangea b l e c h o l e s t e r o l w a s d e c r e a s e d . I n a similar b u t l a r g e r s t u d y , 3 9 more prominent c h o l e s t e r o l d e c r e a s e s and l e s i o n r e g r e s s i o n s were r e p o r t e d . A combination of c l o f i b r a t e and a low s a t u r a t e d f a t d i e t supplemented w i t h corn o i l produced a serum c h o l e s t e r o l d e c r e a s e of 3 3 p e r c e n t i n heterozygous t y p e I1 ~ h i l d r e n . 3 ~These p r e l i m i n a r y f i n d i n g s p r o v i d e a b a s i s f o r optimism i n t h e t r e a t m e n t of heterozygous t y p e I1 c h i l d r e n . However, t h e r e s u l t s o f s t u d i e s w i t h homozygous c h i l d r e n are less promising and add i t i o n a l s t u d i e s w i l l be required t o provide a s a t i s f a c t o r y treatment f o r c h i l d r e n a f f l i c t e d by t h i s malevolent form of h y p e r l i p o p r o t e i n e m i a . Although t h e l i a b i l i t y r e p r e s e n t e d by e l e v a t e d serum c h o l e s t e r o l l e v e l s has been amply s t u d i e d , t h e r i s k of c a r d i o v a s c u l a r d i s e a s e related t o h i g h serum t r i g l y c e r i d e l e v e l s h a s o n l y r e c e n t l y been c h a r a c t e r i z e d . I n view of t h e i n c r e a s e d r i s k of ischemic h e a r t d i s e a s e i n h y p e r t r i g l y c e r i demia, a t t e m p t s have been made t o d e t e c t t h i s a b n o r m a l i t y a t b i r t h . Tsang and Glueck40 f a i l e d t o d e t e c t h y p e r t r i g l y c e r i d e m i a i n c o r d blood of neon a t e s from f a m i l i e s w i t h e l e v a t e d serum t r i g l y c e r i d e l e v e l s . Other s t u d i e s 4 1 ~ 4 2have d e t e c t e d s i g n i f i c a n t l y e l e v a t e d cord blood t r i g l y c e r i d e s . These f i n d i n g s s u g g e s t i n g t h a t h y p e r t r i g l y c e r i d e m i a may b e d e t e c t e d a t b i r t h must be weighed a g a i n s t t h e o b s e r v a t i o n of l i m i t e d p e n e t r a n c e of f a m i l i a l h y p e r t r i g l y c e r i d e m i a and combined h y p e r l i p i d e m i a i n s u b j e c t s below t h e age of 25.25 I n view of d e l a y e d t r i g l y c e r i d e e l e v a t i o n , cord blood t r i g l y c e r i d e a n a l y s e s would b e e x p e c t e d t o d e t e c t only a l i m i t e d p o r t i o n of t h a t p o p u l a t i o n d e s t i n e d t o become h y p e r t r i g l y c e r i d e m i c . Hypolipidemic Drugs - The clear d e s i g n a t i o n of e l e v a t e d serum l i p i d l e v e l s as r i s k f a c t o r s i n t h e development of a t h e r o s c l e r o t i c h e a r t d i s e a s e h a s
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s t i m u l a t e d e f f o r t s t o d i s c o v e r a g e n t s which reduce c i r c u l a t i n g l i p i d l e v e l s . C l o f i b r a t e , t h e most widely used hypolipidemic a g e n t , is t h e subj e c t of4!jyz4 new s t u d i e s e v a l u a t i n g long-term e f f e c t s on m o r b i d i t y and mortality, b u t no r e s u l t s have been d e s c r i b e d . Based on t h e i n i t i a l o b s e r v a t i o n s of an i n v e r s e r e l a t i o n s h i p between plasma t r i g l y c e r i d e l e v e l s and g l u c o s e t o l e r a n c e 4 5 and improved g l u c o s e t o l e r a n c e w i t h c l o f i b r a t e therapy,46,47 a number of s t u d i e s have been undertaken t o d e l i n e a t e t h e e f f e c t s of c l o f i b r a t e on plasma g l u c o s e l e v e l s , g l u c o s e t o l e r a n c e , and glucose- and a r g i n i n e - s t i m u l a t e d i n s u l i n s e c r e t i o n . I n r a t s , c l o f i b r a t e a d m i n i s t r a t i o n reduced c i r c u l a t i n g l e v e l s of plasma i n s u l i n d u r i n g f a s t i n g and a f t e r s t i m u l a t i o n of s e c r e t i o n by g l u c o s e o r glucagon. I n c r e a s e d s e n s i t i v i t y t o i n s u l i n i n v i v o and i n v i t r o w a s obs e r v e d i n c l o f i b r a t e - t r e a t e d animals.48 Eaton and Nye49 found t h a t c l o f i b r a t e administration t o hyperlipemic p a t i e n t s decreased glucose-stimulated i n s u l i n s e c r e t i o n and improved g l u c o s e t o l e r a n c e . I n normal humans, c l o f i b r a t e d e c r e a s e d t h e rate of a r g i n i n e - s t i m u l a t e d i n s u l i n s e c r e t i o n and 51 g l u c o s e t o l e r a n c e w a s e i t h e r improved o r unchanged.50 Fenderson e t a l . r e p o r t e d t h a t c l o f i b r a t e t r e a t m e n t d e c r e a s e d b l o o d g l u c o s e and i n s u l i n e l e v a t i o n s d u r i n g g l u c o s e t o l e r a n c e tests i n normal and h y p e r l i p o p r o t e i n emic s u b j e c t s as w e l l as f a s t i n g blood g l u c o s e a n d / o r plasma i n s u l i n l e v e l s i n t h e l a t t e r . The e v i d e n c e provided by t h e s e i n v e s t i g a t i o n s i n d i c a t e s t h a t c l o f i b r a t e t r e a t m e n t reduces c i r c u l a t i n g i n s u l i n l e v e l s w h i l e e i t h e r d e c r e a s i n g o r h a v i n g no e f f e c t on blood glucose v a l u e s , s t r o n g l y s u g g e s t i n g i n c r e a s e d i n s u l i n s e n s i t i v i t y as t h e b a s i s f o r improved g l u c o s e t o l e r a n c e . A d d i t i o n a l s t u d i e s are r e q u i r e d t o e l u c i d a t e whether c l o f i b r a t e i n c r e a s e s i n s u l i n s e n s i t i v i t y d i r e c t l y o r as a r e s u l t of i t s h y p o t r i g l y c e ridemic e f f e c t . E s t r o g e n , i n i t i a l l y i n c l u d e d i n t h e Coronary Drug P r o j e c t a t d o s e s of 2.5 and 5.0 mglkglday, h a s now been removed from t h e s t u d y b e c a u s e of l a c k of t h e r a p e u t i c e f f e c t and a s u g g e s t i o n of a d v e r s e t r e n d s w i t h r e g a r d t o m o r b i d i t y and m o r t a l i t y . 5 2 The d e l e t i o n of e s t r o g e n l e a v e s c l o f i b r a t e and n i c o t i n i c a c i d as t h e only drugs remaining i n t h e s t u d y . T i b r i c a c i d , a new, s t r u c t u r a l l y d i s t i n c t , hypolipidemic a g e n t , dep r e s s e d plasma t r i g l y c e r i d e l e v e l s of t y p e I V s u b j e c t s f o r up t o t w e l v e months. The drug w a s w e l l t o l e r a t e d w i t h minimal s i d e e f f e c t s . 5 3 0 CH3 00CH2 CH2NHCCH3 ti
p-36;
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Studies with halofenate continue t o e s t a b l i s h t h e u r i c o s u r i c e f f e c t The e a r l i e r p a t t e r n of plasma t r i g l y of t h i s hypolipidemic a g e n t .54-56 c e r i d e lowering w i t h l i m i t e d e f f e c t s on plasma c h o l e s t e r o l l e v e l s c o n t i n u e s t o b e observed and t h e drug a p p e a r s t o be of v a l u e i n t r e a t i n g pat i e n t s w i t h h y p e r t r i g l y c e r i d e m i a and hyperuricemia.
Chap. 1 8
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Holland, P e r e i r a
177
Probucol, a hypocholesterolemic agent which h a s no c o n s i s t e n t e f f e c t on p l a s m a t r i g l y c e r i d e v a l u e s , caused s u s t a i n e d d e c r e a s e s i n plasma c h o l e s t e r o l l e v e l s of approximately 15 p e r c e n t . 5 7 The drug appears t o b e w e l l t o l e r a t e d i n man and t h e unexpected t o x i c i t y of probucol i n dogs appears t o b e due t o s p e c i e s - s p e c i f i c s e n s i t i z a t i o n t o epinephrine-induced v e n t r i c u l a r f i b r i l l a t i o n . 5 8 959 C o l e s t i p o l w a s w e l l t o l e r a t e d and reduced plasma c h o l e s t e r o l l e v e l s d u r i n g a 2 y e a r s t u d y i n t y p e s 11, I11 and I V h y p e r l i p o p r o t e i n e m i c s u b j e c t s . 6 0 I n a n o t h e r s t u d y , c o l e s t i p o l , a t a d a i l y dose of 15 g, decreased p l a s m a c h o l e s t e r o l l e v e l s i n t y p e I1 and I V s u b j e c t s . The addit i o n of c l o f i b r a t e f u r t h e r reduced c h o l e s t e r o l v a l u e s . Serum t r i g l y c e r i d e l e v e l s were e l e v a t e d by c o l e s t i p o l b u t t h e e f f e c t could be r e v e r s e d by c l o f i b r a t e . 6 1 Similar t r i g l y c e r i d e e l e v a t i o n s have been observed w i t h cholestyramine62 which, d u r i n g t h e p a s t y e a r r e c e i v e d FDA a p p r o v a l f o r a lipid-lowering indication. A number of phenoxyacetic a c i d s r e l a t e d t o c l o f i b r a t e were d e s c r i b e d ; two of t h e s e , S-852763 and ~ ~ - 3 0 a8r e, more ~ ~ potent than c l o f i b r a t e i n reducing plasma l i p i d l e v e l s i n t h e r a t . &O-!;YOH c2H5
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u S-8527
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E r i t a d e n i n e is approximately t e n times a s p o t e n t a s c l o f i b r a t e i n rats. D i e t a r y hypercholes t e r o l e m i a was suppressed by e r i t a d e n i n e b u t l i m i t e d e f f e c t s on T r i t o n induced h y p e r l i p i d e m i a were observed. The o r a l r o u t e of a d m i n i s t r a t i o n appears t o produce s u p e r i o r l i p i d r e d u c t i o n s and t h e drug causes a l i m i t e d b u t s i g n i f i c a n t enhancement i n t h e r a t e of removal of cholest e r o l from t h e plasma ~ o m p a r t m e n t . ~ ~
@y) 4, , “I2
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e r i t adenine Treatment During t h e Post-Myocardial I n f a r c t i o n P e r i o d - Although c l i n i c a l emphasis has been d i r e c t e d toward i n f l u e n c i n g t h e major r i s k f a c t o r s p r e d i s p o s i n g t o a t h e r o s c l e r o t i c h e a r t d i s e a s e , s i g n i f i c a n t e f f o r t s are b e i n g made t o a l t e r t h e c o u r s e of e v e n t s immediately f o l l o w i n g an i n f a r c t i o n . The s u r v i v a l of p a t i e n t s h o s p i t a l i z e d w i t h a c u t e myocardial i n f a r c t i o n h a s been improved by p r e v e n t i n g f a t a l arrhythmias through t h e a d m i n i s t r a t i o n of a n t i a r r h y t h m i c drugs.66 The cause of t h e s e arrhythmias h a s come under i n v e s t i g a t i o n and t h e Edinburgh group67 h a s demonstrated t h a t plasma f r e e f a t t y a c i d (FFA) l e v e l s a r e markedly e l e v a t e d d u r i n g t h e f i r s t few hours f o l l o w i n g an a c u t e myocardial i n f a r c t i o n . These f i n d i n g s have been confirmed68 and extended t o show t h a t , i n t h e c r u c i a l , immediate p o s t - i n f a r c t i o n p e r i o d , t o t a l plasma catecholamine, c o r t i s o l and g l u c o s e l e v e l s a r e s i g n i f i c a n t l y i n c r e a s e d . 69 P l a s m a FFA c o n c e n t r a t i o n s have been
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shown t o be p a r t i c u l a r l y e l e v a t e d i n t h o s e p a t i e n t s who develop f a t a l arrhythmias.70 Elevated FFA l e v e l s may, t h e r e f o r e , have p r e d i c t i v e v a l u e i n i d e n t i f y i n g i n f a r c t i o n p a t i e n t s who are a t s p e c i a l r i s k . I n i n f a r c t i o n p a t i e n t s , h e p a r i n , which i n c r e a s e s p l a s m a FFA l e v e l s , does n o t c l e a r l y i n c r e a s e t h e i n c i d e n c e of arrhythmias71 although a r e c e n t study72 s u g g e s t s a p o t e n t i a l r i s k which must be weighed a g a i n s t t h e q u e s t i o n a b l e p o s i t i v e v a l u e of h e p a r i n . The mechanism by which e l e v a t e d FFA l e v e l s l e a d t o a r r h y t h m i a s has been examined and i t has been proposed t h a t , i n t h e p r e s e n c e of myocardial ischemia, i n c r e a s e d FFA l e v e l s l e a d t o i n c r e a s e d uptake and oxygen consump t i o n . 7 3 I t has been claimed t h a t t h e o x i d a t i o n of f a t t y a c i d s r e q u i r e s more oxygen t h a n does glucose o x i d a t i o n . S i n c e oxygen supply i s l i m i t e d i n myocardial ischemia, FFA h a s been viewed as an i n a p p r o p r i a t e s u b s t r a t e d u r i n g t h e p o s t i n f a r c t i o n p e r i o d . I f , however, one c o n s i d e r s t h e e f f i ciency of oxygen u t i l i z a t i o n , i . e . , c a l o r i c y i e l d p e r u n i t of oxygen cons u m d , i t becomes c l e a r t h a t FFA i s o x i d i z e d almost as e f f i c i e n t l y a s i s glucose. On t h i s b a s i s , i t would appear t h a t e x c e s s i v e m o b i l i z a t i o n of o x i d i z a b l e s u b s t r a t e of any s o r t should b e minimized d u r i n g myocardial ischemia. With t h i s o b j e c t i v e , t h e e f f e c t s of a v a r i e t y of l i p o l y s i s i n h i b i t o r s have been examined. 5-Fluoro-3-pyridinemethanol h a s been shown t o s u p p r e s s t h e appearance of arrhythmias produced by h e p a r i n a d m i n i s t r a t i o n t o dogs w i t h l i g a t e d coronary a r t e r i e s . 7 4 I n a d d i t i o n , t h i s agent d e p r e s s e s t h e e l e v a t e d plasma FFA l e v e l s observed i n man immediately f o l l o w i n g a c u t e myocardial i n f a r c t i o n . 75 The r e c e n t f i n d i n g s of Kjekshus and M j & s i n d i c a t e t h a t 3-pyridinemethanol, a n i c o t i n i c a c i d p r e c u r s o r , reduced t h e e x t e n t and s e v e r i t y of myocardial i n f a r c t i o n i n dogs.76 These f i n d i n g s o f f e r encouragement t h a t l i p o l y s i s i n h i b i t o r s may reduce t h e i n c i d e n c e of p o s t - i n f a r c t i o n arrhythmias i n man. C h o l e s t e r o l G a l l s t o n e s - Another a s p e c t of c h o l e s t e r o l metabolism t h a t h a s r e c e i v e d a g r e a t d e a l of a t t e n t i o n i s t h e formation and d i s s o l u t i o n of c h o l e s t e r o l g a l l s t o n e s . More t h a n 70% of t h e c r y s t a l l i n e matter i n most g a l l s t o n e s found i n p a t i e n t s i n t h e United S t a t e s and w e s t e r n c o u n t r i e s i s c h o l e s t e r o l . T h e i r formation i n man i s a s s o c i a t e d w i t h a b n o r m a l i t i e s i n t h e r e l a t i v e c o n c e n t r a t i o n s of t h e major b i l i a r y l i p i d s , c h o l e s t e r o l , b i l e a c i d s and l e c i t h i n . Under normal c o n d i t i o n s s u f f i c i e n t c o n c e n t r a t i o n s of b i l e a c i d s and l e c i t h i n are p r e s e n t i n t h e b i l e s o t h a t t h e c h o l e s t e r o l s e c r e t e d from t h e l i v e r can b e s o l u b i l i z e d by formation of mixed m i c e l l e s . G a l l s t o n e s a r e formed when t h e l i v e r secretes b i l e t h a t i s d e f i c i e n t i n b i l e a c i d s and phospholipids and s u p e r s a t u r a t e d w i t h r e s p e c t t o c h o l e s t e r o l c a u s i n g e x c e s s c h o l e s t e r o l t o p r e c i p i t a t e d u r i n g c o n c e n t r a t i o n and s t o r a g e i n t h e g a l l b l a d d e r . These c h o l e s t e r o l c r y s t a l s , i f n o t emptied from t h e g a l l b l a d d e r o r r e d i s s o l v e d , w i l l a g g r e g a t e , b e cemented t o g e t h e r w i t h b i l i a r y mucous s u b s t a n c e s and grow i n t o macroscopic c h o l e s t e r o l gallstones.77 It i s n o t y e t c l e a r whether t h e primary abnormality i s i n t h e h e p a t i c c e l l s t h a t produce b i l e , o r i n t h e g a l l b l a d d e r i t s e l f . Some s t u d i e s of t h e e f f e c t of cholecystectomy on b i l e a c i d p o o l s , k i n e t i c s and b i l i a r y l i p i d composition i n d i c a t e t h a t t h e formation of l i t h o g e n i c b i l e does n o t depend on t h e presence of a g a l l b l a d d e r , s u g g e s t i n g t h a t t h e g a l l b l a d d e r is merel y a s t o r a g e s i t e c o n t r i b u t i n g t o s t o n e formation, p o s s i b l y by s t r a t i f i c a t i o n of b i l e o r by i t s i n a b i l i t y t o empty completely.78 Others have re-
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p o r t e d however, t h a t b i l i a r y l i p i d composition t e n d s t o r e t u r n t o normal a f t e r c h o l e c y s t e c t o m y . ~ ~I f t h i s be s o , t h e g a l l b l a d d e r must have an act i v e r o l e i n c a u s i n g h e p a t i c s e c r e t i o n of l i t h o g e n i c b i l e . 8 0 9 8 1 I n e i t h e r case, h e p a t i c b i l e s u p e r s a t u r a t e d w i t h c h o l e s t e r o l i s r e q u i r e d f o r g a l l s t o n e formation; t h e r e is ample evidence s u g g e s t i n g t h a t t h e abnormality , at l e a s t i n p a r t , r e s u l t s from i n c r e a s e d h e p a t i c s e c r e t i o n of c h o l e s t e r o l and a d e c r e a s e d b i l e a c i d pool s i z e . A major breakthrough i n u n d e r s t a n d i n g t h e c o n d i t i o n s under which g a l l s t o n e s form was t h e d e f i n i t i o n of c h o l e s t e r 01 s o l u b i l i t y i n b i l e i n terms of t h e t r i a n g u l a r phase diagram. S i n c e t h e o r i g i n a l r e p o r t by Admirand and a number of o t h e r s have shown o v e r l a p between t h e m i c e l l a r zones of normal and abnormal b i l e . By u s i n g more r i g o r o u s s o l u b i l i t y t e c h n i q u e s t o i n s u r e e q u i l i b r a t i o n , i t has now been demonstrated t h a t t h e m i c e l l a r s o l u b i l i t y zone i s smaller t h a n f i r s t p o s t ~ l a t e d . It ~ ~ h a s been r e p o r t e d t h a t some normal b i l e s a r e s u p e r s a t u r a t e d w i t h r e s p e c t t o c h o l e s t e r o l . 8 4 The i n d e x of s a t u r a t i o n u t i l i z i n g r e c t a n g u l a r c o o r d i n a t e s has been proposed a s a method which b e t t e r exp r e s s e s phase diagram d a t a t h a n do t r i a n g u l a r c o o r d i n a t e s . S t u d i e s show t h a t human g a l l s t o n e s d i s s o l v e extremely s l o w l y e i t h e r i n v i t r o o r i n v i v o . Higuchi has p r e s e n t e d evidence o f an i n t e r f a c i a l b a r r i e r t o account f o r t h i s , and has found t h a t a number of q u a t e r n a r y ammonium s a l t s i n t e r f e r e with t h e b a r r i e r and g r e a t l y i n c r e a s e c h o l e s t e r o l d i s s o l u t i o n i n v i t r o . 85 Cholecystectomy is p r e s e n t l y t h e only accepted t r e a t m e n t f o r g a l l s t o n e s . P r o g r e s s toward a chemotherapeutic approach h a s been encouraging. Chenodeoxycholic a c i d (CDC) h a s been shown i n a number of s t u d i e s t o be e f f e c t i v e i n d i s s o l v i n g c h o l e s t e r o l g a l l s tones.86 A d m i n i s t r a t i o n of CDC caused t h e disappearance of s t o n e s o r r e d u c t i o n i n s i z e i n a number of pat i e n t s a f t e r s i x months of t r e a t m e n t . I n one s t u d y , CDC depressed plasma t r i g l y c e r i d e , b u t had no e f f e c t on p l a s m c h o l e s t e r o l l e v e 1 ~ . ~ 7Controv e r s y e x i s t s concerning t h e mechanism whereby CDC d i s s o l v e s g a l l s t o n e s . Expansion of t h e b i l e a c i d p o o l as t h e b a s i s of g a l l s t o n e d i s s o l u t i o n h a s been c h a l l e n g e d . 8 6 ~ ~ ~ CDC , ~ 9 h a s been r e p o r t e d t o d e c r e a s e t h e r e l a t i v e s e c r e t i o n of c h o l e s t e r o l i n t o bile.88990 I t has a l s o been shown t o i n h i b i t h e p a t i c B-hydroxy-6-methylglutaryl CoA r e d u c t a s e , t h e r a t e - l i m i t i n g enzyme of c h o l e s t e r o l s y n t h e s i s . 9 1 , 9 2 I t is tempting t o s u g g e s t t h a t t h e reduced c h o l e s t e r o l s e c r e t i o n observed d u r i n g CDC t h e r a p y i s r e l a t e d t o reduced h e p a t i c s y n t h e s i s . E f f i c a c y s t u d i e s w i t h c h o l i c a c i d produced no change i n g a l l s t o n e s i z e , although sodium c h o l a t e h a s been r e p o r t e d t o d i s s o l v e r e t a i n e d s t o n e s i n t h e common b i l e duct.93 Although CDC i s e f f e c t i v e and w e l l t o l e r a t e d i n manY94 s t u d i e s i n Rhesus monkeys have demonstrated h e p a t i c c h a n g e s . 9 5 ~ 9 6 No comparable l e s i o n s have been observed i n man.97 Another e x p e r i m e n t a l approach t o g a l l s t o n e t h e r a p y i s t h e u s e of h e n o b a r b i t a l . I n Rhesus monkeys i t i n c r e a s e s b i l e s a l t and p h o s p h o l i p i d E e c r e t i o n i n t o b i l e w i t h o u t s i g n i f i c a n t l y changing c h o l e s t e r o l s e c r e t i o n . 9 8 Furthermore, i t has been r e p o r t e d t o d e c r e a s e c h o l e s t e r o l s a t u r a t i o n i n h e p a t i c b i l e i n man.99 P h e n o b a r b i t a l ' s a b i l i t y t o d e c r e a s e t h e r e l a t i v e c h o l e s t e r o l c o n t e n t i n b i l e may allow d i s s o l u t i o n of c h o l e s t e r o l g a l l s t o n e s a f t e r long-term t h e r a p y . N e i t h e r l e c i t h i n n o r cholestyramine are e f f e c t i v e i n human g a l l s tone d i s e a s e . 100
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Chapter 1 9 .
Diabetes Mellitus
A l b e r t Y. Chang, The Upjohn Co., Kalamazoo, M I . This review a t t e m p t s t o survey key developments i n t h e f i e l d of diabetes m e l l i t u s s i n c e 1971, when t h e l a s t review on t h e s u b j e c t appeared i n t h i s series. The concept t h a t d i a b e t e s m e l l i t u s i s a d i s e a s e of d i v e r s e n a t u r e has been a f f i r m e d by f u r t h e r u n d e r s t a n d i n g of i t s e t i o l o g y and a s s o c i a t e d hormonal and m e t a b o l i c d e f e c t s 1 S 2 . Although h e r e d i t y i s known t o p l a y t h e major r o l e i n t h e p a t h o g e n e s i s of d i a b e t e s 3 , o t h e r p o s s i b l e causat i v e a g e n t s such as v i r u s e s 4 have a l s o been i m p l i c a t e d . While i n s u l i n def i c i e n c y remains as t h e primary hormonal imbalance i n d i a b e t i c p a t i e n t s , s e v e r a l o t h e r e n d o c r i n e f a c t o r s a l s o c o n t r i b u t e t o d i a b e t i c abnormalitie^^-^. An i n c r e a s i n g number of m e t a b o l i c derangements, as i n t h e syntheses of s o r b i t o 1 9 , g l y c o p r o t e i n s l O , c h o l e s t e r o l l l , p r o t e i n s 1 2 a n d r i b o n u c l e i c a c i d s 1 3 , have been r e p o r t e d i n t h e s t a t e of d i a b e t e s . Insulin P r o i n s u l i n b i o s y n t h e s i s . S i n c e t h e d i s c o v e r y of p r o i n s u l i n by S t e i n e r and Oyer i n human i s l e t adenoma14, t h e e x i s t e n c e of a p r e c u r s o r i n i n s u l i n b i o s y n t h e s i s h a s been demonstrated i n i s o l a t e d i s l e t s from many s p e c i e s , i n c l u d i n g c o d l 5 , Chinese hamster16, rat17, a n g l e r f i s h 1 8 , b u l l h e a d 1 9 , and i n bovine f e t a l p a n c r e a t i c s l i c e s 2 0 . C e l l - f r e e s y n t h e s i s of i n s u l i n w a s shown f o r t h e f i r s t t i m e i n membrane-bound polysomes from r a t i s l e t s 2 1 . Polypept i d e s produced i n t h i s system were i d e n t i f i e d as i n s u l i n by immunological a s s a y 2 2 and g e l f i l t r a t i o n chromatography2 l. Using t h i s c e l l - f r e e preparat i o n and a r a p i d and s p e c i f i c immunoprecipitation a s s a y t o monitor amino a c i d i n c o r p o r a t i o n i n t o p r o i n s u l i n and i n s u l i n , Permutt and K i p n i s concluded t h a t g l u c o s e exerts b o t h t r a n s c r i p t i o n a l and p o s t - t r a n s c r i p t i o n a l e f f e c t s i n s t i m u l a t i n g i n s u l i n b i o ~ y n t h e s i s ~ ~S p . e c i f i c a l l y , g l u c o s e promotes t h e o v e r a l l rate of i s l e t RNA s y n t h e s i s and a l t e r s t h e n a t u r e of t h e n a s c e n t RNA s p e c i e s toward i n c r e a s e d guanosine cytosine content, greater ease o f h y b r i d i z a t i o n w i t h DNA, and h i g h e r p r o p o r t i o n s of l a r g e molecular Glucose s u p p l i e s n o t o n l y energy and s u b s t r a t e s f o r t h i s weight RNA24. p r o c e s s , b u t a l s o some " t r i g g e r i n g " f a c t o r ( s ) , presumably produced d u r i n g i t s metabolism2 5.
+
Conversion of p r o i n s u l i n t o i n s u l i n . A f t e r p r o i n s u l i n i s formed i n i s l e t endoplasmic r e t i c u l u m , i t i s q u i c k l y s t o r e d i n t h e s e c r e t o r y g r a n u l e s , where t h e c l e a v a g e of p r o i n s u l i n t o i n s u l i n o c c u r s 2 6 . The c o n v e r s i o n procThe ess seems t o be t h e r a t e - l i m i t i n g s t e p of i n s u l i n b i o ~ y n t h e s i s ~ ~ . conv e r t i n g enzyme a p p e a r s t o b e membrane-bound26 and a p r o t e i n p o s s e s s i n enzymatic a c t i v i t y h a s been p u r i f i e d from a b o v i n e p a n c r e a t i c e x t r a c t However, i t remains t o b e e s t a b l i s h e d whether t h i s p r o t e i n indeed p l a y s such a r o l e i n v i v o i n t h e i s l e t B-cells. Using a d i f f e r e n t approach, S t e i n e r and h i s co-workers demonstrated t h a t t h e conversion can b e achieved -i n v i t r o w i t h a combination of c a r b o x y p e p t i d a s e B and t r y p s i n under condit i o n s where t h e t r y p t i c a c t i v i t y i s r a t e - l i m i t i ~ ~ g ~Furthermore, ~. they have p a r t i a l l y c h a r a c t e r i z e d t h e c o n v e r t i n g p r o t e a s e ( s ) i n a s u b c e l l u l a r f r a c t i o n o f r a t i s l e t s e c r e t i o n g r a n u l e and t h e i r r e s u l t s were i n accord
hSUCh .
Chap. 19
Diabetes Mellitus
Chang
la3
with such a scheme of c o n v e r ~ i o n ~ ~In . angler fish islets, the emergence of insulin also appears to proceed via two sequential steps18. However, the precise nature of the conversion process remains to be clarified. Intermediates and C-peptides. Proinsulin is converted via intermediates g (residues LyS59-Arg60 missing) and )- (Arg31-Arg missing) to form insulin, lys, 3 arg and a connecting-peptide (C-peptidelj?. The biological activities of the intermediates, measured in terms of glucose incorporation into C02 and lipids and the antilipolytic activity in the adipose tissue, were higher than that of proinsulin but lower than that of insulin32. C-Peptide showed neither insulin-like activity nor antagonistic or potentiating effects on insulin action in fat tissues32. C-Peptides have been isolated from the pancreatic extracts and found to be present with insulin in a one to one ratio. The primary structures of C-peptides have been determined in a number o f species31, 33. There is considerable interspecies variability, yet several features of their primary structures appear to have been conserved31. Since C-peptide does not cross-react with its corresponding insulin antibodies, it serves as a convenient handle to measure 6-cell secretory function, especially in diabetic patients with circulating antibodies in response to insulin therapy34. This technique has been applied clinically; serum C-peptide immunoreactivity (CPI) was unmeasurable in juvenileonset diabetic patients and it correlated well with immunoreactive insulin levels35. In all patients, serum CPI increased during remission and decreased during clinical relapse34. Secretion. The process of insulin secretion involves the movement of the secretory granules, in which the conversion of proinsulin to insulin occurs26, to the periphery of the cell through a microtubular-microfilamentous system36. The participation of such a system has been experimentally confirmed by studying the influence of a series of drugs, known to interfere with the function and structure of microtubules and microfilaments, of the microtubular system has also upon the P - c e l l ~ ~ ~ An - ~abnormality ~. been suggested as a cause of the diabetic syndrome found in the spiny mouse 0 A large variety of compounds serve as secretagogues for insulin and these include sugars, amino acids, hormones, cations, metabolic modifiers and hypoglycemic The regulation of this complex process is multifactorial and it plays an immensely important role in maintaining normoglycemia in animalslt0. Although the precise nature of the control mechanism is still far from being understood, two aspects of insulin release have been extensively studied. The first of these is how glucose, the primary secretagogue, stimulates insulin release. A number of investigators championed the theory that metabolism of glucose via pentose phosphate shunt45,49 or g l y c o l y ~ i s ~may ~ , be coupled to excitation of the secretory process. Matschinsky and his co-workers, on the other hand, favored the hypothesis that glucose reacts directly with a glucoreceptor molecule l o cated in the @-cell membrane that excites the secretory process51. They have also showed that the fuel and insulin-releasing functions of glucose in the islets can be dissociated by means of specific blockers52. The presence of a possible glucoreceptor has been demonstrated in the extracts of dog pancreatic islet membranes53. The second well-studied aspect of in-
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s u l i n s e c r e t i o n i s t h e requirement f o r Ca* and CAMP. Malaisse and h i s coworkers have demonstrated a n i n v a r i a b l e r e l a t i o n s h i p between t h e n e t uptake and t h e subsequent release of i n s u l i n i n t h e i s o l a t e d i s l e t s 5 4 . of Ca* There i s a l s o a c o r r e l a t i o n between t h e r a t e of i n s u l i n s e c r e t i o n and t h e l e v e l of CAMP i n t h e i s l e t s 5 5 , 5 6 , a n d CAMP may e x e r t i t s e f f e c t through t h e a c t i o n of CAMP-dependent p r o t e i n k i n a ~ e ~ ~ . Receptors. The i n i t i a l o b s e r v a t i o n by C u a t r e ~ a s a st h~a~t a n i n s u l i n - a g a r o s e complex i n c a p a b l e of p e n e t r a t i n g t h e c e l l s showed b i o l o g i c a l a c t i v i t y h a s l e d t o an o u t b u r s t of r e p o r t s on t h e i s o l a t i o n o f i n s u l i n r e c e p t o r s from c e l l membranes of l i v e r 5 9 , 60,fat60,61,1ymphocytes62,63 and m u s c l e 6 4 . The r e c e p t o r s appear t o b e g l y c o p r o t e i n s c o n t a i n i n g s i a l i c a c i d , which i s i n t i m a t e l y i n v o l v e d i n p r o c e s s e s t r a n s m i t t i n g t h e e f f e c t s of i n s u l i n o n g l u c o s e t r a n s p o r t and l i p o l y s i s i n t h e a d i p o c y t e s 6 The g l y c o p r o t e i n n a t u r e of t h e s o l u b l e r e c e p t o r w a s f u r t h e r s u b s t a n t i a t e d by t h e ease w i t h which i t b i n d s l e c t i n s such as concanavalin A and wheat germ a g g l u t i n i n 6 6 . The s t u d i e s on t h e i n s u l i n r e c e p t o r of human lymphocyte^^^ l e d t o t h e development of a s e n s i t i v e r a d i o r e c e p t o r assay6’ which complements t h e radioimmunoassay i n measuring minute q u a n t i t i e s of s u b s t a n c e s i n plasma w i t h i n s u l i n b i o a c t i v i t y . Using t h i s t e c h n i q u e c l i n i c a l l y , a p a t i e n t w i t h a primary d e f e c t i n i n s u l i n r e c e p t o r d e f i c i e n c y w a s r e p o r t e d 6 8 . I n t h e same s t u d y , a c q u i r e d and r e v e r s i b l e r e c e p t o r d e f i c i e n c y w a s a l s o observed i n obese p a t i e n t s 6 8. I n t h e obese-hyperglycemic mutant mouse, i n s u l i n - r e s i s tance a p p e a r s t o c o r r e l a t e d i r e c t l y w i t h a n i r r e v e r s i b l e d e f i c i e n c y i n i n s u l i n r e c e p t o r a c t i v i t y on t h e a d i p ~ c y t eand ~ ~ hepatocyte70 plasma memb r a n e s . C u a t r e c a s a s and h i s co-workers, on t h e o t h e r hand, r e p o r t e d t h e same number of i n s u l i n - b i n d i n g s i t e s i n a d i p o c y t e s from obese and i n s u l i n r e s i s t a n t r a t s as were p r e s e n t i n t h o s e from normal animals7’. They proposed t h a t t h e m e t a b o l i c d e f e c t i n t h e s e animals r e s i d e s i n t h e c o u p l i n g o f t h e s i g n a l s of t h e i n s u l i n - r e c e p t o r complex t o g l u c o s e t r a n s p o r t .
’.
Mechanism of a c t i o n . I n a d d i t i o n t o t h e w e l l documented e f f e c t s of i n = & on t h e t r a n s p o r t of g l u c o s e , amino a c i d s and i o n s , and t h e metabolism of c a r b o h y d r a t e s and l i p i d s 7 2 , i t h a s a l s o been r e p o r t e d t h a t i n s u l i n r e g u l a t e s t h e ~ y n t h e s i s ’ and ~ a c t i v i t y 1 1 , 7 4 , 7 5 of a number of enzymes and stimul a t e s p r o t e i n 1 2 ’ 76 and n u c l e o l a r and e x t r a n u c l e o l a r RNA s y n t h e s e s 1 4 i n a v a r i e t y of t i s s u e s , It i s u n l i k e l y t h a t i n s u l i n exerts i t s e f f e c t s on such d i v e r s e a c t i v i t i e s by a common mechanism. There i s , however, some exp e r i m e n t a l evidence s u g g e s t i n g t h a t some of t h e e f f e c t s of i n s u l i n can b e a t t r i b u t e d t o changes i n c e l l u l a r c o n c e n t r a t i o n s of c y c l i c n u c l e o t i d e messe n g e r s such as d e c r e a s e s i n CAMP^^,'^ and i n c r e a s e s i n C G M P ~ ~ , Devradation. I n a series of s t u d i e s on i n s u l i n d e g r a d a t i o n 8 0 - 8 6 ’Varandani proposed t h a t i n s u l i n i s degraded i n a s e q u e n t i a l manner: a p r e l i m i n a r y s p l i t t i n g i n t o A and B c h a i n s by g l u t a t h i o n e - i n s u l i n transhydrogenase, which i s followed by p r o t e o l y s i s of s e p a r a t e d chains82,83. The transhydrogenase h a s been d e t e c t e d i n a number of t i s s u e s from v a r i o u s s p e c i e s 8 0 , 8 1 and i t i s l o c a t e d i n t h e microsomal membrane84,87. T h i s enzyme h a s been p u r i f i e d from r a t l i v e r 8 6 , 89. I t degrades i n s u l i n v i a sulfhydryl-disUlfi.de i n t e r c h a n g e between t h e d i s u l f i d e bonds of i n s u l i n and a t h i o l donor; a v a r i e t y of t h i o l p r o t e i n s , i n a d d i t i o n t o g l u t a t h i o n e , s e r v e as e f f e c t i v e
-et al.
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c o - s u b s t r a t e s f o r t h e p u r i f i e d enzyme86. T h i s enzyme a p p e a r s t o b e i d e n t i c a l t o t h e s u l f h y d r y l - d i s u l f i d e i n t e r c h a n g e enzyme8a. A p r o t e a s e which s p e c i f i c a l l y d e g r a d e s i n s u l i n i n t o small m o l e c u l e s h a s b e e n p u r i f i e d and c h a r a c t e r i z e d i n liver8’ and muscle9 3 l. T h i s enzyme i s s u l f h y d r y l - d e p e n d e n t q 0 and i t shows r e l a t i v e l y low a c t i v i t y toward p r o i n s u l i n and v a r i o u s D e g r a d a t i o n of 1 2 5 1 - i n s u l i n i n t h e medium by proinsulin intermediates’ i n c u b a t e d f a t c e l l s w a s a l s o r e p o r t e d q 2 , b u t t h e n a t u r e of t h i s d e g r a d a t i o n is not clear.
’
’.
Other Endocrine F a c t o r s Glucagon. An i m p l i c a t i o n of glucagon i n d i a b e t e s m e l l i t u s h a s l o n g been s u g g e s t e d by i t s opposing b i o l o g i c a l e f f e c t s t o t h o s e of i n s u l i n . R e c e n t l y , i t h a s been r e p o r t e d t h a t p a t i e n t s i n d i a b e t i c coma f r e q u e n t l y have v e r y h i g h c o n c e n t r a t i o n s of plasma glucagon5. S t u d i e s i n t h e i s o l a t e d p e r f u s e d -- a l s o showed a p a t t e r n o f nonp a n c r e a s of g e n e t i c a l l y d i a b e t i c mice (db/db) s u p p r e s s i b l e e x c e s s i v e glucagon release, i n s p i t e of t h e h i g h l e v e l s o f endogenous i n s u l i n ’ 3. Buchanan and McCarroll o b s e r v e d i n t h e i r i n v e s t i g a t i o n w i t h newly d i a g n o s e d and u n t r e a t e d human d i a b e t i c p a t i e n t s t h a t t h e s e p a t i e n t s responded t o a r g i n i n e i n f u s i o n i n glucagon s e c r e t i o n and concluded t h a t i n s u l i n d e f i c i e n c y i s t h e major c a u s e of abnormal glucagon s e c r e t i o n i n d i a b e t e s m e l l i t u s q 4 . Gerich e t a l . confirmed t h e s e r e s u l t s b u t f u r t h e r d e m o n s t r a t e d t h a t , d u r i n g i n s u l i n - i n d u c e d hypoglycemia, plasma glucagon d i d n o t r i s e i n j u v e n i l e - o n s e t d i a b e t i c p a t i e n t s whereas glucagon i n t h e cont r o l s r o s e s i g n i f i c a n t l y q 5 . Thus a n i n t r i n s i c a l p h a c e l l d y s f u n c t i o n may b e o p e r a t i v e i n j u v e n i l e - o n s e t d i a b e t e s m e l l i t u s ; a common d e f e c t i n t h e s e c r e t o r y a p p a r a t u s i n t h e a l p h a c e l l s c a n b e excluded s i n c e t h e d i a b e t i c s s t i l l r e t a i n glucagon r e s p o n s e t o a r g i n i n e i n f u s i o n . P o t e n t i a l l y u s e f u l a n t i d i a b e t i c t h e r a p y w i t h glucagon a n t i b o d i e s res u l t e d from t w o l i n e s o f e x p e r i m e n t a l e v i d e n c e . F i r s t , Epand and Douglas o b s e r v e d t h a t r a b b i t s immunized w i t h glucagon show h y p o i n s u l i n e m i a and y e t t h e y are normoglycemic and a b l e t o r a p i d l y u t i l i z e i n t r a v e n o u s l y i n j e c t e d g l u c o s e q 6 . These r e s u l t s d e m o n s t r a t e d t h a t normoglycemia c a n b e m a i n t a i n e d w i t h glucagon a n t i b o d i e s d e s p i t e h y p o i n s u l i n i s m . Secondly, S t a h l e t a l . r e p o r t e d t h e p r o d u c t i o n of glucagon a n t i b o d i e s i n human s u b j e c t s q r P i t u i t a r y hormones. I n c r e a s e d serum l e v e l s of growth hormone have been rep o r t e d i n j u v e n i l e d i a b e t i c p a t i e n t s q 8 and may b e a c a u s a t i v e f a c t o r i n d i a b e t i c a n g i o p a t h y g 9 . The d i a b e t o g e n i c e f f e c t s of growth hormone have been p o s t u l a t e d t o b e mediated by a n i n s u l i n - a n t a g o n i s t i c p e p t i d e , s o m a n t i n , which i s r e l e a s e d a t t h e c e l l membranes of growth-hormone s e n s i t i v e tiss u e s 7 . D i a b e t o g e n i c p e p t i d e ( s ) i s o l a t e d from u r i n e l o 0 and p i t u i t a r i e s 1 0 1 , 1 0 2 h a s a l s o been r e p o r t e d t o p o s s e s s i n s u l i n - a n t a g o n i s t i c e f f e c t s . However, t h e s i g n i f i c a n c e o f t h e s e f i n d i n g s t o t h e e t i o l o g y of d i a b e t e s remains t o b e demonstrated. Hypothalamic f a c t o r s . A growth hormone-release i n h i b i t o r ( s o m a t o s t a t i n ) h a s been i s o l a t e d from hypothalamic e x t r a c t s of sheep and i t s primary T h i s t e t r a d e c a p e p t i d e can s u p p r e s s e x e r c i s e s t r u c t u r e determinedlo i n d u c e d plasma growth hormone rise i n normal and d i a b e t i c s u b j e c t s q q . However, t h e t h e r a p e u t i c p o t e n t i a l of s o m a t o s t a t i n i n t r e a t i n g d i a b e t i c
’.
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a n g i o p a t h y i s c o m p l i c a t e d by t h e o b s e r v a t i o n t h a t i t a l s o i n h i b i t s i n s u l i n s e c r e t i o n by a c t i n g d i r e c t l y on t h e B-cellslo4. Diabetic Complications Glomerular basement membrane. D i a b e t i c r e n a l l e s i o n s i n c l u d e t h e t h i c k e n i n g of t h e p e r i p h e r a l g l o m e r u l a r - c a p i l l a r y basement membrane and a n i n c r e a s e i n t h e mesangium, t h e i n t e r s t i t i a l t i s s u e between g l o m e r u l a r c a p i l l a r i e s . S p i r o e t a l . r e p o r t e d a marked i n c r e a s e i n h y d r o x y l y s i n e c o n t e n t and i n t h e number of g l u c o s y l g a l a c t o s e d i s a c c h a r i d e u n i t s i n basement memb r a n e i s o l a t e d from d i a b e t i c human g l o m e r u l i o They a l s o a t t r i b u t e d t h e s e changes t o h i g h l e v e l s of g l u c o s y l t r a n s f e r a s e , t h e enzyme r e s p o n s i b l e f o r a t t a c h i n g g l u c o s e o n t o basement membrane g l y c o p r o t e i n s 6 . Kef a l i d e s , however, r e p o r t e d no s i g n i f i c a n t i n c r e a s e i n t h e amount o f h y d r o x y l y s i n e o r i n t h e h y d r o x y l y s i n e - l i n k e d d i s a c c h a r i d e i n d i a b e t i c human k i d n e y s l o 7 . The d i f f e r e n c e i n t h e r e s u l t s o b t a i n e d by t h e s e two groups of i n v e s t i g a t o r s may b e due t o t h e d e g r e e of c o n t a m i n a t i o n i n t h e basement membrane p r e p a r a t i o n s by t h e s u r r o u n d i n g e p i t h e l i a l o r e n d o t h e l i a l c e l l s ; t h e c a u s e f o r t h e t h i c k e n i n g o f g l o m e r u l a r basement membrane remains t o b e d e t e r m i n e d .
’.
S o r b i t o l metabolism. E x c e s s i v e s o r b i t o l f o r m a t i o n from g l u c o s e h a s been s u g g e s t e d as a c a u s e f o r d i a b e t i c c o m p l i c a t i o n s , e s p e c i a l l y n e u r o p a t h i e s O8 and c a t a r a c t s 9 . One a p p r o a c h t o t h e c o n t r o l of t h e s e d i a b e t i c complicat i o n s would b e t o l i m i t s o r b i t o l p r o d u c t i o n u s i n g i n h i b i t o r s o f a l d o s e red u c t a s e , t h e enzyme which c o n v e r t s g l u c o s e t o s o r b i t o l . A s t u d y i n s t r e p t o z o t o c i n - i n d u c e d d i a b e t i c rats w i t h AY-22,284 ( I ) , a n o r a l l y a c t i v e a l d o s e r e d u c t a s e i n h i b i t o r , h a s been r e p o r t e d t o b e s u c c e s s f u l 1 0 9 .
Oral Hypoglycemic Agent Sulfonylureas.
E x t e n s i v e c l i n i c a l s t u d i e s have been c a r r i e d o u t w i t h sev1 I ) l lo, u1 g l i p i z i d e (K-4024, 111) ’12-114, and g l i b o r n u r i d e (Ro 6-4563, IV) 11’, l 1 6 . A new h i g h l y e f f e c t i v e s u l f o n y l u r e a , g l i c l a z i d e (S-1702, V ) , showed some unique p h a r m a c o l o g i c a l p r o p e r t i e s i n a n i m a l s t u d i e s 1 1 7 , 18. I t i n h i b i t s p l a t e l e t a g g r e g a t i o n and i n d u c e s d i s a g g r e g a t i o n , I t a l s o shows f i b r i n o l y t i c p r o p e r t i e s and d i m i n i s h e s t h e p e r m e a b i l i t y of c a p i l l a r i e s . The a n t i d i a b e t i c a c t i o n of s u l f o n y l u r e a s i s s t i l l a t t r i b u t e d mainly t o t h e s t i m u l a t i n g e f f e c t on i n s u l i n release. However, several e x t r a p a n c r e a t i c a c t i v i t i s have b e e n d e m o n s t r a t e d w i t h t o l b u t a m i d e . I t w a s found t o a l t e r i n t r a c e l l u and t o i n h i b i t CAMP p h o s p h o d i e s t e r a s e 1 2 0 . l a r p a t t e r n of carbon The s i g n i f i c a n c e of t h e s e f i n d i n g s t o i t s t h e r a p e u t i c a c t i o n i n t h e t r e a t ment of m i l d a d u l t - o n s e t d i a b e t e s remains o b s c u r e .
e r a l h i g h l y p o t e n t s u l f o n y l u r e a s , i.e. G l y b u r i d e (HB-419, U-26452,
Biguanides. Mechanism of a n t i d i a b e t i c a c t i o n of b i g u a n i d e s h a s been a t t r i b u t e d t o t h e i n h i b i t i o n of g l u c o s e a b s o r p t i o n 1 2 1 , of endo enous t r i g l y c e r i d e p r o d u c t i o n 1 2 2 and of d i g e s t i v e a b s o r p t i o n of l i p i d s 1 2 5 A differ-
.
e n t i a l i n h i b i t i o n of phenformin on g l u c o n e o g e n e s i s i n normal and d i a b e t i c rat l i v e r s was a l s o r e p o r t e d and i t is p a r a l l e l e d by t h e s e l e c t i v e hypoglycemic a c t i o n of phenformin i n d i a b e t i c s u b j e c t s l Z 4 . I n a series of s t u d i e s , L o s e r t e t a l . c o n c l u s i v e l y d e m o n s t r a t e d t h e two d i s t i n c t hypoglycemic
.
Chap. 19
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Chang
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effects of b i g ~ a n i d e s l ~ ~At. high doses, biguanides decrease cellular ATPIAMP ratio by inhibiting respiration; as a result, anaerobic glycolysis increases and gluconeogenesis decreases. This explains the hypoglycemic action of biguanides in alloxan-diabetic animals. On the other hand, the therapeutic effect o f chronic administration of low doses of biguanides in maturity-onset diabetes is mediated mainly by potentiating insulin action on glucose uptake and oxidation in peripheral tissues. Others. An iminopyridine, MK-270 ( V I ) , has been reported to be orally active in diabetic subjects not well controlled with sulfonylureas126. 3-Methylisoxazole carboxylic acid (VII) showed a short-term hypoglycemic effect in maturity-onset diabetic patients by inhibiting lipolysis and reducing plasma free fatty acid levels127. Diisopropyl ammonium dichloracetate is effective in lowering blood sugar and clearing ketosis in insulin-deficient diabetic rats128. Several new classes of compounds were also reported to possess oral hypoglycemic activity in animals and these include camphidines ( V I I I ) 129, quarternary 4-(5-)azolylpyridazinium salts ( I X ) 1 3 0 , cycloalkyl lactaminides (RMI 11,894, X) 131, monoguanidines' 32, 2-piperazino-4- (3H) -quinazolinone monoacetate ( X I ) and diphenyleneiodonium ( X I I ) 3 4 . (+)-Decanoylcarnitine inhibits ketone body formation and the combined therapy of insulin and (+)-decanoylcarnitine may yield great benefits t o patients with diabetic ketoacidosis135.
'
0
R
(I) AY-22,284
(11) glyburide
(111) glipizide
HO
0
CH3e S-o 2 N H C o N H - .
CH3
( I V ) glibornuride
( V I ) MK-270
0
-
(V) gliclazide
( V I I ) 3-methylisoxazole
carboxylic acid
( I X ) l-methyl-4-(3-methyl-5-
isoxazoly1)pyridazinium iodide
( V I I I ) 3-(2-dipropargylamino-
ethy1)camphidine
(X) R M I 11,894
188 -
Sect. I V
-
Metabolic Diseases, Endocrine Function
( X I ) Z-piperazino-4(3H)-quinazol i n o n e monoacetate
Moreland Ed.
(XII) diphenyleneiodonium
UGDP s t u d i e s . The c o n t r o v e r s i a l r e p o r t s by t h e U n i v e r s i t y Group D i a b e t e s Program r e l a t i n g t h e t r e a t m e n t of d i a b e t e s w i t h hypoglycemic a g e n t s t o t h e i n c i d e n c e of v a s c u l a r complications’ 3 6 c o n t i n u e t o draw c r i t i c i s m s 1 3 7 9 38 and d e f e n s e s ’ 3 9 , 1 4 0 . A more c a u t i o u s assessment of t h e c a u s e - e f f e c t relat i o n s h i p h a s a l s o been o f f e r e d 9 42.
’’’
I n s u l i n and o r a l a g e n t s . A comprehensive review on t h e p h y s i o l o g i c a l , pharmacological and c l i n i c a l a s p e c t s of i n s u l i n and o r a l hypoglycemic a g e n t s h a s been p u b l i s h e d i n two p a r t s 1 4 3 . Spontaneous D i a b e t e s i n Laboratory Animals Study of human d i s e a s e s i n small l a b o r a t o r y animals w i t h similar p a t h o l o g i c a l f e a t u r e s o f f e r s many advantages, e s p e c i a l l y i n t h o s e d i s e a s e s which i n v o l v e b o t h g e n e t i c and environmental d e t e r m i n a n t s a s i n d i a b e t e s m e l l i t u s . These animals have r e l a t i v e l y s h o r t g e n e r a t i o n t i m e which f a c i l i t a t e s t h e s t u d y of t h e mode of g e n e t i c t r a n s m i s s i o n of t h e d i s e a s e . The i n t e r p l a y of g e n e t i c and environmental f a c t o r s i n p a t h o g e n e s i s may a l s o b e u n r a v e l e d by m a n i p u l a t i n g b o t h t h e g e n e t i c and p h y s i c a l backgrounds o f t h e animal populat i o n . The i n t e n s i v e s e a r c h f o r a n i d e a l animal model f o r human d i a b e t e s i n t h e p a s t few y e a r s h a s r e s u l t e d i n t h e d i s c o v e r y of spontaneous d i a b e t e s i n many s p e c i e s of animals. The s t r i k i n g resemblance of t h e d i a b e t i c f e a t u r e s i n some of t h e s e animals t o t h o s e i n humans g r e a t l y enhances t h e p r o s p e c t s f o r u n d e r s t a n d i n g t h e p a t h o g e n e s i s of d i a b e t e s and f o r d i s c o v e r i n g n o v e l approaches t o t r e a t t h e d i s e a s e . Mus musculus. S e v e r a l s t r a i n s of mice e x h i b i t symptoms s i m i l a r t o d i a b e t i c humans. Severe d i a b e t e s , c h a r a c t e r i z e d by marked hyperglycemia, hy erphaon t h e g i a , and i s l e t d e g e n e r a t i o n , w a s produced by mutant genes & and I n c o n t r a s t , mild d i a b e t e s w i t h t r a n s i t o r y C57BLfKsJ b a c k g r o u n d l “ ~ 145. hyperglycemia and i s l e t hypertrophy was found i n animals produced by mutant l ~ ~ ~The markedly d i f f e r e n t genes db2J and ob on t h e C57BLI6.J b a c k g r o ~ n d 146. s y n d r o m e s r e s u l t i n g from t h e same gene, &, i n two c l o s e l y r e l a t e d i n b r e d s t r a i n s p o i n t t o t h e importance of g e n e t i c background on t h e e x p r e s s i o n of t h e mutant gene. Other mutant s t r a i n s showing d i a b e t i c symptoms i n c l u d e t h e New Zealand Obese (NZO) 1 4 7 9 1 4 9 y t h e yellow K K l s 0 and t h e K K 1 5 1 .
&
C r i c e t u l u s g r i s e u s . C o n s i d e r a b l e d a t a have been accumulated i n t h e epidemi o l o g y , g e n e t i c s , physiology, morphology and b i o c h e m i s t r y of t h e d i a b e t i c Both j u v e n i l e - and m a t u r i t y - o n s e t syndromes i n t h e Chinese hamsters 152. t y p e s have been observed. The complexity i n t h e mode of g e n e t i c transmiss i o n of t h e d i s e a s e and i n t h e d i a b e t i c syndromes i n t h i s animal c l o s e l y
Chap. 1 9
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resembles t h a t i n humans. O t h e r s . A colony o f Acomys c a h i r i n u s ( s p i n y mouse) showed g l u c o s e i n t o l e r ance, o b e s i t y and d e f e c t i v e i n s u l i n s e c r e t i o n 153. L a t e n t d i a b e t e s h a s been r e p o r t e d i n Psammomys obesus (sand r a t ) which developed hyperglycemia and o b e s i t y on c a l o r i c l o a d i n g 1 s;4. Spontaneous d i a b e t e s a l s o arises i n o t h e r animals such as Macaca n i ra ( b l a c k Celebes a p e s ) 1 5 5 , Mystromys a l b i c a u d a t u s (South A f r i c a n Hams*56 , Meriones u n g u i c u l a t u s (Mongolian gerbil)157, and Cavia p o r c e l l u s (guinea pig)158, REFERENCES
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- Metabolic Diseases, Endocrine Function
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41. D.J. Deery and K.W.
T a y l o r , Biochem. J . , lj4,557 (1973) P.E. Lacy, N . J . K l e i n and C . J . F i n k , E n d o c r i n o l . , 2, 1458 (1973) 43. P.J. L e f e b v r e and A.S. Luyckx, Biochem. Pharmacol., 2,1773 (1973) 44. J . M . M a r t i n , C.C. Mok, J . P e n f o l d , N . J . Howard and D. Crowne, J . E n d o c r i n o l . , 42.
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6 8 1 (1973) 352 (1973) 45. H.P.T. Ammon, T.N. P a t e 1 and J. S t e i n k e , Biochim. Biophys, Acta, 46. E. vanobberghen, G. Somers, G. D e v i s , G.D. Vaughan, F. Malaisse-Lagae, L. O r c i and W . J . M a l a l s s e , J . C l i n . I n v e s t . , 2, 1041 (1973) 47. J . S e h l i n , D i a b e t o l o g i a , 2. 89 (1973) 4 b . B. Hellman, A. Lernmftrk, J . S e h l i n , M. S o d e r b e r g , I . B . T a l j e d a l , Arch. Biochem. 435 (1973) Biophys., 257 (1969) 49. K.W. T a y l o r , Biochem. J . , 50 R.H. Georg, K.E. Sussman, J. Wayne-Leitner and W.M. K i r s c h , E n d o c r i n o l . , 3, 169 (1971) 5 1 * F.M. Matchinsky, R . L a n d g r a t , J. E l l e r m a n and J. K o t l e r - B r a j t b u r g , D i a b e t e s , 1, SuPPl. 2 , 555 (1972) 193 (1973) 52 * F.M. Matchinsky and J. E l l e r m a n , Biochem. Biophys. Res. Comm., 2, 459 (1973) 53 * S. P r i c e , Biochim. Biophys. A c t a , 54. W . J . Malaisse, I s r a e l J. Med. S c i . , g, 244 (1972) 343 (1973) 55 * S.L. Howell, I . C . Green and W. Montague, Biochem. J . , &l 56. I . C . Green, S.L. Howell, W. Montague and K.W. T a y l o r , Biochem. J., 174,481 (1973) 3 2 1 (1973) 57. W. Montague and S.L. Howell, Biochem. J . , 450 (1969) 58 P. C u a t r e c a s a s , Proc. Nat. Acad. S c i . , U.S.A., 1277 (1972) 59. P. C u a t r e c a s a s , Proc. Nat. Acad. S c i . , U.S.A., 60. P. C u a t r e c a s a s , Fed. P r o c . , 32, 1838 (1973) 61. P. C u a t r e c a s a s , Proc. Nat. Acad. S c i . , U.S.A., &, 318 (1972) 62. J . R . Gavin, 111, D.L. Mann, D.N. B u e l l and J . Rosh, Biochem. Biophys. Res. Comm., 2, 870 (1972) 63. J.R. Gavin, 111, P. Gorden, J. R o t h , J . A . Archer and D.N. B u e l l , J. B i o l . Chem., 248, 2202 (1973) 64. R. R e n n e r , FEBS L e t t e r s , 2, 8 7 (1973) 65. P. C u a t r e c a s a s and G. I l l i a n o , J . B i o l . Chem., 246, ‘ 9 3 8 (1971) 4E5 (1973) 66. P. C u a t r e c a s a s and G.P.E. T e l l , Proc. Nat. Acad. S c i . , U.S.A., 61. J . R . Gavin, 111, C.R. Kahn, P. Gorden, J . Roth, D.M. N e v i l l e , J r . , and R.E. Humbel, Diabetes, 22, S u p p l . 1, 306 (1973) 68. J . A . A r c h e r , J. C l i n . I n v e s t . , zl No. 6, 4a (1973) 69 P. F r e y c h e t , M.H. Laudat. P. Laudat, G. R o s s e l i n , C . R . Kahn, P. Gorden, and J . Roth, FEBS L e t t e r s , 339 (1972) 70 * C.R. Kahn, D.M. N e v i l l e , J r . , and J. Roth, J . B i o l . Chem., 24s. 241 (1973) 71* J . N . L i v i n g s t o n , P. C u a t r e c a s a s and D.H. Lockwood, S c i e n c e , 177, 626 (1972) 72. M.E. K r a h l , Diabetes, 2l, S u p p l . 2 , 695 (1972) 73. H.D. S o e l i n g and K.O. Unger, Europ. J. C l i n . I n v e s t . , 2, 199 (1972) 74. V. S i c a and P. C u a t r e c a s a s , Biochem., l2, 2282 (1973) 75. R.S. B e r s t e i n and D.M. K i p n i s , D i a b e t e s , 22, 923 (1973) 7 6 , E.B. Chain, P.M. Sender and P . J . G a r l i c k , Biochem. J . , C e l l . A s p e c t s , 132, 593 (1973 77. T.R. S o d e r l i n g . J . D . Corbin and C.R. P a r k , J. B i o l . Chem., 24-8, 1822 (1973) 266 (1972) 78. K.D. Hepp, Europ. J . Biochem., 1, 79 * G. I l l i a n o , G. P.E. T e l l , M . I . S i e g e 1 and P. C u a t r e c a s a s , Proc. Nat. Acad. S c i . , U.S.A., 2443 (1973) 80. P.T. V a r a n d a n i , Biochim. Biophys. A c t a , 286, 126 (1972) 81. M.L. Chandler and P.T. V a r a n d a n i , Biochim. Biophys. A c t a , 286, 136 (1972) 82. P.T. V a r a n d a n l , L.A. S h r o y e r and M.A. Nafz, Proc. Nat. Acad. Sol., U.S.A., 3, 1 6 8 1 (1972) 83. P.T. Varandani. Blochim, Blophys. Acta, ?.9+, 630 (1973) 84. P.T. Varandanl. Blochim. Blophys. Acta, 3 0 y 642 (1973) 85. P.T. V a r a n d a n i , Biochim. Biophys. Acta, E , 249 (1973) 86. M.L. Chandler and P.T. V a r a n d a n i , Blochim. B i o p h p . Acta, 258 (1973)
z,
m,
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2,
m,
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a, a,
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a,
z,
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Chang
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87. S . Ansorge, P. Bohley, H. Kirschke, J. Langner, B. Wiederanders and H. Hanson, Eur. J. Biochem., 2, 27 (1973)
88.
S . Ansorge, P. Bohley, H. Kirschke, J. Langner, J. Marquardt, B. Wiederanders and H. Hanson, FEBS Letters, 238 (1973) 89. G.A. Burghen, A.E. Kitabchi and J.S. Brush, Endocrinol., 633 (1972) 90. W.C. Duckworth, M.A. Heinemann and A.E. Kitabchi, Proc. Nat. Acad. Sci., U . S . A . , 6% 3698 (1972) 91. F.K. Baskin and A.E. Kitabchi. Eur. J. Biochem., 489 (1973) 92. S. Gammeltoft and J. Gliemann, Biochim. Biophys. Acta, E , 16 (1973) 93. H. Laube, R.D. Fussganger, V. Maier and E.F. Pfeiffer, Diabetologia, 2, 400 (1973) 94. K.D. Buchanan and A.M. McCarroll, Europ. J. Clin. Invest., 3, 218 (1973) 95. J.E. Gerich, M. Langlois, C. Noacco, J.H. Karam and P.H. FoTsham, Science, ,&I 171 (1973) 96. R.M. Epand and R.J. Douglas, Biochim. Biophys. Acta. 741 (1973) 97. M. Stahl, P.W. Nars, G. Herz, J.B. Baumann and J. Girard, Horm. Metab. Res., 5, 224 (1972) 98. A.P. Hansen, J. Clin. Endocrinol. Metab., 6, 638 (1973) 99. A.P. Hansen, H. Orskov, K. Seyer-hansen and K. Lundbaek, Brit. Med. J., 3, 523 (1973) 100. M. Okuyama and D.R. Challoner, Clin. Res., 0 , 754 (1972) 101. L.H. Louis, J.W. Conn and M.M. Appelt, Metabolism, 20, 326 (1971) 102. T.B. Miller and J. Larner, Proc. Nat. Acad. Sci., U.S.A., @, 2774 (1972) 103. R. Burgus. N. Ling, M. Butcher and R. Guillemin, Proc. Nat. Acad. Sci., U.S.A., 684 (1973) 104. K.G.M.M. Alberti, N.J. Christensen, S.E. Christensen, A.P. Hansen, J. Iversen, K. Lundbaek, K. Seyer-hansen and H. Orskov, The Lancet, No. 7841, 1299 (1973) 105. P.J. Beisswenger and R.G. Spiro, Science, 168,596 (1970) 106. R.G. Spiro and M.J. Spiro, Diabetes, 0, 641 (1971) 403 (1974) 107. N.A. Kefalides, J. Clin. Invest., 108. E. Pitkaenan and C. Servo, Clin. Chim. Acta, 44, 437 (1973) 109. D. Dvornik, N. Simard-Duquesne, M. Krami, K. Sestanj, K.H. Gabbay, J.H. Kinoshita, S.D. Vaima and L.O. Merola, Science, 182, 1146 (1973) 110. J.M. Feldman and H.E. Lebovitz, Diabetes, 20, 745 (1971) 111. E.F. Pfeiff‘er and S. Raptis, Diabetologia, 8, 41 (1972) 112. S. Lentini, A. Bossini and L.C. Pirola, Arzneimittel-Forsch., 2.1169 (1972) 113. A. Emanueli, E. Molari, L.C. Pirola and G. Caputo, Arzneimittel-Forsch., g,1881 (1972) 114. Investigations with GlibenesD. Results of European Pre-clinical and Clinical Studies. Diabetologia, 2, 309-364 (1973) 115. W. Ewald, Med. Klin., 68, 376 (1973) 116. G. Valenti, Clin. Ter., 23 (1973) 117. J. Duhault, M. Boulanger, F. Tisseraud and L. Beregi, Arzneimittel-Forsch., 22, 1682 (1972) 118. P. Desnoyers. J. Labaume, M. Anstett. M. Herrera, J. Pesquet and J. Sebastien, Arzneim. -Forsch. 2,1686 (1972) 119. T. Liang, G.L. Rangi and J.J. Blum. J. Biol. Chem., 248, 8073 (1973) 120. G. Brookers and M. Fichman, Biochem. Biophys. Res. Comm., 9, 824 (1971) 121. C. Arvanitakis, V. Lorenzsonn and W. Olsen. Gastroenterology, &, 837 (1972) 122. R.W. Stout. J.D. Brunzell, E.L. Bierman and D. Porte, Jr., Eurcp. J. Clin. Invest., 3, 271 (1973) 123. E. Agid and G. Marquie, Compt. Rend., Ser. D, 1787 (1972) 124. J.J. Connon, Diabetologia, 9, 47 (1973) 125. W. Losert, E. Schillinger, W. Kraaz, 0. Loge and P. Jahn, Arzneim.-Forsch., 2, 1157. 1413, 15@, 1752 (1972) 126. J.R. Ryan, F.G. Perez, A. Jain, G. Maha and F.G. McMahon, Clin. Pharmacol. Ther., 131 1 5 1 (1972) 127. Geyer and B. SokopL. Acta Endocrinol., 2,Suppl. 173, 127 (1973) 128. H.L. Eichner, P.W. Stacpoole and P.H. Forsham, Diabetes, 21, Suppl. 1, 358 (1972)
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1 2 9 . P. Schenone, A. Tasca and G. B i g n a r d i , I1 Farmaco, 2J. 194 (1971) 130. G.E. Wiegand, V . J . Bauer, S.R. S a f i r , D.A. Blickens and S . J . Riggi. J . Med. Chem., 3,1326 (1972) 131. J . M . G r i s a r . G . P . Claxton, A.A. Carr and N . L . Wiech, J . Med. Chem.. 679 (1973 132. K . G . M . M . A l b e r t i , H.F. Woods and M.D. Whalley, Europ. J . C l i n . I n v e s t . , 3, 208 (1973) 133. S.K. Mukherjee and S.T. Husain, Biochem. Pharmacol., 22, 2205 (1973) 6056 134. P.C. Holland, M.G. C l a r k , D.P. Bloxham and H.A. Lardy, J . B i o l . Chem., (1973) 135. J . D . McGarry and D.W. F o s t e r , J . C l i n . I n v e s t . , 2, 877 (1973) 136. M . G . Goldner, G . L . Knatterud and T . E . P r o u t , J . Am. Med. Assoc., 218, 1400 (1971) 137. H.S. S e l t z e r , D i a b e t e s , 21. 976 (1972) 138. J . M . Moss and D.E. DeLawter, J . Am. G e r i a t r i c s S O C . , 21, 7 2 (1973) 139. T . E . P r o u t , G . L . K n a t t e r u d , C.L. Meinert and C . R . K l i m t , D i a b e t e s , 21, 1035 ( 1 9 7 2 ) 140. R . H . Gray, Med. J . A u s t r a l i a , 1,594 (1973) 141. D . R . Hadden, D . A . D . Montgomery and J . A . Weaver, The Lancet, No. 7746, 335, ( 1 9 7 2 ) 142. D. Boyle. S.K. B h a t i a , D.R. Hadden. D . A . D . Montgomery and J . A . Weaver, The Lancet, No. 7746. 338 (1972) 143. H.D. B r e i d a h l , G.C. Ennis, F.I.R. Martin, J . R . S t a w e l l and P . T a f t , Drugs, 3 , 79, and 204 (1972) 144. D.L. Coleman and K.P. H u m e l , D i a b e t o l o g i a , 6, 263 (1970) 145. K . P . H u m e l , D.L. Coleman and P.W. Lane, Biochem. G e n e t i c s , 1, 1 (1972) 146. Symposium: Obese-hyperglycemic Mice, A b s t r a c t s . D i a b e t o l o g i a , 48-53 (1972) 147. R.G. L a r k i n s , D i a b e t e s , 22, 2 5 1 (1973) 148. C . J . Lovell-Smith and J.G.T. Sneyd, J . E n d o c r i n o l . , 1 (1973) 149. R . G . L a r k i n s , E n d o c r i n o l . , 2, 1052 (1973) 150. H. Iwatsuka, A. Shino and Z . Suzuoki, Endocrinol. Japon., 3, 23 (1970) 151. W.E. Dulin and B.M. Wyse. D i a b e t o l o g i a , 317 (1970) 1 5 2 . Second Brook Lodge Workshop on Spontaneous D i a b e t e s i n Laboratory Animals. Brook Lodge, D i a b e t o l o g i a , 6 , 154-206 (1970) 153. D . P . Cameron, W. S t a u f f a c h e r , L. O r c i , M. Amherdt and A.E. Renold, D i a b e t e s , 11, 1060 (1972) 154. G. F r e n k e l , P.F. K r a i c e r and J. Shani, D i a b e t o l o g i a , 313 (1972) 155. C.F. Howard, D i a b e t e s , 1 . 1077 (1972) 156. R . A . Stuhlman, J.T. Packer and R . E . Doyle, D i a b e t e s , 21, 715 (1972) 157. L. B o q u i s t , D i a b e t o l o g i a , 8, 274 (1972) 158. B.L. Munger and C.M. Lang, Laboratory I n v e s t . , 2,685 (1973)
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Chapter
X).
Non-steroidal Antiinflammatory Agents
Marvin E. Rosenthale, Wyeth Laboratories Inc., Radnor, Pa.
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Introduction Many new agents continue to appear, some in advanced clinical trials, but the most exciting developments are in the advancing understanding of the interrelationships and roles of immunological factors, prostaglandins and control of degradative enzyme release. Several excellent textsl-3 reviews and symposia4-8 have appeared which s u m r i z e these trends in inflammation and non-steroidal antiinflammatory agents ( N A A ) .
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Pathogenesis The continued inquiry into the nature of infections of bacterial and viral origin and their involvement in initiating degenerative diseases9 has been useful in identifying a number o f potentially answerable questions which encourage speculation and provide direction for future research. Speculation continues that slow viruses (myxovirus) may cause a variety of degenerative diseases including rheumatoid arthritis by inducing persistent new antigens. 10311 Additionally, there are studies which show that mice systemically infected with M co lasma pulmonis develop ce1lular hypersensitivity to synovia1 tis a-us t rabbits, given a single lntraarticular injection of herpes simplex virus, develop a chronic explosive arthritis.13 Yet, detailed studies have failed to demonstrate the presence of mycoplasmas, viruses or a transmissible agent in human rhematoid synovial tissue14 or in cell cultures derived from nodular tissue, cartilage or synovia of patients with arthritic diseases. 15, 16 Perpetuation of the inflammatory res onse by a vari ty of imunologic mediators such as rheumatoid factorj87 lymphokineslg and complementl9 remains of great potential importance and its relationship to other facets of inflammation continue to be elucidated. More recent findings have shown that cellular immune mechanisms,while active in the synovial lesion of rabbits with antigen-induced synovitis,a are not necessarily active in humans with rheumatoid arthritis as has been demonstrated by an anergy to delayed hypersensitivity skin tests in almost half of 84 rheumatoid arthritic patients tested with 5 different antigens. 21 Other studies have shown that a demonstrable inverse relationship exists between the percentage of blood T-cells and clinical disease in rheumatoid patients,22 that the majority of small lymphocytes which infiltrate the rheumatoid synovium are B-cells,‘3 and that microtubular subunits are essential for lymphocyte-mediated cytotoxicity thus adding another example of modulation of this phenomena by cyclic AMP.24 Finally, an interesting in-vitro model designed to mimic cartilage destruction in the rheumatoid joint has demonstrated that human leukocytes release neutral proteases which are capable o f degrading the noncollagenous protein mucopolysaccharide matrix of cartilage.25 Current knowledge and newer concepts of immunology26-28 as well as supportive evidence of a pathogenetic concept of rheumatoid arthritis29 lend direction and promise to future research efforts into areas
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concerned with antibody, delayed hypersensitivity, immune complex formation, complement activation, chemotaxis, leucocytic infiltration and release of tissue-destroying enzymes.
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Mechanism of Action Studies on the effects of existing drugs on deleterious intracellular enzymes indicate that prostaglandins, cyclic AMP, and catecholamines inhibit, but cyclic GMP and cholinergic agents enhance lysosomal enzyme release from rat tissues,30 or guinea pig leucocytes3l while colchicine and gold can inhibit degradative neutral proteases released from human leucocytes.25 Other biochemical effects of the NAA include: a lack of inducing effects on rat hepatic tryptophan pyrrolase, an enzyme thought to play a role in the antiinflammatory action of steroids;32 a profound effect on glycolysis in tissue cultur&3 and an ability to.restore the impaired serum SH-SS interchange reaction of adjuvant arthritic rats. 34 Involvement of the prostaglandins (PG) in the inflammatory process revolves around 3 major areas of activity. ( a ) An inflammatory effect: when PGE1 is injected intradermally in volunteers, a cutaneous inflammatory reaction characterized by a moderate mononuclear cell infiltrate is apparent.35 PGE also lowers the threshold of human skin to histamineevoked itching,g and potentiates the effects of bradykinin on vascular permeability in the rat skin.37 Similar effects on bradykinin or histamine are obtainable with PGE1, E2, A2 and F2a in guinea pig skin.% The involvement of PGs in periodontal and eye inflammations and their general role as mediators of inflammation was reviewed.39 (b) Antiinflammatory effects of the PGs: histopathologic studies of joints from rats with adjuvant arthritis show complete suppression of the disease when rats are treated prophylactically with PGE1. The established disease is also favorably affected.40 Further investigation of this activity shows that at least part of this effect is not mediated by the adrenal-pituitary axis, but may be due to inhibition of chemotaxis andfor inhibition of lysosomal enzyme and inflammatory mediator release.41. ( c) Inhibition of PG biosynthesis as a mechanism of the analgesic activity of NAA : intrasplenic injection of bradykinin in the dog increases the concentration of endogenously released PGs resulting in facilitation of hyperalgesia. Inhibition of this response by indomethacin is proposed as a mechanism of the analgesic activity of
k-
A review of the role of the PGs in inflammation and a discussion of the various sensitivities of different tissue synthetases to NAA, underscores the fact that only the most preliminary aspects of this fascinating subject have been uncovered.43 It has been speculated that the inhibition of PG synthetase of brain,but not o f several peripheral tissues, by acetominophen is responsible for its analgesic effect and lack of antiinflammatory activity,and serves to suggest new approaches in the search for more specific agents.44( See Chap. 17 for additional discussion).
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Test Systems The development of more valid systemically induced imunopathologic model systems of human disease suitable for detection of new therapeutic agents, remains of primary importance. Some inroads
Chap. 20
Non-steroidal Antiinflammatory Agents
Rosenthale
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were accomplished in investigating established models but no significant new system was evident. The essential role of certain complement components in the adjuvant-induced arthritic ( A I A ) rat was studied in cobra venom factor treated animals,45 as was impaired liver microsomal drug metabolism in the A I A rat and the pitfalls of new drug testing in animals with impaired liver function.46 Observations also indicate AIA rats to be more sensitive to the ulcerogenic potential of indomethacin during the later stages of the diseaseY47that polyarthritis can be induced in rats by the intra-lymph node injection of a variety of cell wall preparations from gram-positive bacteria,48 that a paradoxical increase in polyarthritis induced in rats by Mycoplasma arthritides occurs with salicylate treatmentY49 and that arthritis due to mycoplasma in rats and swine is not to be considered a specific model of the human c0ndition.5~ Oral feeding of &sulfanilamidoindazole and intravenous endotoxin induces both local and generalized Shwartzman reactions in rats,51 and may serve as a unique model for further exploration with drugs. Using experimental allergic encephalomyelitis (EAE) in guinea pigs as a model, it was shown that human serum contains a component capable of neutralizing the ability of the highly antigenic encephalitogenic protein which hypothetically might be released after brain inj~ry.5~A localized form of hyperacute EAE which was produced in rats within 1 day of passive transfer,53 might conceivably be adopted as a rapid screen.
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Locally induced models of rheumatoid joint disease received considera le attention this year. Chronic arthritic knee joints were produced5 and treated with antibiotics55 in rabbits and monkeys who received intra-articular injections of bedsonia isolated from a patient with Reiter's syndrome, in rabbits receiving cell free extracts of group A streptococci56 or the membrane disrupting antibiotic filivin, 57 in guinea pigs or rabbits administered immune complexes,58953 and in dogs given M. butyricum in Freund's adjuvant.60 Acute arthritis was induced in sensitized rabbits by the intra-articular injection of horseradish peroxidase,61 and in chickens with sodium urate or calcium pyrophosphate which were then treated with colchicine.62 Clearance of radio-labeled technetium was used as a non-invasive measure of joint inflammation in rats, rabbits and human~.~3
t
The effects of several agents on an experimental model of hyperuricosuria and gouty nephropathy in the rat were described.@+ The relationship between the ability of an agent to displace urate from protein -in vitro and uricosuric properties in vivo was proven by clinical testing.65 This represents a rare and extremely valuable validation of an experimental hypothesis and it is hoped that further such studies will appear in the future. Miscellaneous test systems of more than routine interest included a test for topical agents on external ocular inflammation in rats,66 a test for immunosuppressants in localized graft-versus host in rats,@ two in vitro systems for assaying granulocytic chemotaxis@ or
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macrophage phagocytosis and cytotoxicity69 using radiolabeling techniques, a short-term clinical screen utilizing patient preference,70 and two clinical assays using suppression of tetrahydrofurfuryl nicotinateinduced skin erythema,Tl or analgesia72 as the criterion €or antiinflammatory activity. Compounds Under Investipation
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Encouragement from recent disclosures concerning Immunos uDDr es s selective effects of both antigens and drugs on cell populations responsible for the production of circulating antibodies ( B cells) and those involved in cellular immunity (T cells)73r 74 have brightened the prospects for uncovering useful, specific immunoregulants. A number of immunosuppressants were tested in guinea pigs and mice for their differential effects on B and T lymphocyte functions75~76and the selective effect of cyclophosphamide was assessed in patients having a variety of immunopathic diseases,77 rheumatoid arthritis,@ or multiple sclerosis.79 Contradictory results in both species indicate that more studies, using similar procedures are required before definitive conclusions can be reached. It appears, however, we aresucceeding in more accurately defining the different effects of agents on basic immune mechanisms and further efforts to apply these findings clinically may provide the key to development of superior therapeutic agents.80
T e linical use of immunosuppressant drugs in nonmalignant diseaseB1-83 and their inherent dangers,84-86underscore the need for more specific agents. The therapeutic usefulness of cy lophosphamide azathioprine and 6-MP in systemic lupus, polymy0sitis,~7 rheumat0id~~-9~ and psoriatic arthritis,gl is apparent but is severely limited by side effects. Chemical, phamcological and pharmacokinetic properties of immunosuppressants to a great ext nt determine their relevance for future use in man.80,92,93 Oxisuran,g& the selective suppressant of cell mediated immunity,% was found to be rapidly absorbed, negligibly bound to plasma proteins and to havea long= half-life in man than the rat or dog. Interestingly, the differential immunosuppressive properties of oxisuran reside in the reduced metabolites, the alcohols, which are found in higher concentrations in man than the dog or rat.96 One of a series of arylhydroxytriazenes, PR-H-286 BS (1)was effective in both humoral and cell-mediated immune responses and in rat adjuvant-induced arthritis.97 The orally active antiviral agent effectively inhibited symptoms of adjuvant arthritis tilerone and allergic encephalomyelitis in rats but paradoxically, it potentiated the humoral immune response to the thymus depend nt and independent antigens as well as to homocytotropic antibody.9%
(z),
Chap. 20
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Arylalkanoic Acids and Related Compounds In a number of clinical studies the therapeutic ratios for newer agents were compared to the standard drugs. In a 16 week double-blind crossover study in 30 patients with rheumatoid arthritis (RA) aspirin was slightly more effective than fenoprofen but substantially more toxic.99 Using a new trial designed to minimize the unpredictable course of RA, naproxen (400-500g daily) was found effective without significant gastrointestinal side effects in 117 patients. loo Ketoprofen (Orudis,RP-19583, 100 mg daily) had significant activity with few side effects in 14 patients with RA and had no effect on the microsomal drug-metabolizing enzymes of the liver as shown by lack of effect on antipyrine clearance.101 In a double blind crossover study in 35 patients with RA, the analgesic and antiinflammatory activity of 150 mg daily of ketoprofen was superior to 1,200 mg of ibuprofen.lO* Aspirin antagonized the effects of various NAA in the arthritic rat,1°3 and lowered the plasma concentrations of fenoprofen and indomethacin in humans,104 and naproxen in the rat,l05 again emphasizing the need to assess the presence of salicylates in the clinical evaluation of new agents, particularly aryl acids.
X
=
C02H, C02Na
2
X
=
H, Na
An analog of tolmetin,106McN-;i981 (3)was found to have similar antiinflammatory properties to the parent drug. 1.07 The antiinflanunatoryl08 and analgesic109 properties of clonixin,l06 were demonstrated in rats and had sufficient acmonkeys. Despite initial clinical failure, 61137 tivity and safety in animals to warrant further testing.110 Small ring compounds synthesized in the hope of altering metabolism and thus biological profiles, resulted in development of several active agents ( 5 ) in the potency range of phenylbutazone. 111
(4)
R - CHCOOH
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K 4277 (5)112 and the naphthaleneacetic acid derivatives (1) ,113 were more potent than phenylbutazone when compared in several pharmaco,114 were less potent, logic assays. The benzothiazolacetic acids (8) while GP 45840 (Voltaren, 9),115 was as potent as indomethacin but much less acutely toxic in mice.
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Less toxicity and potency thag Heterocyclic Alkanoic Acid Derivatives the parent indomethacin was reported for the analogs cinmetacin (lo),l l t j and 11,117 in rodents.
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Heterocyclic Carboxamide Derivatives In the carrageenin rat paw assay carboxanilides of the oxindole ring (2) demonstrated antiinflammatory activity somewhat less potent than indomethacin, having long dog plasma half -lives. 118
R
1
:
lsopropyl
R2, 3, 5 Rn
i
H
Ctij
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Quinazolines SL-512 (3) caused minimal gastrointestinal toxicity, was equipotent to phenylbutazone in acute models of inflammation, and was less effective in chronic models such as the adjuvant rat.119 Comparisons of & with indomethacin and phenylbutazone (most potent analog is shown) were sufficiently favorable to warrent further testing of members of this series.1m
-
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Salicylates Gastrointestinal Side Effects Benorylate (6 g daily) was superior to aspirin (4 g daily) as an antiinflammatory a ent with less gastrointestinal side effects in 33 outpatients with RA.521 Previously
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described tolerance in dogs to the gastrointestinal side effects of aspirin was reinvestigated in 9 volunteers who showed no evidence of such tolerance during a 4 week test period. However, a daily dose of 4 or 5 g aspirin induced a uniform rate of gastrointestinal blood loss. 122 Using a labeled erythrocyte assay, fenoprofen and aspirin at equipotent doses caused measurable gastrointestinal bleeding; bleeding was significantly less with fenoprofen. 123 When the labeled erythrocyte method was modified for the rat, aspirin (321 mg/kg) and phenylbutazone (155 mg/kg) but not seclazone (W-2354,a, 321 mg/kg) were shown to increase gastrointestinal bleeding. 124 There was less gastrointestinal blood loss from sodium salicylate tablets than from comparable doses of aspirin over a 7 day period in 13 volunteers in a crossover study.125 The continued interest in the gastrointestinal side effects of NAA emphasizes the difficulty which exists in attempting to dissociate activity from toxicity. The continuing effort to quantitate this side-effect in man is a welcome addition to laboratory studies which thus far have not been as predictive as desired.
-
Miscellaneous Agents The potent antiinflammatory activity of U-245C8 16) correlated closely with plasma concentrations in arthritic rats. 1 2 ~ k a double-blind multicenter trial Abbott a59o,g4 ( 1200 mg/day was equivalent to aspirin (3900 mg/day) in the control o f RA in lo7 patients. are marked by lack of 127 The pharmacologic properties of seclazone ulcerogenic activity in rats and diuretic activity in rats and beagles.1B Seclazone is almost completely metabolized to 5-chlorosalicylic acid in the rat, dog and rhesus monkey.13 The oxazole, ditazol possesses potency similar to that of aspirin, has a low level of toxicity, an absence of gastrointestinal irritation in rats and dogs, and a long halflife in man.130
(a)
(x),
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.-
Disease activity was suppressed in an uncontrolled study in seven patients with RA treated with penicillamine; initial doses of 300 rng/day were increased to 1.8 g/day. Side-effects eventually demanded cessation of treatment.131 At doses up to 1.5 g/day, penicillamine resulted in significantly lowered serum IgG and IgM but not IgA levels at 3 and cj months in patients with RA, suggesting that at least part of its imunosuppressive effect is at the cellular level, and may be specific for cells which produce certain classes of immunoglobulins.132 Radiation synovectomy with intra-articular injections o f yttrium-90 in 44 rheumatoid knee joints benefited 50% o f the patients, however the long-term danger
200
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of this therapy is unknown. 133
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Natural Products and Related Substances A basic peptide fraction of bee venom exhibits powerful antiinflammatory effects in the carrageenin paw and adjuvant rat tests;1% whole bee venom is ineffective in adjuvant arthritis in adrenalectomized rats ;135 lymphocyte chalone concentrate from spleen exhibits immunosuppressive properties;136 vitamin A stimulates humoral and cell-mediated response137 and vitamin E exerts antiinflamnatory effects in rats and on human skin.138 Improved methods of purification, preparation and characterization of immunosuppressive material der ved from bovine serum,l39 antiinflammatory material from human plasma, lto or gram-negative bacteria ( Pseudomona aeruginosa),141 were described.
-
Gold In a multiclinic trial by the Cooperating Clinics Committee of the American Rheumatism Association, suggestive evidence indicated beneficial effects for RA patients treated with gold thiomalate but the overall therapeutic effects were disappointin erhaps because of the small number of patients who completed the study. K4E
-
Comment Some initial advances in 1973 towards a more scientific approach in the search for new antiinflammatory agents suggest an end of the !'me too" era. The recognition of, and specific testing for, compounds capable of individually affecting T and B cells, heralds the development of a long-sought-for class of selective, and thus safer "immunoregulants" which are effective against the basic aspects of inflammatory disease, i. e. the immunologic component. Similarly, the recently disclosed description of substances affecting specific inhibition of various prostaglandin synthetases should speed up the development of superior NAA at a rate more responsive to the needs of research and medicine. Initial clinical reports on several of the agents described in this and past reviews are encouraging and will continue to influence the direction of future endeavors. REFERENCES Harper and Row, Md., 1972. S. Sell, "Immunology Immunopathology and Immunity". Academic P r e s s . New York, 1972. F. H. Each, and R. A. Good, " C l i n i c a l Immunobiology". Williams and Wilkins Co., Baltimore, Md., 1973. 3. M. deV. Cotten, "Immunopharmaco1ogy". 4. H. E. P a u l u s and M. W. Wnitehouse, A n n . Rev. Pharmacol., 1 3 , 107 (1973). 5. J . S. Johnson, J. H. Vaughan, P. K. Henoh, and S. E. Blomgren, Ann. I n t . Med. 78, 937 (1973). 6. '?rimer on t h e Rheumatic Disease'' J.A.M.A. 224 (Supplement No. 5 ) , 661 (1973). 7. G . Nuki and R. Brooks, B u l l . Rheumat. Dis. 23, 726 (1973). 8. R. H. Kahn and W. E. M. Lands, ' F r o s t a g l a n d i n s and C y c l i c AMP". Academic P r e s s , New York,l973 9. E d i t o r i a l , B r i t , Med. J., Oct. 27, p. 186 (1973). 10. J . L. Marx, S c i e n c e , 180, 1 3 5 1 11. P. Barland, Amer. J. Mad., 54, 1 % 7 ? b 7 3 ) . 1 2 . H. J. Harwick, G . M. Kalmanson, M. A. Fox, and L. B. Guze, Proc. SOC. Exper. B i o l . Med., 144, 561 (1973). 13. F. W. Webb, R. Blueston, L . S. Goldberg, S. D. Douglas, and C. M. Pearson, Arth. Rheumat., 16, 241 (1973). 191 (1973). 14. B. C. Cole, J. R. Ward, and C. B. Smith, A r t h . Rheumat., 15. D. A. Pearson, J. T. Sharp, and W. E. Rawls, Arth. Rheumat., l6, 677 (1973). 16. R. M. Wilkes, J . P. Simsarian, H. E. Hopps, H. Roth, J. L. Decker, R. G. Aptekar, and H. M. Meyer, Jr., Arth. Rheumat., E : 4 4 6 (1973). 1 7 . D. E. S t a g e , and M. M a n n i k , B u l l . on t h e Rheumat. D i s . , 23, 720 (1973). 18. E d i t o r i a l , Lancet., June 30, p. 1490 (1973). 1.
2.
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Chap. 20 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. 41. 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.
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M. M. Mayer, S c i e n t i f i c American, 229, 54 (1973). P. S t a s t n y , T . D. Cooke, and M. Z i f f , C l i n . exp, Immunol., 14, 141 (1973). J. Waxman, M. D. Lockshin, J . J . Schnapp, and I. N. Doneson, Arth. Rheumat., 16, 499 (1973). R. C. Williams J r . , J. R. DeBoard, 0. J. Mellbye, R. P. Messner, and F. D. Lindstrbm, J . C l i n . I n v e s t . , 52, 283 (1973). I. Kobayashi, and M. Ziff, Arth. and Rheumat., 16, 471 (1973). T. B. Strom, M. G . Garovoy, C. B. Carpenter, and J. P. M e r r i l l , Science, 181, 1 7 1 (1973). A . Oronsky, L. I g n a r r o , and R. P e r p e r , J . Exper. Med., 138, 461 (1973). M. Z i f f , Fed. Proc., 3 2 , 131 (1973). P. M. C a r t e r , Ann. Rheum. D i s . , 32, 265 (1973). A . C. A l l i s o n , A n n . Rheum. Dis., 32, 283 (1973). N. J. Z v a i f l e r , Advances i n Immunology, 16, 265 (1973). L. J. I g n a r r o , A. L. Oronsky, and R. J. P e r p e r , L i f e Sci., 12, 193 (1973). L. J . I g n a r r o , and C . 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F e r r e i r a , and J. R. Vane, I n "Les P r o s t a g l a n d i n s " P. 345 Inserm Seminaire, P a r i s , 1973. R. J . Flower, Am. H e a r t J . , 86, 844 (1973). L. Kourounakis, R. A. Nelson, Jr. and M. A . Kupusta, Arth. and Rheumat., 16, 71 (1973). M. W. m i t e h o u s e and F. J . Beck, Drug Metab. and D i s p o s i t i o n 1, 251 ( 1 9 7 3 x G. DiPasquale, and P. Welaj, J . Pharm. Pharmacol., 25, 831 (1973). T. Koga, C. M. Pearson, T. N a r i t a , and S. K o t a n i , Proc. SOC. Exper. B i o l . and Med., 824 (1973). V. Eisen, and C. Loveday, B r i t . J . Pharmacol., 47, 2 7 2 (1973). L. S o k o l o f f , Amer. J. P a t h o l . , 73, 261 (1973). J. R. Ward, M. L. M i l l e r , and F. Burch, Arth. and Rheumat., l6, 491 (1973). B. G e r s t l , C. T. Uyeda, H. Hunt, and L. F. Eng, Immunol., 23, 395 (1972). S. Levine, and R. Sowinski, Amer. J. P a t h o l . , 73, 247 ( 1 9 7 q . D. E. Smith, P. G . James, J. Schachter, E. P. Engleman and K. F. Meyer, Arth. and Rheumat., 1 6 , 2 1 (1973). R. J . G i l b e r t , J. S c h a c h t e r , E. P. Engleman, and K . F. Meyer, Arth. and Rheumat., l6, 30 (1973) H. S t e i n , R. Yarom, S. Levin, T. Dishon, and 1. Ginsburg, Proc. SOC. Exper. B i o l . and Med., 143, 1106 (1973). C D . Muirden, and M. P h i l l i p s , A n n . Rwumat. Dis., 32, 251 (1973). B. Belovic and T . D. K i n s e l l a , Ann. Rheumat. D i s . , 167 (1973). J . R. H o l l i s t e r , G . C. Liang, and M. Mannik, Arth. and Rheumat., l6, 10 (1973). R. P . Carlson, G. E. Dagle, C. G. Van Arman, and P. J . K l i n g , Am. J. Vet. Res., 34, 515 (1973). R. C. Graham, Jr. and S. L. Shannon, Amer. J. P a t h o l . , 73, 147 (1973). G. L. Floersheim, K. Brune, and K. S e i l e r , Agents and Actions 3, 20 (1973). H. B e r r y , J . P. Browett, E. C. Huskisson, P. A. Bacon and D. A. Willoughby, A n n . Rheumat. D i s . , 3 2 , 95 (1973). J . R. Klinenberg, R. Bluestone, L . S c h l o s s t e i n , J . Waisman, and M. W. Whitehouse, A n n . I n t e r . Med., 7 8 , 99 (1973). L. H. S c h l o s s t e i n , I. Kippen, M. W. F&itehouse, R. Blueston, H. E. Paulus, and J. R. Klinenberg J . Lab. C l i n . Med., 82, 412 (1973). I. M a i s t r e l l o , G . Rigamonti, C. Frova, and P. de R u g g i e r i , J . Pharm. S c i . , 6 2 , 1455 (1973). K . F. Swingle, T. G . Grant, and P. M. Valle, Proc. S O C . Exper. B i o l . and Med., 142, 1329 (1973) J. I, G a l l i n , R. A. Clark, and H. R. Kimball, J . Immunol., 110, 233 (1973). P. Hersey, T r a n s p l a n t , , l5, 282 (1973). A . S . R i d o l f o , W. M. Mikulaschek, C. M. Gruber, J r . N. E. Scholz, Am. J. Med. S c i . , 265, 375 (1973). C. D. Brooks, C. A. S c h l a g e l , N. C. Sekhar, and J. T. Sobota, C u r r e n t Therap. Res., l5, 180 (1973). P. Lee, J. Webb, J. Anderson, and W. W. Buchanan, B r i t . Med. J., June 23, P. 685 (1973). N. Talal, Arth. and Rheumat., l6, 422 (1973). M. C. R a f f , Nature 19 (1973). G. D. Stockman, L. R. Heim, M. A. South, and J. J. T r e n t i n , J. Zmmunol., 2 ,277 (1973). A Winkelstein J . C l i n . I n v e s t 52 2293 (1973 I: R. Mackay, j . M. Dwyer, and I?K'Rowley, Arti. and Rheumat., 16, 455 (1973).
185,
143,
Lq
242,
.
202 I
Sect. IV
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Metabolic Diseases, Endocrine Function
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A. W i n k e l s t e i n , J. M. M i k u l l a , H. R. Nankin, B. H. Pollock, and B. L. S t o l z e r , J . Lab. C l i n . Med., 3,506 (1972). 79. N. I. Abdou, B. Zweiman, and S. R. C a s e l l a , Clin. Exp. Immunol., 13, 55 (1973). 80. G . W. Camiener, and W. J . Wechter, Prog. Drug. Res., If?, 67 ( 1 9 7 2 r 81. A. D. S t e i n b e r g , J . A l l e r g y C l i n . Immunol., 52, 242 (1973). 82. A. Winkelstein, D. Segel, B. L. S t o l z e r , M. Fogel, R. K. Shadduck and M. Lewis, Amer. J . Med. S c i e n c e , 265, 92 (1973). 83. J. L. Decker, J . R. B e r t i n o , E. R. Hurd, and A. D. S t e i n b e r g , Arth. and Rheumat., l6, 79(1973) 84. D. C. Dale, A. S. Fauci, and S. M. Wolff, Arth. and Rheumat., l6, 657 (1973). 85. B. H. P o l l o c k e t a l . , Arth. and Rheumat., l6, 524 (1973). 86. R. G . Aptekar J. P. Atkinson, J. L. Decker, S. M. Wolff, and E. W. Chu. Arth. and Rheumat., 1 6 , 461 (1971j. 87. J. F. F r i e s , G . C. Sharp, H. 0. McDevitt, and H. R. Holman. Arth. and Rheumat., l6, 154 (1973) J . T. S h a r p and S. B i l l i n g s Arth. and Rheumat 16, 148 1973 88. M D Lidsk ALth. and Rheumat. 89. M: B: Urowifzf D. A. Gordon, H. A. Smythe: W. P r u z a n s k i and I?KT Ogryz!o, 16, 411 (19733. 90. J. Levy E. V. B a r n e t t , N. S. MacDonald, J . R. K i i n e n b e r g , and C. M. Pearson. J . C l i n . I n v e s t , 51, 2234 (1972). 91. J . Baum E. Hurd D Lewis J. L. Ferguson and M. Z i f f Arth. and Rheumat., 2, 139 (1973). A. H. Ckhlmers, 1. i.Burggyne, and A. W. Murray, Drugsf 3, 2 2 7 (1972). 92. 93. E. M. Lanoe I n " C l i n i c a l Immunobiology." p. 193, Ed. F. H. Each and R. A. Good, Academic P r e s s , New York Reports i n Medicin 1 Chemistry, 1972" Vol. 8, p. 214, Ed. 94. M. E. R~senthale',(:~~~)L;lrmal R. V. Heinzelrnan Academic P r e s s New York (1973f. Freedman, I n f e c t . and Immunity 8, 549 (1973). 95. A. E. Fox, J. L.'Gingold and H.'H. 96. F. J . DiCarlo, M. D. Melgar, L. J. BayneS, and M. C. Crew, J . Reticuloend. 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Chem., 16-1237 121. P. N. Sperryn, E. D. Hamilton a n d . Parsons, A&. Rheumat. D i s . , 157 (1973). J R Leonards G Levy, and R. Njemczura. New Eng J Med 289 1020 (1973). A: S: R i d o l f o B t HI., C i i n Pharmaool. and Ther,ap.', 1 4 , 226 (Id). 124. J . Edel;on, and J. F. Douglas, 8. Pharmaool. Exper. TEerap. 184, 449 (1973). 1.25. J. R. Leonards, and G. Levy Clin. Pharmacol. and T h e r a 1 4 6 2 (1973). 126. D. G . K a i s e r and E. M. G l e d , J. Pharmaceut. S c i . 61 !;d8+972). 1 2 7 . J. R. Ward e t al. J. Clin. Pharmacol. 1 3 218 ( i 9 7 7 j . 128. F. M. B e r g e r e t a i . , Pharmacology, 9, i 6 a j l 9 7 3 ) . 129. J . Edelson, J. F. Douglas and B. J. Ludwig, J. Pharm. S c i . . 62, 229 (1973). 130; L. Caprino. F. B o r r e l l i and R. Falchetti, Arzneim. Forsch 1 2 2 (1973). 131. E. C. Huskisson and F. 6. H a r t . Ann. Rheumat. Dis 31, 3 2 1972 , 1 3 2 . R. Bluestone and L. S. Goldberg Ann. Rheumat. Dis:: 3 7 , 50 (1973)! 133. E d i t o r i a l , B r i t . Med. J . Aug. 247 (1973). 134. M. E. J . Billingham e t Nature, 245, 163 (1973). 135. R. B. Zurier, H. Mitnick, D. B l o c m g a z n and G. Weissmann Ann. Rheumat. D i s . , 32, 466 (1973). 136. J . C . Houck A M A t t a l a h , and J . R. L i l l y N t u r e 245, 148 (1973). 137. M. J u r i n and I: F: Tannock, I m u n o l . 23 283 r1972j.138. M. Kamimra, J . Vitaminol., 18, 204 139. A. B. Karpas, and D. Segre, FOG. SOC. Exper. Biol. and Med. 144 141 (1973). 140. A. W. Ford-Hutchinson e t a l . , J . Pharm. Pharmacol 25, 881 141. G . L . Floersheim e t a l . , Agents and A c t i o n s , 2, 2'3i v 9 7 2 ) . 142. Cooperating C l i n i c s Committee A.R.A. Arth. and Rheumat., l6, 353 (1973).
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Chapter 21. M.
Cyclic Nucleotides and Drug Discovery
S a m i r Amer and Gordon R. McKinney, Mead Johnson Research Center E v a n s v i l l e , Indiana
The explosive growth of c y c l i c n u c l e o t i d e research i s s i g n i f i c a n t l y advancing our understanding of a v a r i e t y of d i s e a s e s and of t h e mechanisms o f a c t i o n o f both new and o l d drugs. I n drug discovery, it i s opening up important new o p p o r t u n i t i e s f o r t h e design of new agents expected t o a c t via r e g u l a t i o n of c y c l i c n u c l e o t i d e s , p a r t i c u l a r l y a s evidence of t h e i r apparent involvement i n an ever-expanding number of d i s e a s e s t a t e s accumul a t e s and g r e a t e r understanding of t h e f a c t o r s r e s p o n s i b l e f o r c o n t r o l l i n g t h e i r i n t r a c e l l u l a r concentrations i s achieved. Primarily because of space l i m i t a t i o n s only r e p r e s e n t a t i v e examples o f research i n t h i s a r e a d i r e c t l y concerned with drug discovery w i l l be discussed i n t h i s a r t i c l e . References t o o t h e r p e r t i n e n t a r t i c l e s can be found i n t h e t h r e e a l r e a d y published volumes of Advances i n CycZic NucZeotide Research' and t h e proceedings of two symposia on c y c l i c nucleotides i n d i s e a s e 2, 3 . The r o l e t h a t c y c l i c n u c l e o t i d e s may p l a y i n t h e e t i o l o g y and/or maintenance of a number of d i s e a s e states i s becoming c l e a r e r . I n asthma, t h e a b i l i t y of p a t i e n t s with t h i s d i s e a s e t o e x c r e t e increased amounts of c y c l i c AMP i n u r i n e i n response t o epinephrine is g r e a t l y diminished, p o s s i b l y due t o a d i s e a s e - r e l a t e d decrease i n t h e s e n s i t i v i t y of adenylyl c y c l a s e (AC) t o t h e ~ a t e c h o l a m i n e ~ .This lack of AC s e n s i t i v i t y however could be reversed by c o r t i c o i d t h e r a p y 5 r 6 i n accord with the long e s t a b l i s h e d permissive e f f e c t s of t h e s e hormones on c y c l i c AMP synt h e s i s . I n nephrogenic d i a b e t e s i n s i p i d u s , diminished u r i n a r y production of c y c l i c AMP i n response t o ADH w a s a l s o r e p o r t e d 7 . Other conditions t h a t may involve d e f i c i e n t c y c l i c AMP production include hypertension', p s o r i a s i s g and stroke". On t h e o t h e r hand, c y c l i c AMP production appears t o be excessive i n mania' , cardiomyopathy' , Down's Syndrome' , e p i l e p s y ' 4 1 , t h y r o t o x i c o s i s ' , magnesium deficiency' and d i a b e t e s ' O . Cyclic GMP, which appears t o share with c y c l i c AMP t h e secondary messenger function i n many systems2r 3 , 2 1 , a l s o appears t o be e l e v a t e d i n p s o r i a s i s g and hypertension22-2 4. C l i n i c a l l y t h e determination of c y c l i c n u c l e o t i d e l e v e l s i n b i o l o g i c a l f l u i d s is beginning t o y i e l d promise a s a d i a g n o s t i c t o o l f o r a v a r i e t y of d i s e a s e s 2 5 and a s a r e l i a b l e i n d i c a t o r of f e t a l health26.
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Cyclic nucleotides now appear t o be involved i n t h e c o n t r o l of sperm a c t i v i t y and m o t i l i t y 2 7 and sperm c a p a c i t a t i o n 2 ' and as mediators of v i r a l i n f e c t i o n 2 3 0 , c e l l u l a r growth3' (including t h a t of b a c t e r i a 3'1 , t h e immune response 3 3 - 3 6 , inflammation37, anaphylaxis3" 3 9 , histamine r e l e a s e 4 0 , 4 1 , h y p e r ~ e n s i t i v i t y ~ ~and ' neurotransmitter r e l e a s e 4 4 , 4 6 , among t h e many o t h e r important c e l l u l a r processes previously known. The p o t e n t i a l of t h e c y c l i c n u c l e o t i d e s o r t h e i r d e r i v a t i v e s as drugs i s a l r e a d y appar8 ,~4 9 , i n h i b i e n t i n many a r e a s i n c l u d i n g contraception4 , r a d i o p r ~ t e c t i o n t i o n of drug metabolism5' and antagonism of a n e s t h e t i c s 5 1 t o mention a few.
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Although c y c l i c AMP and c y c l i c GMP appear t o mediate a n t a g o n i s t i c events i n many systems support f o r t h e s i m p l i s t i c view t h a t they a l w a y s have a n t a g o n i s t i c functions i s dwindling. N e w e r s t u d i e s appear t o s t r e s s t h e independent r o l e played by each c y c l i c n u c l e o t i d e i n t h e mediation of s p e c i f i c c e l l u l a r responses. Thus, the demonstration of t h e e x i s t e n c e and r e c i p r o c a l responsiveness of c y c l i c GMP i n a p a r t i c u l a r s y s t e m i s no longer s u f f i c i e n t t o a s s i g n t o i t merely a r o l e a s a n a n t a g o n i s t of c y c l i c AMP i n t h a t system. The s i t e s a t which a p p r o p r i a t e l y designed drugs could a c t t o a l t e r c y c l i c nucleotide-mediated events have been previously o u t l i n e d 5 and a r e shown i n t h e accompanying f i g u r e 1. The s i t e s of p o s s i b l e drug a c t i o n a r e enumerated i n t h e f i g u r e and w i l l be discussed i n t h e same order a s they appear t h e r e i n . I.
CONTROL O F THE INTRACELLULAR LEVELS OF CYCLIC NUCLEOTIDES
A. Synthesis: The s y n t h e s i s of each of t h e two n a t u r a l l y - o c c u r r i n g c y c l i c n u c l e o t i d e s i s catalyzed by a s p e c i f i c cyclase t h a t uses t h e corresponding nucleoside t r i p h o s p h a t e (ATP o r GTP) a s s u b s t r a t e . The a c t i v i t i e s of these cyclases determine t o a g r e a t e x t e n t t h e i n t r a c e l l u l a r l e v e l s of t h e s e messengers. Consequently, s t i m u l a t i o n of t h e c y c l a s e s r e p r e s e n t s an important s i t e f o r p o s s i b l e drug e f f e c t s ( S i t e 1) on t h i s system. S t i m u l a t i o n of t h e cyclases has long b e e n l i m i t e d t o only hormones and t h e i r immediate d e r i v a t i v e s s i n c e t h e s e cyclases appear t o r e p r e s e n t themembrane s i t e s f o r hormonal r e g u l a t i o n and have c o n s i s t e n t l y demonstrated marked Although some drugs, s p e c i f i c i t y t o c e r t a i n hormones i n t a r g e t t i s s u e s . e . g . , i s o p r o t e r e n o l , a r e known t o s t i m u l a t e t h e cyclase enzymes, t h e s e f o r t h e most p a r t have represented only minor modifications i n t h e s t r u c t u r e s of t h e p a r e n t hormones. However, compounds l e s s c l o s e l y r e l a t e d t o t h e n a t u r a l hormones now appear t o be capable of cyclase s t i m u l a t i o n i n a v a r i e t y of t i s s u e s , e n l a r g i n g t h e p o s s i b i l i t i e s f o r drug discovery. Among on t h e s e are a group of i n s e c t i c i d e s i n c l u d i n g a-chlordane and heptchlor and t h e long l i v e r AC, tolbutamide and ethanol on p a n c r e a t i c i s l e t AC3' described e f f e c t s of f l u o r i d e i o n s . S i m i l a r l y d i r e c t i n h i b i t i o n of t h e a c t i v i t y of t h e cyclases i s an important s i t e f o r t h e c o n t r o l of t h e i n t r a c e l l u l a r c y c l i c n u c l e o t i d e l e v e l s ( S i t e 2 ) . Lithium i o n s were reported t o d i r e c t l y i n h i b i t AC i n b r a i n s 5 , p l a t e l e t s s 6 and t h y r o i d 5 7 . Valinomycin5' and p o s s i b l y o t h e r a n t i b i o t i c s a s w e l l 5 9 , 6 o a l s o appear t o i n h i b i t AC. Adenosine a c t s a t l e a s t p a r t i a l l y Via a s i m i l a r mechanism61r62. The 6a d r e n e r g i c blocker propranolol a l s o i n h i b i t e d AC i n c a t myocardium6 and turkey e r y t h r o c y t e s 6 ' , suggesting t h a t AC i n h i b i t i o n may u n d e r l i e i t s membrane e f f e c t s u n r e l a t e d t o 6-adrenergic r e c e p t o r blocking a c t i v i t y . S o t a l o l , a 6-adrenergic r e c e p t o r blocker devoid of t h e s e membrane e f f e c t s , d i d not i n h i b i t AC under s i m i l a r conditions. Alloxan6' and e t h a c r y n i c a c i d 6 6 apparently a c t v i a AC i n h i b i t i o n i n t h e pancreas and kidney, r e s p e e t i v e l y . There a r e i n d i c a t i o n s from s t u d i e s with b r a i n t i s s u e i n v i t r o t h a t c o b a l t i o n s may i n h i b i t guanylyl cyclase (GC) a c t i v i t y 3 . I n a d d i t i o n t o e i t h e r s t i m u l a t i n g o r i n h i b i t i n g cyclase a c t i v i t y , blockade of enzyme s t i m u l a t i o n by n a t u r a l s t i m u l a n t s r e p r e s e n t s a major s i t e f o r t h e a c t i o n of p o t e n t i a l drugs ( S i t e 3 ) . Hormone antagonist^^^-^' and 6-adrenergic7' ,
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as w e l l as a-adrenergic, blockers71 probably a c t a t t h i s s i t e . I n general, compounds t h a t a r e capable of antagonizing hormone-induced e f f e c t s i n i n t a c t systems are a l s o capable of antagonzing hormonal e f f e c t s on t h e c y c l i c n u c l e o t i d e system. This a p p l i e s e q u a l l y w e l l t o those d e r i v a t i v e s of p e p t i d e , s t e r o i d and o t h e r hormones t h a t antagonize hormonal e f f e c t s . Frequently, hormonal d e r i v a t i v e s devoid of hormone-like a c t i o n s f a i l t o induce responses s i m i l a r t o those induced by t h e a c t i v e hormones on t h e c y c l i c nucIeoti.de system. S t u d i e s continue t o advance t h e understanding of t h e mechanisms i n volved i n c y c l a s e a c t i v a t i o n and/or i n h i b i t i o n . I t now appears t h a t n e t AC a c t i v i t y r e p r e s e n t s an equilibrium between two forms of t h e enzyme, a phosphorylated i n a c t i v e and a dephosphorylated a c t i v e form7 Phosphor y l a t i o n of AC is catalyzed by a cyclase k i n a s e , t h e p r o p e r t i e s of which a r e under i n v e s t i g a t i o n and may hold t h e answer t o many q u e s t i o n s r e l a t e d t o t h e a c t i o n s of drugs on AC a c t i v i t y . I t appears, f o r example, t h a t f l u o r i d e and prostaglandins (PDE's) a c t i v a t e AC by s h i f t i n g the cyclase k i n a s e r e a c t i o n toward t h e a c t i v e , dephospho-form. Other drugs stimulating o r i n h i b i t i n g cyclase a c t i v i t y may w e l l be expected t o a c t on t h e c y c l a s e kinase s h i f t i n g t h e r e a c t i o n toward t h e phospho- o r t h e dephospho-forms, r e s p e c t i v e l y . I t i s a l s o p o s s i b l e t h a t GC may be s i m i l a r l y regulated.
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S t u d i e s on AC s e n s i t i v i t y in V i V O a r e more d i f f i c u l t and less conc l u s i v e because of t h e complex p a t t e r n s of i n t e r a c t i n g f a c t o r s a f f e c t i n g t h e AC response. Ethanol i s thought t o induce lower AC a c t i v i t y i n c e r t a i n a r e a s of t h e m u s e b r a i n mainly by i n h i b i t i n g adenosine r e l e a s e . Although e t h a n o l does not seem t o a f f e c t AC a c t i v i t y d i r e c t l y , i t s in vivo e f f e c t s could be e a s i l y mistaken f o r a d i r e c t e f f e c t on t h e enzyme. A h y p o t h e t i c a l , t h r e e component model f o r AC was proposed and appears to f i t w e l l with t h e known a s p e c t s of t h e enzyme a c t i o n 7 3 . According t o t h i s model t h e enzyme i s composed of a d i s c r i m i n a t o r , a transducer and a n a m p l i f i e r . The d i s c r i m i n a t o r u n i t i s t h a t p a r t exposed on t h e e x t e r i o r s u r f a c e of the c e l l and may be i n d i s t i n g u i s h a b l e from t h e long sought hormonal r e c e p t o r . I t embodies t h e s p e c i f i c s t r u c t u r a l f e a t u r e s necessary f o r t h e r e c o g n i t i o n of and binding with t h e n a t u r a l hormone. The amplifier u n i t i s t h a t p a r t f a c i n g t h e c e l l i n t e r i o r which c a t a l y z e s t h e conversion I t i s a l s o c a l l e d t h e c a t a l y t i c subunit. The of ATP t o c y c l i c AMP. d i s c r i m i n a t o r o r r e c e p t o r u n i t i s coupled to t h e a m p l i f i e r o r c a t a l y t i c u n i t via t h e transducer o r coupling u n i t . This l a t t e r subunit t r a n s m i t s t h e information from t h e r e c e p t o r a f t e r hormonal i n t e r a c t i o n t o t h e amplif i e r u n i t leading t o increased o r decreased c a t a l y t i c a c t i v i t y . Although t h e s t r u c t u r a l requirements f o r i n t e r a c t i o n w i t h t h e r e c e p t o r s u b u n i t a r e well-defined i n many systems and t h e c o f a c t o r and o t h e r requirements f o r optimal a c t i v i t y of t h e c a t a l y t i c subunit a r e a l s o w e l l - i d e n t i f i e d , l i t t l e , i f any, information i s a v a i l a b l e about t h e s t r u c t u r e and/or function of t h e transducer o r coupling subunit. It appears t o be t h e locus where c a t i o n i n t e r a c t i o n s with t h e AC system occur. Furthermore, most d i s e a s e r e l a t e d a b e r r a t i o n s i n t h e c y c l i c AMP system seem t o be c l o s e l y - r e l a t e d t o abnormal function of t h i s s u b u n i t , s i n c e it has t h e main r o l e of r e g u l a t i n g t h e o v e r a l l responsiveness o r s e n s i t i v i t y of t h e e n t i r e system.
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Drugs could a c t on any of t h e t h r e e AC components. As examples, most hormones appear t o a c t a t the d i s c r i m i n a t o r , Ca++ a t t h e transducer and Fa t t h e a m p l i f i e r l e v e l . The s i t u a t i o n with GC may be more complex s i n c e i t s a c t i v i t y i s n o t l i m i t e d t o c e l l u l a r membranes and s o l u b l e GC a c t i v i t y appears t o e x i s t i n a number of t i s s u e s ’ . The s e n s i t i v i t y of AC and p o s s i b l y a l s o GC t o s t i m u l a t i o n by n a t u r a l hormones appears t o be an important component of a number of d i s e a s e states I i n c l u d i n g hypertension’, 1 e ~ k e m i - a,’ ~hepatoma7 5 r 7 6 . p s o r i a s i s 7 ’ , asthma7’ and p o s s i b l y aginge0-’ I n a d d i t i o n , decreased AC responsiveness may u n d e r l i e t h e process of c a r c i n o g e n e s i s e 4, b a c t e r i a l i n f e c t i o n ’ 5, and p o s s i b l y morphinee6 and a l c o h o l dependence”. I n a l l t h e s e conditions, t h e f a c t o r ( o r f a c t o r s ) e s s e n t i a l f o r t h e optimal function of t h e coupler u n i t apparently involved i n t h i s l o s s of c y c l a s e s e n s i t i v i t y i s ( a r e ) unknown. The importance of t h e permissive o r maintenance hormones, i n p a r t i c u l a r growth, t h y r o i d and s t e r o i d hormones i n maintaining AC and/or GC s e n s i t i v i t i e s should be explored. GTP a l s o appears t o be an o b l i g a t o r y requirement i n t h e s t i m u l a t i o n and i n h i b i t i o n of AC a c t i v i t y i n p l a t e l e t s 8 8 , r a t a n t e r i o r p i t u i t a r y g l a n d ” , adipose t i s s u e ” I , and l i v e r g 2 , apparently by enhancing t h e d i s s o c i a t i o n of t h e a c t i v e l i g a n d s a f t e r a c t i o n on t h e enzymeg3. Many o t h e r f a c t o r s appear t o i n f l u e n c e t h e sens i t i v i t y of AC t o s t i m u l a t i o n b u t t h e i r e x a c t mechanisms are less understood. These f a c t o r s include a r e v e r s i b l e macromolecular i n h i b i t o r g4, p h o s p h o l i p i d s g 5 ‘9 6 1 adenosineg7, c o r t i c o i d s g ’ , f a t t y a c i d s g 9 and t h e p r o s t a g l a n d i n s . The p o s s i b l e involvement of these and o t h e r f a c t o r s i n d i s e a s e - a s s o c i a t e d l o s s of cyclase s e n s i t i v i t y r e p r e s e n t s an important a r e a f o r f u t u r e research toward understanding of t h e d i s e a s e process a s a whole and may a l s o provide a d d i t i o n a l i n s i g h t s i n t o new mechanisms f o r drug a c t i o n .
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Another important f a c t o r t h a t should be s t r e s s e d i s t h a t AC s e n s i t i v i t y a l s o appears t o r e f l e c t t o a c e r t a i n e x t e n t t h e r e c e n t h i s t o r y of the enzyme. Perkins2 showed t h a t b r i e f exposure of human astrocytoma c e l l s i n c u l t u r e t o concentrations of norepinephrine r e s u l t e d i n decreased responsiveness of t h e same c e l l s t o t h e a d d i t i o n of t h e same o r higher c o n c e n t r a t i o n s of t h e hormone. This l o s s of AC s e n s i t i v i t y i s ligands p e c i f i c s i n c e t h e response t o PGEl i s u n a l t e r e d a f t e r exposure t o norepinephrine. The responsiveness of t h e s e c e l l s t o PGEl i s , however, reduced a f t e r b r i e f exposure t o t h e prostaglandin while t h e response t o norepinephrine remains i n t a c t . The e x a c t mechanisms involved here a r e unknown. B. Loss t o E x t r a c e l l u l a r Fluid: The loss of t h e c y c l i c n u c l e o t i d e s from c e l l s i s an important f a c t o r i n t h e c o n t r o l of t h e i r i n t r a c e l l u l a r l e v e l s . Released cyclic n u c l e o t i d e s can be a valuable i n d i c a t o r of hormonal a c t i v i t y 1O 0 s i n c e they r e f l e c t t h e “ f r e e “ , metabolically-active pool ( s ) O ’ . The s y s t e m r e g u l a t i n g c y c l i c n u c l e o t i d e leakage r e p r e s e n t s an important s i t e f o r drugs t h a t can a c t by s t i m u l a t i n g o r i n h i b i t i n g t h e r e l e a s e of c y c l i c nucleotides t o t h e e x t r a c e l l u l a r f l u i d s and t h u s appreciably a f f e c t ing t h e s i z e of t h e i r f r e e a c t i v e pools ( S i t e 4 ) . Whether t h e same f a c t o r s are involved i n t h e t r a n s p o r t of t h e c y c l i c nucleotide i n t o t h e c e l l s ’ 0 2 i s not a t a l l c l e a r . Present evidence seems t o i n d i c a t e t h a t l o s s of c y c l i c
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nucleotides t o e x t r a c e l l u l a r f l u i d accounts f o r t h e readily-assayable l e v e l s of these n u c l e o t i d e s i n plasma and t o t h e assignment of a p o s s i b l e primary messenger function t o them. However, t h i s view i s complicated by t h e notorious i n a b i l i t y of these n u c l e o t i d e s t o c r o s s c e l l u l a r membranes n e c e s s i t a t i n g extremely high e x t r a c e l l u l a r l e v e l s , f a r above those encount e r e d in v i v o , t o accomplish such a r o l e . L o s s of c y c l i c n u c l e o t i d e s t o t h e o u t s i d e of c e l l s may a t l e a s t p a r t l y e x p l a i n t h e r a p i d decay of t h e hormonally-induced rises i n i n t r a c e l l u l a r c y c l i c n u c l e o t i d e s i n some systems even i n t h e continued presence of t h e s t i m u l a t i n g hormone and complic a t e s c o r r e l a t i o n s t u d i e s between c y c l i c nucleotide l e v e l s and hormonal responses‘. This leakage of c y c l i c nucleotides t o t h e e x t r a c e l l u l a r f l u i d appears t o be r e g u l a t e d t o a c e r t a i n e x t e n t and t o be a f f e c t e d by drugs. I n man, probenecid caused a s u b s t a n t i a l i n c r e a s e i n t h e c y c l i c AMP l e v e l i n lumbar s p i n a l f l u i d l o 3 . Adenosine enhanced t h e r e l e a s e of c y c l i c AMP from c o r t i c a l t i s s u e and t h a t e f f e c t was i n h i b i t e d by C a + + ’ 0 4 .
C. Degradation: Stimulation and i n h i b i t i o n of cyclic n u c l e o t i d e degradation ( S i t e s 5 and 6 ) a r e very important mechanisms f o r c o n t r o l l i n g t h e i r i n t r a c e l l u l a r l e v e l s , and t o d a t e r e p r e s e n t t h e most f e r t i l e sources of prototype drugs. The v a r i e t y of s t r u c t u r e s t h a t can i n f l u e n c e t h e a c t i v i t y of t h e phosphodiesterase enzymes ( P D E ’ s ) and t h e importance of Since these enzymes i n drug development have been r e c e n t l y reviewed” P D E ’ s are influenced by a number of hormones, e . g . , i n s u l i n and cholecystok i n i n , i t i s a t l e a s t t h e o r e t i c a l l y p o s s i b l e t h a t some drugs could block t h e a c t i o n of these hormones on P D E ’ s ( S i t e 7). However, no such drug has y e t been found.
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N e w e r advances i n t h i s a r e a s t r o n g l y suggest t h e p o s s i b l e presence of s p e c i f i c c y c l i c AMP-PDE’s and c y c l i c GMP-PDE’s i n c o n t r a s t t o what was p r e v i o u s l y believed’ E a c h c y c l i c n u c l e o t i d e appears t o s i g n i f i c a n t l y i n f l u e n c e t h e r a t e of hydrolysis of t h e o t h e r . Thus a drug a c t i n g on t h e p a r t i c u l a r PDE of e i t h e r c y c l i c n u c l e o t i d e would be expected t o change i t s i n t r a c e l l u l a r concentration, which could i n t u r n i n f l u e n c e t h e rate of h y d r o l y s i s and t h e i n t r a c e l l u l a r concentration of t h e o t h e r c y c l i c nucleot i d e . This may explain a t l e a s t i n p a r t t h e opposite responses of t h e two naturally-occurring c y c l i c nucleotides i n a v a r i e t y of circumstances, e . g. , during c a r d i a c c o n t r a c t i o n ‘ and under the i n f l u e n c e of 6-adrenergic s t i m u l a n t s ’ 7 . Therefore , t h e e f f e c t s of PDE i n h i b i t o r s and/or a c t i v a t o r s could be more complex than appears a t f i r s t s i g h t . S e l e c t i v i t y of drugs f o r e i t h e r c y c l i c AMP-PDE’s o r c y c l i c GMP-PDE’s i s beginning t o be recognized” and may e x p l a i n t h e previously nonconforming pharmacoloqic e f f e c t s of some well-known PDE-inhibitors’ This i s underscored by t h e apparentl y o p p o s i t e events mediated by t h e two c y c l i c nucleotides i n many systems 1,71 An example i s t h e d i v e r g e n t e f f e c t s of t h r e e PDE-inhibitors on gast r i c a c i d s e c r e t i o n by the mammalian stomach. While theophylline poteninhibits t i a t e s , papaverine has no e f f e c t on, and g l y c y r r h e t i n i c a c i d ” a c i d s e c r e t i o n . Theophylline was found t o be more s e l e c t i v e i n i n h i b i t i n g c y c l i c GMP-PDE’s than c y c l i c AMP-PDE’s. Glycyrrhetinic a c i d showed t h e r e v e r s e s e l e c t i v i t y , being more a c t i v e on c y c l i c AMP-PDE. Although a p o t e n t i n h i b i t o r f o r both enzymes, papaverine showed no s e l e c t i v i t y f o r
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e i t h e r . Since c y c l i c GMP appears t o mediate t h e s t i m u l a t i o n of a c i d s e ~ r e t i o n ’ ’and ~ c y c l i c AMP i t s i n h i b i t i o n 2 ’ , t h e s e l e c t i v i t y of PDE i n h i b i t o r s f o r the enzymes s p e c i f i c f o r t h e two n u c l e o t i d e s determines t o a l a r g e e x t e n t t h e r e l a t i v e in V i V O rise i n t h e l e v e l s of t h e s e mediators and consequently t h e e f f e c t on mammalian a c i d s e c r e t i o n . This i s very important, s i n c e PDE-inhibitors, p a r t i c u l a r l y t h e methylxanthines, a r e used e x t e n s i v e l y as t o o l s i n t h e e l u c i d a t i o n of mechanisms involving t h e c y c l i c n u c l e o t i d e s . I n most systems I t h e methylxanthines e x h i b i t g r e a t e r a b i l i t y to i n h i b i t t h e c y c l i c GMP-PDE’s, r e s u l t i n g i n a g r e a t e r rise i n c y c l i c GMP than c y c l i c AMP l e v e l s and should be expected t o mimic those e f f e c t s mediated by c y c l i c GMP r a t h e r t h a n by c y c l i c AMP i n most systems studied. 11.
CONTROL OF THE INTRACELLULAR ACTIVITY O F THE CYCLIC NUCLEOTIDES
A. A l t e r a t i o n i n Free and Bound Pools: Several l i n e s of evidence s t r o n g l y i n d i c a t e t h e presence of a t l e a s t two pools of c y c l i c AMP: one s i t u a t i o n may a l s o p r e v a i l f o r f r e e and one b ~ u n d ’ ’ ~ ~ ~ ~ A- ’similar ’~. Bound c y c l i c n u c l e o t i d e s a r e apparently unavailable f o r c y c l i c GMP’ PDE h y d r o l y s i s ” ’ and t h e r e i s evidence f o r t h e e x i s t e n c e of a t l e a s t one membrane-bound pool‘ Methods of measuring both bound and f r e e pools are becoming a v a i l a b l e ” ‘1 and c y c l i c nucle0ti.de e f f e c t s appear t o be r e l a t e d t o t h e s i z e of t h e f r e e pools. Although no drugs a r e y e t known t h a t can change t h e s i z e s of these pools ( S i t e 8 ) , t h i s remains an import a n t p o t e n t i a l mechanism f o r drug a c t i o n .
”.
B. A l t e r a t i o n of Cyclic Nucleotide-dependent P r o t e i n Kinase Activi t i e s : The r e l a t i v e i n a b i l i t y of the n a t u r a l c y c l i c nucleotides t o c r o s s c e l l u l a r membranes l i m i t s t o a g r e a t e x t e n t t h e i r u t i l i t y as drugs i n c y c l i c nucleotide-deficiency d i s e a s e s . This has s t i m u l a t e d a s e a r c h f o r c y c l i c n u c l e o t i d e d e r i v a t i v e s t h a t can p e n e t r a t e c e l l u l a r membranes with g r e a t e r ease, while r e t a i n i n g the a b i l i t y t o a c t i v a t e t h e c y c l i c nucleotide-dependent p r o t e i n k i n a s e s ( S i t e 9 ) . The importance of t h i s p o t e n t i a l drug mechanism i s underscored by t h e p o s s i b l e involvement of p r o t e i n and i n t h e mechanism of a c t i o n of a t l e a s t some kinase i n d i s e a s e 2 ‘ ’I7, hormones Several c y c l i c n u c l e o t i d e d e r i v a t i v e s have been prepared and t e s t e d f o r a c t i v i t y a g a i n s t p r o t e i n kinases from a v a r i e t y of sources Some well-known drugs, includwith e x c e l l e n t drug p o s s i b i l i t i e s ’ i n g tolbutamide’ furosemide’ and mefenamic a c i d and phenylbutazoneln, were shown t o d i r e c t l y a f f e c t p r o t e i n kinase a c t i v i t y . Thus, t h e p o s s i b i l i t i e s of t h i s s i t e f o r drug development appear q u i t e promising.
‘ ’.
”’
’ ’”.
C. Antagonism of Cyclic Nucleotide E f f e c t s : Agents capable of antagonizing t h e i n t r a c e l l u l a r e f f e c t s of t h e c y c l i c nucleotide ( S i t e 10) r e p r e s e n t a p o t e n t i a l approach t h a t would be p a r t i c u l a r l y u s e f u l i n s i t u a t i o n s where excessive c y c l i c nucleotide s y n t h e s i s may r e p r e s e n t a d i s e a s e - r e l a t e d d e f e c t , e . g . , c y c l i c AMP i n d i a b e t e s ’ ? alcoholism’ 2 8 1 cholera’ 2 9 , and mania”, and c y c l i c GMP i n p s o r i a s i s ’ and hypertension2’-‘? This a r e a has been r e c e n t l y reviewed elsewhere 2 .
’’,
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INDIRECT CONTROL OF CYCLIC NUCLEOTIDE METABOLISM
A. Prostaglandins: Cyclic n u c l e o t i a e s appear t o be o b l i g a t o r y intermediates i n t h e a c t i o n o f t h e prostaglandins’ 3 0 3 2 and t h e l a t t e r t h u s r e p r e s e n t an important t a r g e t f o r i n d i r e c t c y c l i c nucleotide modulat i o n ( S i t e 11) as do prostaglandin a n t a g o n i s t s ( S i t e 1 2 ) and prostaglandin s y n t h e s i s i n h i b i t o r s ( S i t e 1 3 ) . Prostaglandins a l s o appear t o be obliqat o r y i n t e r m e d i a t e s i n t h e a c t i o n s of a t l e a s t some hormones on t h e c y c l i c n u c l e o t i d e system’33‘’35. I t i s a l s o of i n t e r e s t t o note t h a t c y c l i c n u c l e o t i d e s can modulate t h e s y n t h e s i s of prostaglandins a I sugg e s t i n g a complex system involving both p r o s t a g l a n d i n s and c y c l i c nucleot i d e s i n t h e r e g u l a t i o n of t i s s u e responses t o hormones. When a p p l i e d exogenously, p r o s t a g l a n d i n s of t h e E type appear t o s t i m u l a t e t h e synthes i s of c y c l i c AMP,whereas those of t h e F type appear t o a c t v i a increased c y c l i c GMP. Thus, t h e r e a r e reasonable grounds f o r t h e assumption of s t r i c t coupling between t h e prostaglandin system on t h e one hand and t h e c y c l i c n u c l e o t i d e system on t h e o t h e r .
‘-’
B. Co-factor Control: Since s e v e r a l enzymes with varying co-factor requirements a r e involved i n t h e c y c l i c nucleotide system, i t is a t l e a s t t h e o r e t i c a l l y p o s s i b l e t o modulate t h e system s i g n i f i c a n t l y by c o n t r o l l i n g t h e a v a i l a b l i l t y of t h e e s s e n t i a l co-factors. Of these f a c t o r s , Ca++ occupies a commanding p o s i t i o n . Ca++ g e n e r a l l y i n h i b i t s AC a c t i v i t y , i s e s s e n t i a l f o r GC a c t i v i t y , modulates PDE a c t i v i t y and i t s e f f l u x appears Furthermore , a c t i v a t i o n of t o be c o n t r o l l e d by t h e prostaglandins p r o t e i n kinase by c y c l i c AMP seems t o enhance Ca++ u p t a k e 1 4 0 . The importance of Ca++ t o t h e functioning of c y c l i c nucleotide system i s i n d i c a t e d by t h e l a c k of hormonal response i n Ca++-free media i n many s y s t e m s i n s p i t e of an observed accumulation of t h e c y c l i c nucleotides.
’.
I n summary and on the b a s i s of t h e above c o n s i d e r a t i o n s , i t seems e v i d e n t t h a t t h e c y c l i c nucleotide s y s t e m p r e s e n t s s i g n i f i c a n t challenge f o r t h e design of more s e l e c t i v e and s p e c i f i c drugs. To e x p l o i t t h a t challenge w i l l r e q u i r e t h e i n t i m a t e cooperation of medicinal chemists and molecular b i o l o g i s t s and pharmacologists. An even f u r t h e r expansion of our knowledge of f i r s t t h e b a s i c l e s i o n s i n c y c l i c n u c l e o t i d e metabolism i n t h e v a r i o u s d i s e a s e s t a t e s and second t h e most l o g i c a l and p e r t i n e n t approaches t o c o r r e c t those l e s i o n s i s fundamental t o t h e s u c c e s s f u l discovery of new and b e t t e r drugs f o r t h e medical armamentarium.
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EXTRACELLULAR
@'+n
: t \
I NT R AC E L L U L AR
CNMP
I
=--/
I I
/
Figure 1:
P o s s i b l e s i t e s of drug a c t i v i t y on t h e c y c l i c n u c l e o t i d e systems c y c l i c nucleotide-3',5'-monophosphate nucleotide triphosphate nucleotide diphosphate nucleotide monophosphate c o f a c t o r , e . g . , Mq++ and Mn++ l i g a n d , e . g . , norepinephrine o r a c e t y l c h o l i n e p o s s i b l e drug a c t i o n s i t e stimulation inhibition modulation blockade bound c-NMP regulatory u n i t C = catalytic unit Km = Michaelis Menten Constant
Note t h e m u l t i p l e receptor s i t e s on n u c l e o t i d y l cyclase and t h e membranous l o c a t i o n of t h e low Km phosphodiesterase. Two bound (one membrane-assoc i a t e d ) and one f r e e pools of t h e c y c l i c nucleotides a r e a l s o shown.
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Zapf a n d A. J a k o b , FEBS L e t t e r s 2, 7 3 ( 1 9 7 3 ) . W. L. Ryan a n d M. A , D u r i c k , S c i e n c e , 1002 ( 1 9 7 2 ) . H . Cramer. L. K. Y. Ng a n d T. N. Chase, Arch. N urol.. 2.1 9 7 ( 1 9 7 3 ) . I . P u l l a n d H. M c l l w a i n , Biochem. J., 136, 8 9 3 ( 1 9 7 3 ) . M. s. Amer a n d w. E. Kreigbaum, J. Am. Phann. sci., 1 9 7 4 , i n press. A. W o l l e n b s r q e r , E. B. B a b s k i i , E.-G. K r a u s e , S . Genz, D. Blohm a n d E. V. Boqdanova, Biochem. Biophys. R e s . COm8.. 55. 446 ( 1 9 7 3 ) . M. S. Amer, K . W . Dunqan a a d G. R . McKinney, J. Pharmacol. E x p t l . T h e r a p . , 1974, i n press. G. S c h u l t z , J. G . Hardman. K. S c h u l t z , J. W. Davis a n d E. W. S u t h e r l a n d , P r o c . N a t l . Acad. S c i . , 70, 1721 (1973). M. S. Amer, Am. J. D i g e s t . D i s . , 17, 945 ( 1 9 7 2 ) . M, S. Amer, A. Akcasu a n d G. R. McKinney, Biochem. P h a m a c o l . , 1974. i n press. J. S c h u l t z a n d J. W. Daley, J. B i o l . Chem., 248, 8 4 3 ( 1 9 7 3 ) . M. Z a k a r i j a a n d J. M. McKenzie, L i f e S c i . , 225 ( 1 9 7 3 ) . L. D. Khac, S. Harbon a n d H. J. C l a u s e r , Eur. J. Biochem., 40, 1 7 7 ( 1 9 7 3 ) . E. F. F a l l o n , R . Agrawal, R. Cowden, E. F u r t h a n d A . L. S t e i n e r , C l i n . Res., 21, 490 ( 1 9 7 3 ) . W. Y. Cheung, B i o c h e m . B i o p h y s . Res. Corn.. 46, 99 ( 1 9 7 2 ) . J. A. Kant a n d T. L. S t e c k , Biochem. Biophys. R e s . Corn., 116 ( 1 9 7 3 ) . R. K S h a r m a , C a n c e r Res., 2, 1734 ( 1 9 7 2 ) . M. D r e z n e r , F. A . Neelon a n d H. E. L e b o v i t z , New Eng. J. Med.. 1056 ( 1 9 7 3 ) . 0. Walaas, E. Walaas a n d 0. G r l n n e r # d , E u r . J. Biochem., 40, 465 ( 1 9 7 3 ) . L. N . S i m n , D. A. Shrnnan a n d R. K. Robins. "Advances i n C y c l i c N u c l e o t i d e R e s e a r c h " , 001. 3, P . G r e e n q a r d and G. A. Robison, Eds., Raven P r e s s , New York, 1 9 7 3 , p. 225. L. I . Rebhun a n d C. V i l l a r - P a l a s i , Biochim. Biophys. A c t a , 321, 165 ( 1 9 7 3 ) . J. Nagyvary, R. N. G o h i i , C. R. K i r c h n e r and J . D . S t e v e n s , Biochem. Biophys. Res. C o r n . , E, 1072 ( 1 9 7 3 ) . F. A. Neelon and 8. M. B i r c h , J. B i o l . Chem., 248, 8361 ( 1 9 7 3 ) . G. C e h o v i c , N-8. Giao a n d T. P o s t e r n a k , C. R. C. D. Acad. S c i . , 277, 2057 11973). H. L. Wray a n d A . W. Harris, Biochem. Biophys. Res. C o r n . , 2, 2 9 1 (19731. D. R. F e r g u s o n a n d 8. R. mite, B r . J.. P h a n n a c o l . , 288 ( 1 9 7 3 ) . V. D i n n e n d a h l , H. D. P e t e r s a n d P. S. S c h o n h o f e r , Naunyn-Schmiedeberq's Arch. Phannacol., 278, 2 9 3 ( 1 9 7 3 ) . M. A. Israel, H. Kumura a n d K. Kuriyama, E x p e r i e n t i a . 2, 1322 ( 1 9 7 2 ) . M. F i e l d , New Enq. J. Med., 2BQ. 1137 ( 1 9 7 1 ) . W. Braun a n d C. Shiozawa, " P r o e t a g l a n d i n s and C y c l i c Am'*, R. H. Kahn a n d W. E. M. Lands, Eds., Academic P r e s s , New York, 1 9 7 3 , p. 21. F. A. Kuehl, Jr., J. L. Hums, L. R. Mandel, V. J . C i r i l l o , M. E. Z a n e t t i a n d E. A. Ham, Biochem. Biophys. Res. C o r n . , 44. 1464 ( 1 9 7 1 ) . R. H.Kahn and W. E. M. Landa. ( 6 d s . l " p r o s t a g l a n d i n s a n d C y c l i c m", Academic p r e s s . New York, 1 9 7 3 . G. B u r k e , E n d o c r i n o l o g y , 94, 91 ( 1 9 7 4 ) . S. G a l l a n t a n d A . C. Brownie, Biochcm. Biophys. Res. Comm., 55, 8 3 1 ( 1 9 7 3 ) . G. Burke, L-L. Chang and M. S r a b o , Z c i c n c e 872 ( 1 9 7 3 ) . G. Burke, P r o s t a g l a n d i n s , 2, 291 ( 1 9 7 3 ) . C. J. L i m a a n d J. N . Cohn. P r o c . SOC. E x p t l . B i o l . Ned., 142. 1230 ( 1 9 7 3 ) . B. Hamprecht, 8 . M. J a f f a and G. W . P h i l p o t t , FEBS Letters, 6, 1 9 3 ( 1 9 7 3 ) . E. C a r a f o l i a n d F. C r o v e t t i , Arch. Biochem. Biophys., %, 4 0 ( 1 9 7 3 ) . P. J. L a r a i a , L. J. Z w e r l i n g and E. Morkin, Fed. P r o c . , X , 346 ( 1 9 7 3 ) .
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213 Section V Editor:
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W i l l i a m J . Wechter, The Upjohn Co., Kalamazoo, Michigan
Section Editorial During my t e n u r e as e d i t o r of t h e s e c t i o n Topics i n Biology, I have t r i e d t o emphasize new developments i n Biochemistry and Biology, which s h o u l d impact on t h e p r a c t i c e of m e d i c i n a l c h e m i s t r y . This w a s p a r t i c u l a r l y e x e m p l i f i e d by t h e c h a p t e r s on t r a n s i t i o n s t a t e a n a l o g s , gene therapy, r e v e r s e t r a n s c r i p t i o n , and t h e r e c e n t c h a p t e r on Enzyme S u i c i d e . I n more b i o l o g i c a l terms I have t r i e d t o raise t h e l e v e l of u n d e r s t a n d i n g by t h e m e d i c i n a l chemist of t h e g e n e r a l s u b j e c t of immunology. T h i s , I hope, w a s accomplished through t h e two c h a p t e r s on immediate t y p e h y p e r s e n s i t i v i t y , a c h a p t e r on t h e complement system, a c h a p t e r on t h e d e l a y e d m e d i a t o r s , and f i n a l l y , t h e r e c e n t c h a p t e r on immune a d j u v a n t s . The o n l y s u b j e c t t h a t is n o t i c e a b l y l a c k i n g h e r e is t h a t of t h e b i o l o g y of immunosuppress a n t s . This s u b j e c t h a s been a d e q u a t e l y covered i n a number of r e c e n t a r t i c l e s ( f o r example, see P r o g r e s s i n Drug Research, 16:67(1972)). I cann o t overemphasize t h e importance of immunology, a f i e l d now approaching i t s golden age. It i s n o t , however, t h e continuous advances i n immunology t h a t s h o u l d excite t h e i m a g i n a t i o n of t h e m e d i c i n a l c h e m i s t , b u t r a t h e r t h e i m p l i c a t i o n s of t h e r o l e of immunology i n t h e m a j o r i t y of d i s e a s e s t a t e s w i t h which w e d e a l . Immunologic mechanisms are i m p l i c a t e d i n asthma and o t h e r a t o p i c d i s e a s e s , l u p u s , rheumatoid a r t h r i t i s , g l o m e r u l o n e p h r i t i s , myasthenia g r a v i s , p e r n i c i o u s anemia, immune i n f e r t i l i t y , d i a b e t e s m e l l i t u s , pemphigus, c i r r h o s i s , u l c e r a t i v e c o l i t i s , myocardial d i s e a s e and a h o s t of less common syndromes. Unquestionably, t h e advances i n immunology w i l l p r o v i d e a means w i t h which t o d e a l w i t h many of t h e s e d i s e a s e s . Cons e q u e n t l y , I l o o k t o t h e s u b j e c t s of t h e c h a p t e r s which have appeared i n t h e l a s t t h r e e y e a r s i n Topics i n Biology as a p r o p e r f o c u s of a c t i v i t y i n academic Medicinal Chemistry. These are t h e areas from which w e , i n t h e a p p l i e d s e c t o r of h e a l t h s c i e n c e s h o u l d take o u r l e a d . By no means do I see t h e problem of communicating t h e s e advances t o o u r community as complete. I l e a v e immunology and o t h e r s u b j e c t s t o t h e c a t h o l i c tastes of my s u c c e s s o r s . Thus, Annual Reports can s e r v e as a means of p r o v i d i n g a b i o l o g i c a l d i r e c t i o n f o r m e d i c i n a l c h e m i s t r y . Doubtl e s s , i t is a working u n d e r s t a n d i n g of what i s new i n b i o l o g y , which prov i d e s n o t o n l y impetus € o r i n n o v a t i o n , b u t a l s o a w e a l t h of p u r e l y chemical problems t h a t c h a l l e n g e b i o l o g y . These are e x e m p l i f i e d by q u e s t i o n s such as what are t h e chemical s t r u c t u r e s of SRS-A and t h e m e d i a t o r s of delayed t y p e h y p e r s e n s i t i v i t y (Volume 8 , Chapter 9 ) . I make a p a r t i c u l a r p o i n t of t h e importance of t h i s t y p e of work as " t r u e " academic m e d i c i n a l c h e m i s t r y , because i t c l e a r l y h a s p o t e n t i a l f o r r e l e v a n c e i n human medicine. Thus, I leave t h e charge t o my s u c c e s s o r s t o show u s t h e way.
Section V - Topics in Biology Editor: William J. Wechter, The Upjohn Company Kalamazoo, Michigan
Chapter 22. Anti-aging Drugs Jasjit 5. Bindra Pfizer Inc., Groton, Connecticut introduction - Research on aging has long been regarded as an impractical pseudoscience, and proven prolongation o f life by pharmacological means apparently still remains in the realm of science fiction. Only in recent years, with the application of scientific method and control, has experimental gerontology begun to be taken more seriously.’ A major stimulus to this increasing interest has come from social and considerations. One welcome consequence o f this interest has been an improvement in the quality o f research on senesence phenomena and a discernible shift from purely descriptive studies to studies on mechanism. The present discussion focuses on some recent studies and theories in gerontology which have yielded significant results, and the one aspect o f gerontological research which perhaps most immediately concerns the medicinal chemist, viz., the current status o f agents purported to affect the aging process. Theories o f Aging - Theories o f aging are legion. While several books highlighting various aspects and individual theories have appeared in recent years, the host of theories proposed to explain why aging occurs have been dealt with in comprehensive Several of the theories simply attempt to explain obvious deleterious changes associated with senesence and therefore must be approached with caution. Aging processes are increasingly being thought o f as originating a t the cellular or subcellular level, even though there is no general agreement as to the nature of these processes. However, there i s no reason to believe that there i s only one fundamental type of cellular or subcellular aging process. Currently attractive hypotheses encompassing similar or identical cellular or molecular processes have been consolidated together by La Bella8 in the coalescence-accretion-stabilization hypothesis of aging. There have been other attempts at developing integrated theories of aging.’,’’ Aging as impaired Cellular information Phenomenon - The most attractive general hypothesis o f aging is that senesence i s primarily a cellular information loss phenomenon originating a t the molecular level, and that d yshomeostasis of other kinds through loss of neuronal, endocrine, and immunological information is o f a secondary nature.2 Taken in the broadest sense this hypothesis may be related to any theory of aging. Interference with the flow of cellular information could occur anywhere in the sequence from DNA to RNA to protein synthesis, building’up t o Orgel’s cytoplasmic “error catastrophe” due to impaired protein synthesis.’ Alternatively, impaired cellular function due t o breakdown of essential cellular components, rampant lysosomal enzyn?e activity, autoimmune reactions, increased cross-linking,
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accumulation of lipofuscin pigments and waste products could also result in cellular information loss. Programmed vs Unprogrammed Cellular Death - Goldstein’ has reviewed the two opposing, though not necessarily mutually exclusive, views on whether loss o f cellular mitotic potential and information flow is caused by a random accumulation of faults in informational macromolecules or an inbuilt genetic control mechanism related to differentiation. Although experiments have been designed to distinguish between these two possibilities, the results can be interpreted in favor of either theory. 15 Aging of Tissue Culture Ce/1sl6 - Diploid fibroblasts of human’ 7-20 and chick e m b r y ~ ~ ’ - ~ ~ origin have a finite capacity for replication in vitro and are therefore being used increasingly as a model for studying aging. H a y f l i ~ kattributed ~~ this limited life-span (50 +, 10 doublings) to an intrinsic programmed expression of aging at the cellular level (clonal aging theory), and has suggested that this finite limit i s rarely, i f ever, reached by cells in vitro, and that viral transformations may be a prerequisite for conversion of such cells into permanent lines. 26 Evidence in support of clonal aging is based on the inverse correlation existing between age of donor in vivo and life-span obtained in Moreover, physiologic rather than chronologic age appears to be important since fibroblasts from the premature-agingsyndrome of progeria28 and Werner’s syndrome26 have decreased growth potential in comparison with age-matched controls. Support for the view that genetic material of a given tissue is programmed to undergo a given number of cell divisions has been obtained by Daniel e t al. 29-31 who studied the growth rate of mouse mammary epithelium and found it declines during serial transplantation in vivo. These authors compared transplant lines in which cells proliferated continuously, with lines in which growth was restricted and showed that the decline in growth rate was related primarily to the number of cell divisions undergone rather than to the passage o f metabolic time. There have been reports confirming the f a c t that in vitro finite l i f e span of human diploid cells is related to the number of cumulative population doublings and occurs independently of metabolic time. 20 M a ~ n a r d - S m i t hhas ~ ~ summarized some results from serial transplantation technique which seem t o argue against the view that clonal aging of mitotic cells is an important cause of aging. Nevertheless, limited life-span of fibroblasts has now been observed sufficiently often in different laboratories for it to be regarded as a phenomenon that calls for an explanation, whether or not aging of individuals can be attributed directly to the limited life-span of stem lines. A ging Mechanisms have dealt with programmed senesence rather Theoretical Models - Strehler e t extensively a t the theoretical level in their “codon-restriction theory” of aging. They propose that DNA is read differentially because of changes in transfer RNA and aminoacyl-tRNA synthetase in various tissues during differentiation and aging. Other cellular factors could also
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affect these changes in codon usage, i.e., feed-back loops alter the cell’s language, and negative feed-back effects (repressors) may be the ultimate cause of aging.
In all tissues examined thus far, the rate of production of new cells is controlled by a tissue-specific antimitotic messenger molecule called a chalone (negative feedback system). B ~ l l o u g hsuggests ~~ that the onset o f aging and the rate at which it proceeds is probably controlled through a chalone mechanism by a non-mitotic tissue which acts as a pacemaker. There is reason to believe that deterioration of post-mitotic cells may well be a major cause of aging in mammals.32 Evidence has been presented that changes in neurons and in cells in the microvascular system are the main cause of aging in mammals.35 Since the antimitotic power of chalones is known to be strengthened by stress hormones, such as adrenaline and glucocorticoid hormones, one test of the chalone theory would be to discover whether it is possible to affect the life-span by strengthening or weakening the chalone action. The marked increase in life-span o f rats on restricted caloric intake14 has been explained on basis of continued high level of stress hormone secretion due to strengthened chalone action. 34 Gelfant and Smith36 have recently presented a model in which cellular and tissue aging is regarded as the result of progressive conversion of cycling to noncycling cells in tissues capable of proliferation. Cycling cells are regarded as cells that are actively moving through the cell cycle of periods of nuclear DNA synthesis and mitosis. A cell may be blocked a t any of the intervening gaps.37 These authors suggest that immune mechanisms may be involved in cellular aging by keeping noncycling cells in restraint. Reversal of cellular aging by hydrocortisone, an immunosuppressive agent, has been rationalized as a release of noncycling cells. 36,38 Hormonal Control of Aging - The diversity of regulatory functions performed by the hypothalamus has led to the suggestion that age-dependent changes in the hypothalamus are responsible for the functional and metabolic changes associated with aging. F r o l k i ~ has ~ ~ , ~ ~ presented evidence that the hypothalamus undergoes structural changes and exhibits a decrease in functional activity with age. There is a reduction in the activity of neurosecretory processes and the hypothalamic structures show an increased sensitivity to such physiologically active agents as catecholamines, acetylcholine and insulin.39 Dilman4’ suggests that the key process in the genetic program of development and aging is a gradual reduction in sensitivity of the hypothalamus to feed-back control. This leads to increased secretion of “aging hormones”, and disruption of the two basic homeostatic systems regulating reproduction and energy metabolism. E ~ e r i t has t ~ ~further examined the relationship between hormones and aging, stressing the role of hypothalamic-pituitary regulation of the rate of aging and onset of age-related pathology. lmpaired Protein Synthesis - Aging may affect protein synthesis either at the DNA level by mutation, single strand breaks or other structural changes, or at subsequent stages. 5,43,44 However,questions of age-associated changes in DNA have been particularly difficult to pursue because of the problem of detecting subtle molecular changes at that level. Animals receiving sublethal doses of X-irradiation have been shown to become prematurely senile and susceptible to fatal diseases. X-irradiation has been used to induce single strand breaks and to apparently
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duplicate age-associated changes that occur naturally in DNA of senescent brain cells.45 However, in view of the rather remarkable ability of cells to rejoin broken strands and to replenish altered base units in DNA46,47the relevancy of radiation to aging is debatable. This does not exclude the ossibility, of course, that damage could build up if DNA-repair enzymes are progressively lostI4 or if an age-associated rampant increase in the release of hydrolytic enzymes occurs. Cellular aging has also been attributed to an irreversible nuclear protein-DNA interaction 48,49 or RNA polymerase inactivation by arginine-rich his tone^.^' Orgel’ has recently reviewed how errors in the synthesis of RNA and information-handling enzymes can accumulate progressively, decrease the fidelity of protein synthesis and eventually jeopardize Frolkis has discussed the entire protein-synthesizing machinery (“error catastrophe”).”,’ the interrelationship between cell function and protein biosynthesis in the process of aging. 51
*
Agents Affecting Protein Synthesis - Agents that enhance learning and memory5* have been examined in geriatrics since they supposedly act on brain RNA and protein synthesis. 5,5-Diphenyl hydantoin reportedly prevents polyribosomal disaggregation in the aging brain tissue and renders RNA more resistant to hydrolysis by RNAse. The drug has been shown to improve the avoidance response and learning retention in aged rats. 53-55 Procainamide has been tested for learning enhancement activity in experimental animals of different ages and found to improve behaviour and learning deficits in aging rats.53 Procainamide was shown to increase brain RNA content and to redistribute intracellular amino acids from the free pool to an RNA-bound form. 54
Impaired Cellular Function Accumulation of Cell Debris - Lipofuscin granules are fluorescent lipoprotein masses, appropriately called age-pigments, which tend to accumulate with advancing age, especially in postmitotic cells such as those of the brain, myocardium, skeletal muscle and adrenals. 56 Lipofuscin pigments may be debris l e f t over from free-radical attacks on cell components , and might be an important indicator of the degree of cellular insufficiency in aging.57,58 However, there is as yet no direct evidence that they interfere with normal cell processes. Meclofenoxate (centrophenoxine) (I)is a geriatric therapeutic agent useful for treatment of patients suffering from confusional states, Parkinsonism and other senile mental disorders. Subsequently, Nandy and Bourne59,60 showed that meclofenoxate treatment could reverse the accumulation of lipofuscin pigments in neurons of senile guinea pigs, and suggested that reduction of lipofuscin may be one of the ways by which the drug exerts i t s beneficial effects on the CNS. There was also a marked reduction in the activity of a number of oxidative enzymes, as determined histochemically.60 The drug is said to promote an increase in glucosed-phosphate dehydrogenase activity by diversion of glucose metabolism to the pentose cycle, the common pathway of glucose metabolism in cells undergoing rapid growth or multiplication, which results in an unexplained elimination of the accumulated pigments.
Free-Radical Damage - Free-radicals can arise from a number of sources in the living system.
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Harman has suggested that free-radical mediated damage of macromolecules and other cellular components can result in cumulative degeneration o f cellular function which expresses itseif as aging. -63 Several antioxidants and free-radical scavengers, such as cysteine, 2-mercaptoethylamine (2-MEA), 2,2’-diaminodiethyIdisuIfide, butylated hydroxytoluene (BHT) (It) and ethoxyquin (Ill) have been tested for antiaging effects. BHT and 2-MEA were amongst the most effective agents in increasing the mean life-span in animal colonies, but had no significant effect on maximum life-span.63~64Antioxidants which are ineffective in increasing survival may have achieved inadequate tissue distribution or concentration. The role of unsaturated fatty acids as a source of free-radicals has been stressed.56 Vitamins C and E, cysteine, glutathione and selenium compounds which have been shown to inhibit peroxidation and can act as free-radical scavenger^^^ are being investigated as antioxidant therapy in humans.65
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Lysosomes and Membrane Damage - Labilization of the lysosomal membrane and rampant lysosomal action have been suggested as a cause o f cellular death during aging.66 A detailed discussion of the possible involvement of membrane breakdown in aging has been presented. 67 H o ~ h s c h i l dnotes ~ ~ that leakage of lysosomal enzymes into the cytoplasm could, in principle, be responsible for several processes suggested as primary aging mechanisms, such as reduced fidelity of protein synthesis. In this context membrane damage in inositol-starved mutants of Neurospora has been shown to result in synthesis of altered proteins.68 Several agents are known to stabilize lysosomal membranes (e.g., c o r t i c ~ s t e r o i d s ) . ~Cortisone7* ~ and hydroc~rtisone~ have ~ been shown to prolong the mitotic capacity of human embryonic fibroblasts. However, it is as yet unclear whether corticosteroids act on aging systems by stimulating protein synthesis or by stabilizing lysosomal membranes. Dimethylaminoethanol (DMAE), another agent believed to stabilize membranes, when administered to senile mice in drinking water resulted in a significant increase in their life span.72~73FDA-approved clinical studies with DMAE are underway.74 Cross-linking of Connective Tissue - Agents which interfere with crosslinking of peptide chains in fibrous connective tissue macromolecules and collagen are expected to retard biological aging. 75-79 Lathyrogens inhibit the oxidative deamination of free €-amino groups of lysyl residues t o aldehydes which is the first step in the cross-linking of lysine residues on different peptide chains. Therefore, laythrogenic compounds, such as J-aminopropionitrile, peni~illamine~’and semicarbazide, which prevent maturation of collagen, have been suggested as anti-aging drugs. La BeIla8,75 has obtained some evidence that these drugs will extend the average life-span (but not the maximum life-span) of rats if given in doses sufficient to slow down crosslinking but not enough to cause undesirable side-effects. The use of penicillamine was accompanied by an inhibition of wound healing and by increased skin fragility.80,81 Autoimmunity
- W a l f ~ r and d ~ B~ ~~r ~n ~e t have ~ ~ elaborated , ~ ~ on the idea that aging is an
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autoimmune phenomena. They propose that aging is a consequence of increasing immunogenetic diversification of dividing cell populations of the body. The age-dependent-decline of immunocompetence is manifested in a progressive failure of the organism’s own cells to distinguish self from foreign cells with damaging immunological reaction against them (“unleashing of self-destroying process9,831, or the development of damaging autoimmune cell clones (forbidden clones) due to somatic mutation of immunocytes and a decreased responsiveness to extrinsic antigens. B ~ r n e t *has ~ emphasized that decreased immunological surveillance may originate in the thymus-dependent segment of the immune system, and the latter, in turn, may be dependent on exhaustion of the Hayflick limit (inbuilt-metabolic clock) in thymus cells and dependent tissues, Based on experiments with “nude” mice (a C3H derived strain of mice born without a thymus which suffer from immunodeficiencies similar to, but more acute than those produced experimentally by thymectomy), Pantelouris86 suggests that senescent normal animals have an excess of IgM antibodies over IgG antibodies. The IgM antibodies mainly produced in response to bacterial polysaccharides often cross-react with the mammal’s own tissue (cross-reaction autoimmunity). This gradually increasing dependence on IgM antibodies has been correlated with involution of the thymus8’ and may be an important process in “immunological aging” independent, though not exclusive of forbidden clones and somatic mutations. In general, immune function does show a decline in intensity with advancing age. There is a progressive incidence of autoantibodies such as antinuclear, rheumatoid, antithyroid and antiparietal factors in the aged. 82,88-90 This occurs despite a decrease in the numbers of antibody-forming cell precursors in both the spleen and bone marrow with increasing age. 91 On the basis o f the autoimmune theory, Walford tested the effect of an immunosuppressant, azathioprine (Imuran) (IV), on the life-span of aging mice.92 Animals receiving a daily dose of 100 mg/kg showed a mean survival time 10 weeks longer than controls, but the maximum life-span (135-140 weeks) was not affected. Similar results were obtained with another immunosuppressant, cyclophosphamide (V).92 It seems probable that anti-aging therapy a t this level may be too late in the chain of events. Moreover, suppression of the immune system may accelerate death from other causes.
Co‘p”” T d N‘ H
(CHzCHZCI)Z
V Conclusion - The enigma of human aging continues to persist, although it is becoming increasingly evident that aging may be caused by a number of different mechanisms operating simultaneously in the individual. There is growing research emphasis on some theories of aging that are especially promising. Most impressive has been the accumulation of evidence that aging i s caused, at least in part, by a progressive breakdown of the body’s immunological defenses - whether programmed or random. While on the basis of present experimental evidence it is difficult to recommend any treatment to inhibit aging, a clearer understanding of
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the fundamental aging process will surely lead to a rational approach to retarding human aging during the next two decades. Acknowledgment - Thanks are due to Dr. G. R. Evanega for several stimulating discussions, to Drs. G. M. Milne and J . R. Tretter for helpful comments and Dr. H.-J. Hess for continued encouragement and guidance.
References 1. A. Comfort, Nature, 21 7, 320 (2968). 2. F. Verzar, Triangle, 8 7 9 3 (1968). 3. I. 5. Wright, J . Am. Geriat Soc., L9, 737 (1971). 4. A. D. Bender, C. G. Kormendy and R. Powell, Exp. Gerontol, 97 (1970). 5. C. G. Kormendy and A. D. Bender, J . Pharm. Sci.,AO, 167 (1971). 6. See also reports on aging, Chem. Eng. News, July 24, p. 13 (1971);March 28, p. 15 (1974). 7. H. P. von Hahn, Mech. Aging Develop.,2, 245 (1973). 8. F. S. La Bella in “Search for New Drugs’; ed. A. A. Rubin, Marcel Dekker, New York (1972) p. 347. 9. D. G. Carpenter and J . A. Loynd, J. Am. Geriat. Soc., 16, 1307 (1968). 10. T. R. Griffiths, Mech. Aqing Develop., 2 295 (1973).11. L. E. Orgel, Proc. U. S. Not. Acad. Sci., A9, 427 (1963). 12. L. E. Orgel, ibid., L7, 1476 (1970). 13. L. E. Orgel, Nature, 243 441 (1973). 14. S. Goldstein, New EnTdnd J. Med., 25, 1121 (1971). 15. S.Goldstein and C. C. Lin, Exp. Cell Res., LO,436 (1972). 16. L. Hayflick, Amer. J . Med. Sci., x 5 , 432 (1973). 17. L. Hayflick, Exptl. Gerontol., 5, 291 (1970). 18. L. Hayflick and P. S. Moorhead, Exptl. Cell Res., 585 (1961). 19. R. J. Hay, Adv. Gerontol. Res., 2, 121 (1967). 20. R. T. Dell‘Orco, J. G. Mertens aEd P. F. Kruse, Exptl. Cell Res., l7, 356 (1973). 21. R. J. Hay and B. L. Strehler, Exptl. Gerontol., 5 123 (1967). 22. J. Ponten, int. J. Cancer, ~5,323 (1970). 23. L. Lima and A. Macieira-Coelho, Exptl. Cell Res., 70, 279 (1972). 24. L. Lima, E. Malaise and A. Macieira-Coelho, Expti,-Cell Res., 2, 345 (1972). 25. L. Hayflick, Exptl. Cell Res., Lz 614 (1965). 26. G. M. Martin, C. A. Sprague and C. J. Epstein, Lab. invest., 23, 86 (1970). 27. S. Goldstein, J. W. Littlefield and J . S. Soeldner, Proc. U.S. mat. Acad. Sci., &, 155 (1969). 28. S. Goldstein, Lancet, L 424 (1969). 29. C. W. Daniel, K. 5. DeOme, L. J. T. Young, P. B. Blair and L. J. Faulkin, Proc. U.S. Natl. Acad. Sci.,Ll, 52 (1968). 30. C. W. Daniel, L. J. T. Young and D. Medina, Exp. Gerontol., 6, 95 (1971). 27(1971). 31. C. W. Danieland L. J. T. Young, Expt. Cell Res., 32. /. Maynard-Smith in “Cell Biology in Medicine”ed., E. E. Bittar, John Wiley and Sons, New York (1973) p. 681. 33. 8. Strehler, G. Hirsch, D. Gusseck, R. Johnson and M. Bick, J. Theoret. Biol., A3, 429 (1971). 34. W. S. Bullouqh, Nature, a 9 , 608 (1971). 35. L. M. Franks, Expt. Gerontol., J 281 (1970). 36. S. Gelfant and J. G. Smith, Science, 178, 357 (1972). 37. S. Gelfant, Exp. Cell. Res.,A2, 521 11963); J. J. DeCosse and S. Gelfant, Science, s 2 , 698 (1968). 38. V. J. Cristofalo, Advan. Gerontol. Rer,4, 45 (1972). 39. V. V. Frolkis, V. V. Bezrukov, Y. K. Duplenko and E. D. Genis, Exp. Geront.,-7, 169 (1972). 40. V. V. Frolkis, N. Verzhikovskaya and G. V. Valueva, Exp. Geront.,i, 285 (1973). 41. V. M. Dilman, Lancet I/, 121 1 (1971). 42. A. V. Everitt, Exp. GeTont., 8, 265 (1973). 43. H. J . Curtis, “Biological Meczanisms of Aging’; C. C. Thomas, Springfield (1966). 44. K. L. Yielding, Persp. Biol. Med., 201 (1974). 45. S. P. Modak and G. B. Price, Exp. Cell Res., 289 (1971). 46. E. Fahr, Aqnew. Chem. Int. Ed., 3,578 (1969). 47. C. J. Dean, M. G. Ormerod, R. W. Serianniand P. Alexander, Nature, 222, 1042 (1969). 48. J . S.Salser and M. E. Balis, J. Geront., 27, 1 (1972). 49. H. P. von Hahn, J . Gerontol., 21, 291 (1966). SO. T. C. Spelsberq, S. Tankersiey and L. S. Hnilica, Experientia, 25, 129 (1969). 51. V. V. Frolkis, Gerontologia 9 189 (1973). 52. A. Weissman, Ann. Repts. Med. Chem., 1967, C. K. Coin, Ed., Acad. Press New York ( 1 9 6 8 ) ~ 279. . 53. P. Gordon, S. S. Tobin, B. Doty and M. Nash, J . Gerontol. 23,434 (1968). 54. P. Gordon, Recent Adv. Biol. Psychiat., 121 (1968). 55. B. Doty and R. Dolman, Psychon. Sci., 14, 109 (1969). 56. A. L. Tappel, Geriatrics, 97 (1968).57. B. L. Strehler, Adv. Gerontol. Res., 1, 343 (1964). 58. W. Reichel, J. Hollander, J. H. Clark and B. L. Strehler, /. Geront., 3 71 (1968). 59. K. Nandy and G. H. Bourne, Nature 210, 313 (1966). 60. K. Nandy, J . Geront., 23, 82 (1968).61. D. Harmon, J. Geront., 23, 476 (1968).
a,
u,
a,
z,
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62.D. Harman, J. Geront., 26,451 (1971). 63.D. Harmon, Agents A c t z n , 1, 3 (1969). 64.D. Harmon, Am. J. Clin. Nutrition, 2.5,839 (1972). 65.R. A. Passwaterand P. A. Welker, f i e r . Lab.,A 21 (1971). 66.A. Comfort, Lancet, 2 1325 (1966). 67.R. Hochschild, Exp. deront., 6 1.53(1971). 68.J. L. Sullivan and A. G. Deb& Nature New Biol., E3, 72 (1973). 69.G. Weissman in “Lvsosomes in Bioloqy -. and Patholoqy’: -. . Part I, . .I. T. Dinqle and H. B. Fell,. Eds.,. .lohn Wiley and Sons, New York (1969): 70.G. C,Yuan and R. S.Chang, Proc. SOC.Exp. Biol. Med., 130, 934 (1969). 71. V. Gristofalo in “Aging in Cell and Tissue Culture’; E. Holeckova and V. J. Gristofalo, Eds.,Plenum, New York (1970) p. 83. 72.R. Hochschild, Exp. Geront., 8 185 (1973). 73.R. Hochschild, Exp. Geront., $ 177 (1973). 74.Anon., Med. World News, Jan. 11, p. 13 (1974). 7.5.F. 5. LaBella, Gerontologist,J, 13 (1968). 76.R. R. Kohn and A. M. Leash, Exptl. Mol. Pathol., 2, 354 (19671. 77.A. J, Bailey, Gerontologia, 15,65 (1969). 78. F. Verzar, Gerontologia, GT233 (1969). 79.J. Bjoorksten, J. Amer. Geriatrics SOC.,16 408 (1968). 80.A. Ruiz-Torres, Arzneim-Forsch., 5 594 (1968). 81.M. E. Nimmiand L. A. Baretta, S&nce, &O, 905 (1965). 82. R. L. Walford, Lancet,?, 1226 (1970). 83. R. L. Walford, “Immunologic Theory o f Aging’: Munksgaard, CopenhagerjI (196‘9). 84.F. M. Burnet, “Immunological Surveillance” Sydney ( I 970). 85. F. M. Burnet, Lancet 2,358 (1970). 86. E. M. Pantelouris, Exp. Geront.,L 73 (1972). 87.E. M. Pantelouris, Exp. Gemnt.,lJ 169 (1973). 88.“Tolerance, Autoimmunity and Aging‘; M. M. Sigel and R. A. Good, Eds., c. c. Thomas, Springfield I,n-I?l
(IYlLj.
89.S. Whittingham, J. D. Mathews, 1. R. Mackay, A. E. Stocks, B. Ungar and F. I. R. Martin, Lancetl, 763 (1971). 90.J. Prieto Valtuena, M. 1. Gonzalez Guilabert, M. A. del Pozo Perez, F. del Pozo Crespo and R. Velasco Alonso, Biomedicine, g, 301 (1973)and references therein. 91.S. Kishimoto and T. Yanamura, Clin. Exp. Immunol., 4 957 (1971). 92. R. L. Walford, Symp. SOC.Exp. Biol., 2l,351 (1967).
Chapter 23. Affinity Labeling of Hormone Binding Sites John A. Katzenellenbogen, Department of Chemistry, University of Illinois, Urbana, Illinois 61801 Affinity labeling (AL) is a technique for covalently labeling protein binding sites. According to this method, a reactive functional group (attaching function) is incorporated within the structure of a ligand molecule in such a manner that the modified ligand is bound by the protein and reacts with amino acid residues in o r near the binding site, forming a covalent bond through the attaching function. Most AL studies have utilized as attaching functions the alkylating and acylating groups typically used in protein modification studies ("conventional" affinity labeling; CAL) and have been directed at binding sites in homogeneous protein preparations, particularly the active sitf -s7 in enzymes. Several comprehensive reviews of this work are available. (Metabolically-activated irreversible enzyme inhibitors are reviewed in Chapter 24 of this volume.) More recently, the scope of AL studies has expanded to include antibody binding sites and binding sites associated with the allosteric control of enzymes and membrane transport (regulatory sites) and with the action of hormones, drugs, and neurotransmitters (receptor sites). A new dimension in labeling selectivity has been demanded in many of these latter applications, as the binding proteins generally constitute only a very small fraction of the total aggregate of macromolecules. The development of many new attaching functions, most notable of which are those which require photoactivation ("photoaffinity" labeling; PAL), has improved the versatility of this technique. This review will focus on the developments in the area of AL relating principally to hormone binding sites. Fundamental methodological considerations of labeling selectivity in heterogeneous systems will also be covered. Fundamental Considerations - The salient feature of an AL process is that covalent bond formation takes place within a complex of protein and labeling reagent; the high, localized concentration of reagent in the complex results in an enhanced reaction between the reagent and nearby amino acid residues (prqximity effe§t), which is the basis for the labeling selectivity. Baker and others have outlined certain kinetic features that a binding-preceeded labeling process should display: (1) There should be evidence for a reversible equilibrium between reagent and binding site; (2) the labeling reaction should display a rate saturating effect; (3) non-reactive ligands which compete for the same binding site should exert a protective effect, reducing the rate, but not extent of labeling, and (4) there should be a stoichiometric relationship between the extent of labeling and the extent of inactivation. These properties are generally used as the criteria for establishing that a labeling process is indeed occurring by an AL mechanism, as opposed, in particular, to a simple bimolecular mechanism.
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Labeling Selectivity in Heterogeneous Binding Systeqs:,,Conventional versus Photoaffinity Labeling - Metzger, Wofsy, and Singer”” ”- have proposed a kinetic model for the selectivity of an AL process in a homogeneous binding system. This model can be expanded to ’cover heterogeneois systems. ..s K
a
PS + L-x
___c
pns + L-x
__c
PS*L-x
Kns a
c-
pns* L-x
kS
kns _c
P ‘L-x
n
Pns*L-x
....
(1)
. ...
(2)
The AL reagent L-x consists of a ligand portion L and an attaching s function 5 . It is in reversible equilibrium (K ) with a specific binding protein (P ) whose labeling is desired, and covafent attachment proceeds within this complex by a first order process (ks) (eqn 1). Undesired labeling can take place by two mechanisms: The labeling reagent, particularly if non-polar, may associate in a low-affinity, non-specific manner with hydrophobic regions of other proteins, and labeling can take place via this complex (eqn 2 ) . In addition, the labeling reagent may react with exposed residues on any of the proteins in a bimolecular fashion, i.e., without a prior binding equilibrium (eqn 3 ) . Labeling selectivity, thus, constitutes a predominance of process 1 over processes 2 and 3 . An expression for the f9tio of the rates of labeling can be derived in a straightforward manner, and the value of its integral over the time of labeling gives the labeling selectivity. However, it is equally instructive to consider a simpler expression which represents the maximum selectivity (selectivity limit, SL) expected under optimum conditions (low concentration of L-x and low extent of labeling). Three terms can be recognized in this ex ression‘ (a) A binding .K /P:” Kn’) , which describes term (Ps SL = (Po.Ka) .k b (P;”.Kis) .kns + k .Po the dishfhtion 8f bound reagent between the specific and nonspecific sites; (b) an attachment rate o r efficiency term (ks/kns), which relates how rapidly o r efficiently covalent attachment takes place withinbthe two types of complexes, and (c) a bimolecular labeling term (k .Po). s
s
s
r:
A two-point distinction can be made between conventional and photoaffinity labeling processes: (1) Dark vs. irradiation conditions gives one external control over the reactivity of the PAL reagents, and (2) the covalent bond-fomping reactions with CAL reagents (alkylation and acylation) have sufficiently high activation energies to make them slow relative to the rates of complex formation and dissociation, while the reactions of the highly energetic species (ketone triplets, carbenes, nitrenes, etc.) produced by photolysis of the PAL reagents is rapid relative to the rates of complex association and dissociation.
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The e x t e r n a l c o n t r o l can be p u t t o two advantages t o i n c r e a s e l a b e l i n g s e l e c t i v i t y . Once t h e system h a s reached b i n d i n g e q u i l i b r i u m ( i n t h e d a r k ) t h e system can be p e r t u r b e d by r a p i d l y removing t h e e x c e s s f r e e r e a g e n t by molecular s i e v i n g o r a d s o r p t i o n . A s t h e system s t r i v e s t o r e e q u i l i b r a t e , bound r e a g e n t w i l l d i s s o c i a t e more r a p i d l y from t h e low a f f i n i t y , n o n - s p e c i f i c s i t e s , causing a temporary i n c r e a s e i n t h e v a l u e o f t h e b i n d i n g term ( a ) , a t which time t h e system i s i r r a d i a t e d . Removal o f e x c e s s f r e e r e a g e n t a l s o r e d u c e s t h e v a l u e of term ( c ) . Such an e q u i l i b r i u m - p e r t u r b a t i o n approach cannot be a p p l i e d t o a CAL p r o c e s s . The r a p i d , i n d i s c r i m i n a n t r e a c t i o n of t h e PAL r e a g e n t s a l s o has two p o t e n t i a l advantages. The r e l a t i v e l y slow and s e l e c t i v e attachment r e a c t i o n s a s s o c i a t e d w i t h t h e CAL r e a g e n t s may well r e s u l t i n a wide v a r i a t i o n i n t h e r a t e o f c o v a l e n t attachment w i t h i n t h e d i f f e r e n t t y p e s of complexes t h a t w i l l form i n a heterogeneous b i n d i n g p r e p a r a t i o n ( v a l u e o f term b u n c e r t a i n ) , while t h e PAL r e a g e n t s w i l l presumably r e a c t with r e a s o n a b l e e f f i c i e n c y with r e s i d u e s i n any b i n d i n g s i t e , r e g a r d l e s s o f i t s p r e c i s e composition o r s t e r i c l a y o u t (term b % 1 ) . Furthermore, i n PAL, but n o t i n CAL, a scavenger c a p a b l e o f i n t e r c e p t i n g t h o s e r e a g e n t molecules which a r e p h o t o a c t i v a t e d i n s o l u t i o n b e f o r e t h e y r e a c t with p r o t e i n , can b e added t o reduced b i m o l e c u l a r r e a c t i o n .
5 A -3-Ketosteroid isomerase h a s been t h e s u b j e c t of t h r e e r e c e n t s t u d i e s . 66-Bromotestosterone a c e t a t e (BTA), a p o t e n t c o m p e t i t i v e i n h i b i t o r , ca es i r r e v e r s i b l e i n a c t i v a t i o n o f t h e enzyme upon long-term Y3 P r o t e c t i o n a g a i n s t i n a c t i v a t i o n i s a f f o r d e d by 19-norincubation. t e s t o s t e r o n e , and t h e pH-rate p r o f i l e o f i n a c t i y a t i o n s u g g e s t s t h e i n volvement o f h i s t i d i n e . Labeling s t u d i e s with H-BTA Qave shown t h a t each mole o f enzyme can r e a c t w i t h c a . t h r e e moles of H-BTA ( c f . trimer s t r u c t u r e ) . In s t r i k i n g c o n t r a s t t o t h e r e a c t i v i t y of t h e 6P-bromo d e r i v a t i v e , f i v e d i f f e r e n t 2a-, 4a-, and 6a-bromoketoandrostane s u b s t r a t e analogs were shown t o have no c a p a c i t y f o r i r r e v e r s i b l e i n h i b i t i o n y l Z v e n though t h e y a l l are even more p o t e n t c o m p e t i t i v e i n h i b i t o r s t h a n BTA.
In a p a r t i c u l a r l y i n t r i g u i n g apprgach t o l a b e l i n g t h e s t e r o i d i s o merase, " n a t u r a l " l i g a n d s , having t h e A -3-keto chromophore ( t e s t o s t e r ?Ye and 1 9 - n o r t e s t o s t e r o n e acetate,NTA) were used a s p h o t o a f f i n i t y l a b e l s . Enzymatic a c t i v i t y was l o s t when t h e enzyme was i r r a d i a t e d i n t h e presence of t h e s e d e r i v a t i v e s , and t h e r a t e o f i n a c t i v a t i o n p a r a l l e l e d t h e f r a c t i o n a i s a t u r a t i o n o f t h e a c t i v e s i t e . Competitive i n h i b i t o r s l a c k i n g t h e A -3-keto s t r u c t u r e e x h i b i t e d s p e c i f i c p r o t e c t i o n a g a i n s t i r r a d i a t i o n - i n d u c e d i n a c t i v a t i o n by NTA, s u g g e s t i n g t h a t r e a c t n was o c c u r r i n g a t t h e a c t i v e s i t e . I r r a d i a t i o n i n t h e presence of C-NTA r e s u l t e d i n c o v a l e n t l a b e l i n g o f t h e enzyme.
fa
The 176-hydroxysteroid dehydrogenase i s i r r e v e r s i b l y i n h i b i t e d
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10'17 Both estradiol and NADP act as by 16a-iodo-3-acetoxyestrone. protectors against inactivation, and the alkylation process exhibits a rate saturation effect, consistent with an AL mechanism. The 3-0 iodoacetyl derivatives of estfpe (IAE ) and estradiol (IAE ) also rapidly 1 is slowed by f8tradiol 2 inactivate the dehydrogenase. The rate and is also affected by NADP and NADPH. Reaction with C-IAE1 plus NADPH is rapid, giving a labeling stoichiometry of 0.9, and chymotryptic digestion produces a single labeled peptide, which contains a carboxymethyl histidine. The alkBlated enzyme undergoes a slow reactivation upon prolonged incubation at 37 , with 1§oncomitant release of the steroid by hydrolysis of the 3-0 ester bond.
Warren has utilized a number of halogenated progesterone (P) and cortisone (C) derivatiygs2gn a comprehensive study of the 200-hydroxysteroid dehydrogenase. All of the derivatives act as substrates for the dehydrogenase, but prolonged incubation in most cases, results in irreversible inactivation, which is slowed by the addition of progesterone o r cortisone and in some cases also by coenzyme. Reaction using tritiumlabeled derivatives results in each case in the labeling of a single amino acid: 66-bromo- and 66-bromoacetoxy-P -+ cysteine, lla-bromoacetoxyP + methionine, and 16a-bromoacetoxy-P and 21-iodoacetyl-C -+ histidine. Thus, one can establish with considerable precision the relative spatial arrangement of the three nucleophilic residues in the active site. 16a- and lla-Bromoacetoxyprogesterone and 20-acryloxyprogesterone irreversibly2&nactivate the 110- and 21-hydroxylase activities in the rat adrenal; progesterone can protect against the effect o f the 1 6 ~ and 20-derivatives. The diazoketone derivative aroused interest in connection with its apparent, rapi$ irreversible-inactivation of corticosteroid acetyltransferase. The inhibitory activity, however, was traced to the corresponding a-chloroketone which contaminated the preparation. Two straight-chain (non-steroidal7 chloroketone derivatives (2 - and 3) also show considerable irreversible inhibition.
HO
(DZE) __
R'02C (CH2)nCOCH2C1 3 R'=H 5- R'=CH3 (2) Enzymes Affected Allosterically by Steroids - The activity of several metaboiic enzymes is affected by 2geroids: glucose 6-phosp98te dehydrogenase, glu3pate dehydrogenase, aldehyde dehydrogenase, As the steroid is acting as an allosteric effector, and pyruvate kinase. these regulatory binding sites may be more appropriate models for steroid
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hormone r e c e p t o r s i t e s t h a n a r e t h e c a t a l y t i c s i t e s d i s c u s s e d above. The two e s t r o g e n d e r j y a t i v e s , 4 - m e r c u r i e s t r a d i o l (4ME) 30 and 2d i a z o e s t r o n e s u l f a t e (DZE) r e a c t r a p i d l y with c y s t e i n e r e s i d u e s i n g l u t a m a t e dehydrogenase; DZE a l s o r e a c t s with p y r u v a t e k i n a s e . E s t r a d i o l slows t h e r a t e o f r e a c t i o n of t h e s e d e r i v a t i v e s , which i s c o n s i s t e n t with an a f f i n i t y l a b e l i n g mechanism. The r e a c t i v i t y and i n s t a b i l i t y o f t h e s e d e r i v a t i v e s , however, l i m i t t h e i r u t i l i t y . 3
Kallos and S h a found ~ ~ ~t h a t 1 mole o f H-bromoacetyl d i e t h y l s t i l b e s t r o l would r e a c t c o v a l e n t l y with g l u t a m a t e dehydrogenase. Both DES and ADP a f f o r d e d p r o t e c t i o n a g a i n s t t h i s a l k y l a t i o n r e a c t i o n . The modified enzyme was i n s e n s i t i v e t o t h e a l l o s t e r i c e f f e c t o r s ADP and GTP, and c l e a v a g e o f t h e DES group w i t h hydroxylamine f a i l e d t o r e s t o r e normal enzymatic a c t i v i t y . (3) Target T i s s u e - S p e c i f i c , High A f f i n i t y Binding P r q t e i n s ( S t e r o i d Hormone Receptors) - (a) Estrogens - Liarakos and May-" have d e s c r i b e d t h e p r e p a r a t i o n o f some 3 - e s t r a d i o l w-bromoalkyl e t h e r s a s p o t e n t i a l a l k y i a t i n g a g e n t s f o r t h e e s t r o g e n r e c e p t o r i n - r a t u t e r u s . -Although no l a b e l i n g s t u d i e s were done, t h e low b i n d i n g a f f i n i t y and b i o l o g i c a l potency o f t h e s e d e r i v a t i v e s s u g g e s t s t h a t t h e y have l i t t l e p o t e n t i a l a s l a b e l i n g agents.34The 16a, 17a-, and 166, 176-epoxides of e s t r a d i o l were a l s o p r e p a r e d . Both d e r i v a t i v e s have high u t e r o t r o p h i c a c t i v i t y , b u t show no i n d i c a t i o n o f prolonged a c t i v i t y o r i n h i b i t i o n .
The r e p o r t e d s e n s i t i v i t y of t h e u t e r i n e e s t r o g e n r e c e p t o r t o organomercurials prompted Warren and cowortfgrs t o i n v e s t i g a t e 4-mercuriT h i s - g y r i v a t i v e demonstrates e s t r a d i o l (4ME) a s a p o t e n t i a l AL r e a g e n t . which i s conmore p e r s i s t e n t e s t r o g e n a c t i v i t y t h a n e s t r a d i o l s i s t e n t e i t h e r w i t h prolonged occug5ncy of t h e e s t r o g e n r e c e p t o r s i t e o r a slower m e t a b o l i c cle2fjance r a t e , and i t s b i n d i n g a f f i n i t y 3 i s roughly 30% t h a t of e s t r a d i o l . Direct i n c o r p o r a t i o n s t u d i e s u s i n g H-4ME have shown t h a t r a d i o a c t i v i t y becomes a s s o c i a t e d 59 a s o l v e n t - i n e x t r a c t a b l e manner w i t h p r o t e i n s i n r a t u t e r i n e c y t o s o l . E s t r a d i o l can p a r t i a l l y block t h e l a b e l i n g , and t h e p r i n c i p a l l a b e l e d prott&n3§ediments a s an 8 s s p e c i e s on s u c r o s e d e n s i t y g r a d i e n t c e n t r i f u g a t i o n . ' Det a i l e d s t u d i e s u s i n g t h i s d e r i v a t i v e ar25hampered by t h e r e l a t i v e i n s t a b i l i t y o f t h e mercury s u l f u r bond. The p r e p a r a t i o n of a number o f p h o t o s e n s i t i v e a z i d e and d i a z o 40 d e r i v a t i v e s o f e s t r a d i o l , e s t r o n e , and h e x e s t r o l h a s been d e s c r i b e d . T h e i r promise a s s e l e c t i v e p h o t o a f f i n i t y l a b e l s €or t h e r a t u t e r i n e e s t r o g e n r e c e p t o r has been e v a l u a t e d s y s t e m a t i c a l l y by measuring t h e i r r e c e p t o r binding a f f i n i t y i n u t e r i $ ? c y t o s o l ( s e e v a l u e s i n s q u a r e and by determining t h e i r a b i l i t y brackets, % of e s t r a d i o l a f f i n i t y ) t o consume e s t r o g e n b i n d i n g a c t i v i t y i n u t e r i n e c y t o s o l upon irradia&$:g3. Reaction e f f i c i e n c i e s , i . e . , p e r c e n t i n a c t i v a t i o n , r a n g e from 0-21%. Four a n t i e s t r o g e n s o f t h e t r i a r y l e t h y l e n e c l a s s (Upjohn IJ l1.100Ay ParkeDavis CI-628, -680, a g j 9411x27) and 6 - o x o e s t r a d i o l were a l s o shown t o be highly photoreactive.
A f f i n i t y Labeling
Chap. 23
Katzenellenbogen
OH
__ 227
0
R=CH2COCHN2; X,Y=H [1.4]
R=CH2COCHN
X=N3;
R,Y=H
~3.01
X=N3;
Y=N3;
R,X=H
P. 91
X,Y=N3; R=H
2; R,Y=H
X,Y=H
[ 1.81
[69.0] [12.0]
The preparation of several s t e r o i d d e r i v a t i v e s (estrogens, androgens, progestins, c o r t i c o s t e r o i d s ) bearing n i t $8~$9mustard o r ethylene The p- (N,N-bis ( 2 imine a l k y l a t i n g functions has been described. chloroethy1)aminophenylacetic e s t e r s , i n p a r t i c u l a r , showed a c t i v i t y against DMBA-induggd, t r a n s p l a n t a b l e mammary adenocarcinoma and a v a r i e t y o f r a t leukemias. I t i s not c l e a r , however, whether t& s t e r o i d c a r r i e r conveys any p a r t i c u l a r s e l e c t i v i t y t o these d e r i v a t i v e s .
--
(b) Progesterone - Solo and Gardner46-48 have prepared a s e r i e s of progesterone d e r i v a t i v e s bearing r e a c t i v e a l k y l a t i n g functions a t p o s i t i o n s 16a o r 17a ( c f . 1 and 2). The r e l a t i v e l y low l e v e l of prog e s t a t i o n a l a c t i v i t y a s t h e absence of p e r s i s t e n t hormonal o r a n t i hormonal a c t i v i t y of these d e r i v a t i v e s were taken t o mean t h a t no covalent i n t e r a c t i o n with the receptor s i t e was taking place. A number of r e a c t i v e halo, t h i o , diazo, and mercuri-substituted progesterone and t e s t o s t e r o n e derivatjlj;$.,have been synthesized a s p o t e n t i a l a f f i n i t y labeling reagents. The binding a f f i n i t i e s of 6aand 66-bromoprogesterone and 17B-thiol- and 17B-methylthio-4androsten-3-one i n human uterus and chickloviduct a r e s u f f i c i e n t l y high t o .merit f u r t h e r study i n t h e s e systems. OH R 0
N2& 0
;.& clHy!P R=COCHN2; R=SH
(c)
Androgens - A preliminary report describes t h e synthesis of several 7a-substituted t e s t o s t e r o n e s , including t h e 3’-chloropropyl d e r i v a t i v e s . 52
Protein Hormone Binding S i t e s - Bromoacetyloxytocin (BrOXY) has been used t o study t h e neurohypophyseal hormone receptor associated5yith t h e adenyl BrOXY i t s e l f cyclase system of toad bladder and r a b b i t kidney medulla. does not s t i m u l a t e cyclase a c t i v i t y , but it i r r e v e r s i b l y blocks t h e
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stirnulatory effect of the natural vasoactive hormones. Specificity is inferred from the fact that BrOXY has no effect on parathyroid hormone stimulation of adenyl cyclase in the kidney cortex. Several bradykinin derivatives, bearing N-terminal nitrogen mustardtype alkylating agents,5$ause permanent potentiation of bradykinin A possible mechanism involves the irreversible activity in pig ileum. inactivation of kininases by alkylation. Similar angiotensin mustard deriva 'ves act as irreversible antagonists to angiotensin I1 action in ileum,$3 and thus appear to be promising reagents for affinity labeling studies. Serum albumin&as at least two binding sites for oligopeptide Irradiation of photoreactive derivatives of the analogs of gastrin. pentapeptide X-GlyTrpMetAspPheNH2 (X = 4-azidobenzoyl, 2-nitro-5azidobenzoyl, 4-acetylbenzoyl, and 4-benzoylbenzoyl) re3ylts in incorporation of up to two moles of derivative per mole protein. Irradiation of three of the derivatives in radiolabeled form in the acinar cell suspensions results in incorporation of label. Other Binding Sites of Interest: (1) Nucleotide Binding Sites - The preparation of three photosensitive diazomalonyl deriv$gixGs of adenosine 3':5'-cyclic monophosphate (c-AMP) has been described. ' In the dark, 5 is an allosteric activatoij of phosphofructokinase. Irradiation at 354 nm. in the presence of H-5, gjsults in an incorporation of up to 0.7 moles of reagent per mole enzyme; incorporation is blogbed by c-AMP An affinity and the derivatives and 2 are ineffective in labeling. labeling study of the cataiytic site of phosphofructokinazf, using a 6mercaptopurine ribonucleoside triphosphate, has appeared. NHR,
'v
( 2 1
o a
-4o\'
R1
R2
Z-COCN2CO2Et - H 6-COCN2C02Et -COCN2C02Et i-H -COCN2C02Et
R2
Derivative 2 g y alsQ used in a study of c-AMP receptors in human In the dark, it acts as an activator of protein erythrocyte ghosts. kinase activity and competes with c-AMP for binding. Irradiation causes incorporation of label which is partially blocked by c-AMP. Gel electrophoresis reveals a prominent radioactive band which corresponds in mobility to the endogenous substrate of the membrane protein kinase. A number of n cleotide degjvggives bearing alkylating functions were as specific inhibitors of enzymes prepared by and others involved in nucleotide metabolism (e.g., thymidine kinase and phosphorylase, guanine and adenosine deaminase). Related derivatives have been
bake^-^-^
Chap. 23
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Katzenellenbogen
229
usgd-f? label the effector binding site in rabbit muscle phosphorylase b. 7Jhe synthesis of several mercurated nucleosides has been described. These derivatives appear to be quite stable, and they should prove to be interesting reagents for further studies on nucleotide binding sites. ( 2 ) Neurotransmitter Binding Sites - Affinity7jabeling studies on neuro-74 transmitter receptors were reviewed last year. In a preliminary study, the irradiation of two (2-azidobenzy1)trialkyl ammonium salts was reported to irreversibly inactivate the acetylcholine (ACh) esterase of red blood cells and the ACh receptor of frog sartorius muscle. Specific protection by unreactive ligands seemed to imply that a photoaffinity labeling mechanism was operative. However, subsequent work revealed that the photogenerated species was sufficiently long-lived to be in reversible equilibrium with the binding site before reaction; s o , reaction was actually occurring by a conventjgnal affinity labeling process (termed pseudo photoaffinity labeling).
''
Diazoacetylcholine has been prepared, and its pharmacological activity has been shown to be similar to that of acetylcholine (ACh). 77 Irradiation causes 99 irreversible depolarizing block of the7bCh receptor in mouse diaphragm. Its affinity for ACh esterase is low. Despite considerable interest in the use of irreversible a-adrenergic blocking agents of the B-haloethylamine type, as affinity labeling reageq&sgpr adrenergic receptors, little success has been achieved to Most studies have used radiolabeled reagents with lo date. ' specific activity, and non-specific labeling has been extensive.Y3 Catechol-0-methyltransferase is irreversibly &activated by Niodoacetyl-3,5-dimethoxy-4-hydroxyphenylethylamine. Inactivation shows a rate saturating effect and is blocked by 3,4-dihydroxybenzoate. Iodoacetate and iodoacetamide also inactivate this enzyme, but at a much slower rate. 5
Drug Receptors - H-N-B-(2-azidopheny1)ethyl norlevophanol (APENS2was used in a preliminary affinity labeling study of the opiate receptor. Although irradiation of APEN caused incorporation of radiolabel into rat brain homogenates and guinea pig ileum, the propensity of APEN for indiscriminant attachment resulted in high levels of non-specific labeling. (3)
The NaK-ATPase activity of bovine rzit brain homogenates appeared to be irreversibly blfjgked by cardiotonic steroid derivative34strop2anthidine bromoacetate (SBA) and hellebrigenin iodoacetate (HIA), and H-HIA appeared to label the sites with considerable selectivity. However, a recent study 8yggests that the action of SBA on the NaK-ATPase is not irreversible. A variety of reactive anileridineg6 or benzomorphang7 derivatives have been prepared as potential non-equilibrium inactivators of the analgetic receptor. Certain long-term activity of some of these compounds
230 -
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may be ascribed to receptor alkylation. Recent Developments in the Methodology of Photoaffinity Labeling - The potential of a thiocarbonyl group as a photoattaching function is evident from a recent, intriguing photoaffinity labeling 38udy of E. -coli RNA polymerase using mono and poly 4-thiopyrimidines. Irreversible inactivation of polymerase activity was observed when the enzyme was irradiated in the presence of 4-thiourijine triphosphate and polydeoxy-4thiothymidylic acid, and studies with ( P)-derivatives have shown incorporation of label into the B and B l subunits. The use of an iodopyrimidine as a photoattaching function is described86'n an elegant study of the photoinactivation of E. -coli thymidine 5 - Iodo-2 -deoxyuridine (IdUrd) , a substrate analog of thymidine, kinase. enhances the rate of photodegradation of the enzyme; the sensitizing effect is saturable, is protected by substrate, and is also affected by the allosteric ligftds dTTP and dCDP. Incp-fgorationof label occurs upon irradiation with C-IdUrd, but not with I-IdUrd, implicating a pyrimidine radical as the active species. The potential of aryldiaziriggs as photoattaching functions has been suggested by a recent model study. The chemicgl stability and 370 nm, E = 70-100) of these derivatives accessible chromophore (A and the high reactivity oyarhe resulting carbene make them particularly attractive. Photoexcited carbonyl functions (presumably the reactive n-T* appear to have considerable potential as photoattaching functions. The benzophenone triplet was shown to react with AcGlyOMeg$n benzene o r water to give t$s addition product Ph,C(OH)CH(NHAc)C02Me. A series of studies by Elad on the photolytic modification of proteins using toluene o r 1-butene and triplet sensitizers (diacetyl/di-tert-butyl peroxide) contains much work of relevance to photoaffinity labeling.
1sstsy3
A curious phgtoactivation of chlorocyanoethyl colchicine (CCCol) has been observed. In the dark, its interaction with the Col binding site on the 120,000 MW microtubule protein dimer is reversible; however, UV irradiation results in irreversible attachment of labeled CCCol to a protein with an apparent MW of 10-15,000. Conclusion - Although the application of the technique of affinity labeling to hormone binding sites is relatively new, the rapid proliferation o f reports is a testament to its current popularity, i f not its general utility. However, most of the work to date in which highly-specific labeling has been achieved, is confined t o relatively well-defined and homogeneous binding systems. This leaves for the future the delineation of the ultimate selectivity of the conventional, and particularly the photoaffinity labeling techniques, and the successful application of these techniques to the many receptor systems which are available only as heterogeneous preparations.
Chap. 23
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12,
15,
2,
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Chap. 23 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. 93. 94.
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Katzenellenbogen
233
P. Cuatrecasas, J . Biol. Chem., 245, 574 (1970). G. E. Means and R. E. Feeney, J . Biol. Chem., 246, 5532 (1971). F. W. Hulla and H. Fosold, Biochemistry, 11,1056 (1972). R. A. Anderson and D. J. Graves, Biochemistry, 12, 1895 (1973). R. A. Anderson, R. F. Parrish and D. J. Graves, Biochemistry, 12, 1901 (1973). R. M. K. Dale, D. C. Livingston and D. C. Ward, Proc. Nat. Acad. Sci., USA, 70, 2238 (1973). J. S.Bindra, Ann. Repts. Med. Chem., 1972, R. V. Heinzelman, Ed., Academic Press, New York, (1972). p. 262. H. Kiefer, J. Lindstrom, E. S. Lennox and S. J . Singer, Proc. Nat. Acad. Sci., USA, 67, 1688 (1970). A. E. Ruoho, H. Kzfer, P. E. Roeder and S. J. Singer, Proc. Nat. Acad. Sci., USA, 70, 2567 (1973). J. Frank and R. Schwyzer, Experimentia, 26, 1207 (1970). P. G. Waser, A. Hofmann and W. Hopff, Experimentia, 26, 1342 (1970). W. H. Hopff, Experimentia, 29, 763 (1973). W. T. Comer and A. W. Gomollin A. Berger, Ed., "Medicinal Chemistry," (3rd edition), Wiley-Interscience, New York, 1970, part 11, p . 1019. M. Nickerson in L . S. Goodman and A. Gilman, Eds., "The Pharmacological Basis of Therapeutics" (4th edition), MacMillan, New York, 1970, p . 549. R. T. Borchard and D. Thakker, Biochem. Biophys. Res. Commun., 54, 1233 (1973). B. A. Winter and A. Goldstein, Molec. Pharm., 8, 601 (1972). L. E. Hokin, M . Mokotoff and S. M. Kupchan, Proc. Nat. Acad. Sci., USA, 55, 797 (1966). L. E.Hokin, J . Gen. Physiol., 54, 327s (1969). T. Tobin, T. Akera, D. Ku and M T C . Lu, Mol. Pharmacol., 9, 676 (1973). P. S. Portoghese, V. G. Telang, A. E. Takemori and G. Hayashi, J. Med. Chem., 14, 144 (1971). M. May.L. Czoncha, D. R. Garrison and D. J . Triggle, J. Pharm. Sci., 57, 884 (1968). A . M. Frischauf and K. H. Scheit, Biochem. Biophys. Res. Commun., 53, 1227 (1973). R. Cysyk and W. H. Prusoff, J . Biol. Chem., 247, 2522 (1972). R. A. Smith and J . R. Knowles, J. Amer. Chemxoc., 95, 5072 (1973). R. F . R. Church, A. S. Kende and M. J . Weiss, J . A m e r Chem. SOC., 87, 2665 (1965). R. E. Galardy, L. C. Craig and M. P. Printz, Nature New Biology, 242, 127 (1973). 95, M. Schwarzberg, J . Sperling and D. Elad, J. Amer. Chem. SOC., 6418 (1973). S. Hammond and J. Bryan, J . Cell Biology, 55, 103a (1972).
Acknowledgement - The research described in refs. 40-43 was supported by grants from the National Institutes of Health (AM-15556), The Ford Foundation (700-0333), the Eli Lilly Company, and the du Pont Company.
234 Chapter 24
Mechanism-Based I r r e v e r s i b l e Enzyme I n h i b i t o r s Robert R . Rando, Dept. of Pharmacology Harvard Medical School, Boston, Mass.
I n t r o d u c t i o n - T h i s review d i s c u s s e s a g e n e r a l approach t o t h e d e s i g n of i r r e v e r s i b l e enzyme i n h i b i t o r s of e x t r a o r d i n a r y s p e c i f i c i t y . These i n h i b i t o r s are s o c o n s t r u c t e d t h a t t h e y r e q u i r e chemical a c t i v a t i o n by t h e t a r g e t enzyme. Upon a c t i v a t i o n , a chemical r e a c t i o n ensues between t h e i n h i b i t o r and enzyme r e s u l t i n g i n t h e i r r e v e r s i b l e i n h i b i t i o n of t h e l a t t e r . Thus, t h e enzyme by i t s s p e c i f i c mode of a c t i o n c a t a l y z e s i t s own i n a c t i v a t i o n . These i n h i b i t o r s are "kcat i n h i b i t o r s " b o t h because t h e y r e q u i r e c a t a l y t i c conversion by t h e t a r g e t enzyme and because i t i s t o t h i s term of t h e enzymatic p r o c e s s t h a t t h e y owe t h e i r g r e a t s p e c i f i c i t y l . I n t h i s review examples of s y n t h e t i c a s w e l l as n a t u r a l l y o c c u r r i n g i n h i b i t o r s which a c t by t h i s mechanism are d e s c r i b e d . I' I n h i b i t o r s - The paradigm o f t h i s k i n d of cat i n h i b i t o r is t o be found i n s t u d i e s on t h e i r r e v e r s i b l e i n h i b i t i o n of B-hydroxydecanoyl t h i o e s t e r dehydrase by A 6 4, decynoyl N-acetylT h i s enzyme, which i s r e q u i r e d f o r u n s a t u r a t e d f a t t y a c i d cysteamine2. s y n t h e s i s i n E . c o l i , c a t a l y z e s t h e r e v e r s i b l e i n t e r c o n v e r s i o n of hydroxydecanoyl t h i o e s t e r s w i t h t h e i r a,B t r a n s and @ , y counterparts. ( F i g . 1)
Examples of S y n t h e t i c "k
Q
Q
CH3-(CH2)
+
+
CH~-(CH,),-CECK-&!-SR
trans CH3-(CH2)
;CH=CH-m2-kR
cis Fig. 1 The "kcat i n h i b i t o r " o f x i s enzyme , A(34) decynoyl-NAC, i s t r e a t e d as a s u b s t r a t e by t h e enzyme. However, u n l i k e t h e B,y o l e f i n i c s u b s t r a t e , t h e acetylene i s converted i n t o t h e highly r e a c t i v e conjugated a l l e n e which immediately a l k y l a t e s an a c t i v e s i t e h i s t i d i n e r e s i d u e 3 . (Fig. 2 ) En z Irreversible Inactivation' CH3-(CH 2 ) E ; e C - a J-SR 9 (m3(CH,) 5 - C d H - L R )
zs
cH3-(CH2) 5-CH
1
.Ao >!-h Enz
/ Fig. 2 Enz S i n c e t h e enzyme can be t o t a l l y i n a c t i v a t e d by a s t o i c h i o m e t r i c amount of t h i s acetylene, a s i n g l e turnover is a l l t h a t is required f o r i r r e v e r s i b l e i n h i b i t i o n t o o c c u r . The enormous s p e c i f i c i t y of t h i s i n h i b i t o r i n multi-component systems and i n v i v o w a s demonstrated i n t h e c l a s s i c e x p e r iments of Kass and Bloch4 ,S.These experiments showed t h a t B-hydroxydecanoyl t h i o e s t e r dehydrase w a s a b s o l u t e l y r e q u i r e d f o r u n s a t u r a t e d f a t t y a c i d s y n t h e s i s i n E. c o l i .
Chap. 24
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F l a v i n l i n k e d monoamine o x i d a s e s are a l s o i r r e v e r s i b l y i n h i b i t e d by molecules t h a t c o n t a i n an a c e t y l e n i c moiety .6 Compounds such as pargyline , c h l o r g y l i n e 2 , and d e p r e n y l 1 are i r r e v e r s i b l e i n h i b i t o r s of t h e enzyme and i n c e r t a i n i n s t a n c e s t h e y have been used clinically7. (Fig. 3)
/cl C1-•/*=*\ ,*-O-CH
*/*=* \-CH2-;CH2-C=CH \*-/ 1
L*/2
2-CH 2-CH
2-
E(ed2-=c-H
-
3
CH3
Fig. 3 S t u d i e s d e m o n s t r a t e t h a t t h e a c e t y l e n i c moiety must be 8 t o t h e amino group f o r i n h i b i t i o n t o o c c u r and t h a t c o v a l e n t attachment t o t h e f l a v i n r e s u l t s . Furthermore, w e have found t h a t &-3-haloallyl amines a r e i r r e v e r s i b l e i n h i b i t o r s of t h i s enzymeti. As e x p e c t e d , t h e primary acetyl e n i c amine, p r o p a r g y l amine, a l s o i r r e v e r s i b l y i n a c t i v a t e s t h e enzyme. All o f t h e s e r e s u l t s can be understood i n terms of a p l a u s i b l e mechanism f o r f l a v i n cocatalyzed reactionsg. (Fig. 4 ) H+
+
R-CH2-NH
ti
-
When t h e R group is an a c e t y l e n e o r v i n y l h a l i d e , t h e f o l l o w i n g convers i o n s can o c c u r . (Fig. 5)
H
Irreversible) Inactivation
236
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I n b o t h c a s e s , t h e a-proton i s l a b i l i z e d by t h e enzyme, b u t i n s t e a d of t h e e l e c t r o n s flowing toward t h e f l a v i n , t h e y are s u b v e r t e d w i t h res u l t a n t i s o m e r i z a t i o n of t h e molecules. Thus, t h e g e n e r a t i o n of h i g h l y r e a c t i v e a l l e n e s and a l l y l i c h a l i d e s is achieved from t h e i r u n r e a c t i v e counterparts. O t h e r f l a v i n l i n k e d enzymes are a l s o s u s c e p t i b l e t o i r r e v e r s i b l e i n h i b i t i o n by a c e t y l e n e s . Abeles and coworkers have demonstrated t h a t f l a v i n l i n k e d l a c t a t e dehydrogenase i s i r r e v e r s i b l y i n h i b i t e d by 2hydroxy-3-butynoic a c i d l o . I n t h i s c a s e a l s o , t h e i n h i b i t o r reacts w i t h t h e f l a v i n c o f a c t o r . The g r e a t s p e c i f i c i t y of t h i s i n h i b i t o r w a s used t o advantage i n s t u d i e s by t h e s e a u t h o r s d e s i g n e d t o determine t h e physiol o g i c a l r o l e of membrane bound l a c t a t e dehydrogenase i n E. c o l i l l . I n a d d i t i o n , t h e non-flavin l i n k e d plasma monoamine and diamine o x i d a s e s s u f f e r i r r e v e r s i b l e i n h i b i t i o n i n t h e p r e s e n c e of t h e a c e t y l e n e , p r o p a r g y l amine12. These enzymes r e q u i r e two c o f a c t o r s f o r t h e i r a c t i v i t y , t h e f i r s t h a s been p o s i t i v e l y i d e n t i f i e d as c u p r i c i o n and t h e second might b e a p y r i d o x a l d e r i v a t i v e U J 5 T h i s l a t t e r c o f a c t o r i s c a p a b l e of forming a S c h i f f b a s e w i t h t h e primary amino s u b s t r a t e s and, as e x p e c t e d , t h e enzyme is i n h i b i t e d by s t a n d a r d c a r b o n y l r e a g e n t s such as hydraz i n e l 5 . The f o l l o w i n g conversion could m e d i a t e t h e a c t u a l i r r e v e r s i b l e i n h i b i t i o n s t e p . ( F i g . 6)
0
Irreversible Fig. Inactivation' - 6 Consistent with t h i s i n t e r p r e t a t i o n i s t h e observation t h a t pro a r g y l amine h a s no e f f e c t on t h e enzyme when t h e c u p r i c i o n i s a b s e n t P2 T h e r e f o r e , t h e a c e t y l e n e i s n o t i n h i b i t o r y i n and of i t s e l f b u t must b e enzymatically converted i n t o t h e a c t i v e substance.
.
Enzymes which u s e t h e coenzyme p y r i d o x a l phosphate are a l s o q u i t e s u s c e p t i b l e t o t h e s e i n h i b i t o r s . I n 1949, Gunsalus and Wood showed h a t b a c t e r i a l t h r e o n i n e d e h y d r a t a s e was i r r e v e r s i b l e i n h i b i t e d by s e r i n e The mechanism of a c t i o n of t h i s enzyme r e q u i r e s a b s t r a c t i o n of t h e ap r o t o n of t h e amino a c i d w i t h c o n c o m i t a n t e l i m i n a t i o n of H20 t o form t h e
&.
Chap. 24
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I r r e v e r s i b l e Enzyme I n h i b i t o r s
aminoacrylate intermediate
Rando
237
(.& ( F iI g -) 7).
CH
CH 3 4 H 2C-!0
AA
-
2
Fig. 7 When s e r i n e i s t h e s u b s t r a t e t h e h i g h l y r e a c t i v e a m i n o a c r y l a t e t h a t i s g e n e r a t e d a p p a r e n t l y engages i n a Michael r e a c t i o n w i t h an a c t i v e - s i t e amino a c i d r e s i d u e r e s u l t i n g i n t h e i n a c t i v a t i o n of t h e enzyme17. Along s i m i l a r l i n e s , L - s e r i n e s u l f a t e h a s been shown t o be an i r r e v e r s i b l e i n h i b i t o r o f s o l u b l e a s p a r t a t e amino-transferase18 and % - c h l o r o a l a n i n e i r r e v e r s i b l y i n h i b i t s b o t h t h i s enzyme13 and L-aspartate-8-decarboxylaseZ0. The mechanisms of a c t i o n of t h e s e enzymes i n v o l v e t h e i n i t i a l c l e a v a g e of t h e a-C-H bond. Once t h i s o c c u r s , w i t h t h e i n h i b i t o r s as s u b s t r a t e s , t h e r e a c t i o n sequences d e p i c t e d i n F i g . 8 can i n t e r v e n e . ( F i g . 8)
X-CH2-gY2
j/
\*/ I
b
\/ f
I1
*\yo\ Irreversible Inactivation
Irreversible Inactivation
H 1
Fig. 8 The subsequent r e a c t i o n of e i t h e r of t h e s e molecules w i t h c r i t i c a l amino a c i d r e s i d u e s r e s u l t s i n t h e i n a c t i v a t i o n of t h e enzyme. These observat i o n s would l e a d one t o e x p e c t t h a t B,y-unsaturated amino a c i d s ought be p o t e n t i r r e v e r s i b l e enzyme i n h i b i t o r s s i n c e t h e i r enzymatic c o n v e r s i o n s would l e a d t o similar h i g h l y r e a c t i v e i n t e r m e d i a t e s . ( F i g . 9)
Wechter, Ed.
Topics i n Biology
238 -
1
-
H
/
-
2
Fig. 9 We have found t h a t 2-amino-3-butenoic a c i d i s an i r r e v e r s i b l e i n h i b i t o r o f s o l u b l e , p y r i d o x a l phosphate l i n k e d a s p a r t a t e amino t r a n s f e r a s e 2 1 . F i n a l l y , p r o p a r g y l g l y c i n e h a s been r e p o r t e d t o b e an i r r e v e r s i b l e i n h i b i t o r of pyridoxal-linked y-cystathionase. I t i s assumed t h a t an i s o m e r i z a t i o n of t h e a c e t y l e n e t o t h e r e a c t i v e a l l e n e f o l l o w s t h e i n i t i a l a-C-H bond cleavage22. Examples of N a t u r a l l y Occurring "k-_+" I n h i b i t o r s
-
Two n a t u r a l l y occur-
r i n g B,y-unsaturated amino a c i d t o x i n s have been r e p o r t e d i n t h e l i t e r a t u r e which are of i n t e r e s t h e r e . R h i z o b i t o x i n e (2-amino-4-(2-amino-3hydroxypropoxy)-trans-but-3-enoic a c i d ) ( F i g . 1 0 a ) i s an i r r e v e r s i b l e i n h i b i t o r of B-cystathionase23, 24 from b a c t e r i a and p l a n t s . The compound shows s t r o n g a n t i m e t a b o l i c a c t i v i t y , a s e x p e c t e d , and resembles t h e n a t u r a l s u b s t r a t e f o r t h e enzyme, c y s t a t h i o n i n e . (Fig. l o b )
H-CH2-O-CH--QI 2
a
H C0,H
%2
H0,C-g Fig. 10
-CH2-S-CH2-CH b
2
2
T h i s p y r i d o x a l - l i n k e d enzyme degrades c y s t a t h i o n i n e by t h e mechanism shown i n F i g . l l a . We propose t h a t r h i z o b i t o x i n e i s an i r r e v e r s i b l e i n h i b i t o r of t h i s enzyme by v i r t u e of t h e f a c t t h a t i t can be e n z y m a t i c a l l y converted i n t o t h e r e a c t i v e i n t e r m e d i a t e shown i n F i g . l l b .
Chap. 24
Mechanism-Based Irreversible Enzyme Inhibitors
Rando
239 -
In this instance a chemical reaction ensues between the rizobitoxine and the pyridoxal phosphate cofactor. The second compound of this type is L-2-amino-4-methoxy-trans-3-butenoic acid (AMB)25. (Fig. 12)
Fig. 1 2 This compound has been shown to exhibit strong antimetabolic activity against bacteria. We have found that this compound is an irreversible inhibitor of the pyridoxal phosphate-linked aspartate amino transferase26. A mechanism similar to the ones described above can account €or this. (Fig. 13)
+
Irreversible
______,
AH3+
Inactivation
Fig. 13
H
The antibiotics D-cycloserine and 0-carbamyl D-serine are irreversible inhibitors of certain pyridoxal phosphate containing enzymes27 ,28,29. Their utility as inhibitors of cell wall biosynthesis in bacteria is prob-
240
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a b l y a r e f l e c t i o n of t h e f a c t t h a t b o t h are i r r e v e r s i b l e i n h i b i t o r s of a l a n i n e racemase, an enzyme c r u c i a l i n c e l l w a l l b i o s y n t h e ~ i s 3 ~ .The are mechanisms of a c t i o n of D-cycloserine and 0-carbamyl-D-serine summarized i n F i g . 1 4 .
fFB 2
1
L
c-
:Fnz-B :
Chap. 24
Mechanism-Based
I r r e v e r s i b l e Enzyme I n h i b i t o r s
B
__ 241
Rando
BH+
2 4
Carbamyl S e r i n e Fig. 1 4 I n t h e case of c y c l o s e r i n e t h e h i g h l y r e a c t i v e a c y l a t i n g a g e n t 1 i s e n z y m a t i c a l l y formed whereas i n t h e c a s e of carbamyl s e r i n e t h e h i g h l y rea c t i v e Michael a c c e p t o r 2 i s formed. The l a t t e r i n t e r m e d i a t e i s , of c o u r s e , p r e c i s e l y t h e same as t h e a c t i v e i n h i b i t o r of t h r e o n i n e deh y d r a t a s e w i t h s e r i n e as s u b s t r a t e . The d i a z o c a r b o n y l c o n t a i n i n g a n t i b i o t i c s , a z a s e r i n e , 6-diazo-5-0x0L - n o r l e v c i n e (DON), and 5-diazo-4-0x0-L-norvaline (DONV) a l s o f u n c t i o n by a kcat mechanism31. (Fig. 1 5 )
)&CH2xd; 22
Azas e r i n e
3
H C‘ ( ‘
2
1CH DON
*-&: 3
-
y L22 $ c o 2 3 +
Fig. 15
DONV
These molecules f u n c t i o n as i r r e v e r s i b l e enzyme i n h i b i t o r s , w i t h t h e moiety b e i n g c r u c i a l t o t h e i r mechanisms of a c t i o n . I n g e n e r a l , t h e f u n c t i o n i n v i v o by i r r e v e r s i b l y i n h i b i t i n g c e r t a i n enzymes involved t h e b i o s y n t h e s i s of p u r i n e s and p y r i m i d i n e s and hence t h e d r u g s have used a s a n t i-cancer a g e n t s31.
diazo drugs in been
S i n c e t h e d i a z o g r o u p i n g , as s u c h , i s i n e r t towards n u c l e o p h i l i c a t t a c k i t must f i r s t b e p r o t o n a t e d by t h e t a r g e t enzyme t o g e n e r a t e t h e (Fig. 16) h i g h l y r e a c t i v e diazonium ion32.
Wechter, Ed.
Topics i n Biology
242 -
H30+ +-ORH 14
20
HO-CH2 J - O R
2 -
Fig. 16
+ N2
+
H30+
Once g e n e r a t e d t h i s i o n r e a c t s w i t h an a c t i v e - s i t e n u c l e o p h i l e a t an e s s e n t i a l l y d i f f u s s i o n c o n t r o l l e d rate. For example, a z a s e r i n e which i s an i r r e v e r s i b l e i n h i b i t o r of t h e enzyme t h a t c o n v e r t s formylglycinamide r i b o t i d e (FGAR) t o formyl g l y c i n a m i d i n e r i b o t i d e (FGAM) , i s p r o t o n a t e d by t h i s enzyme p r i o r t o a l k y l a t i n g i t % & . T h i s p r o t o n a t i o n is r e q u i r e d t o a c t i v a t e t h e d i a z o compound t o t h e h i g h l y r e a c t i v e diazonium i o n . (2 F i g . 1 7 )
Fig. 1 7 Once a c t i v a t e d , t h e diazonium compound undergoes an a l k y l a t i o n r e a c t i o n w i t h an a c t i v e - s i t e c y s t e i n e 3 3 ~ 3 4 . Conclusion - T h i s a r t i c l e h a s demonstrated t h a t i r r e v e r s i b l e enzyme inh i b i t o r s which r e q u i r e a c t i v a t i o n by t h e t a r g e t enzyme are of a g e n e r a l n a t u r e . I n h i b i t o r s of t h i s t y p e have been found amongst b o t h s y n t h e t i c and n a t u r a l l y o c c u r r i n g molecules. They are e x c e e d i n g l y s p e c i f i c as a consequence of t h e i r r e q u i r i n g chemical conversion t o t h e a c t i v e form by t h e t a r g e t enzyme. Thus, unwanted r e a c t i o n s w i t h f o r e i g n biomolecules a r e circumvented. Because of t h e i r g r e a t s p e c i f i c i t y i n h i b i t o r s of t h i s t y p e ought b e a u s e f u l s t a r t i n g p o i n t i n t h e r a t i o n a l d e s i g n of d r u g s . References 1. 2. 3.
4. 5. 6. 7. 8.
R.R. Rando, S c i e n c e ( i n p r e s s ) K. Bloch, Accts. Chem. Res., 2, 1 9 3 (1969). K. Endo, G.M. Helmkamp, K. Bloch, J. B i o l . Chem., 254, 4293 (1970). L.R. Kass and K. Bloch, Proc. Nat. Acad. S c i . USA, 58, 1168 (1967); L.R. Kass, J. B i o l . Chem., 243, 3223 (1968). L . Hellerman and V.G. Erwin, J . B i o l . Chem., 243, 5234 (1968). R.F. S q u i r e s i n Adv. i n Biochem. Psychopharm., 5, 356 (1972) ed. by E. Costa R.R. Rando, J . Amer. Chem. S O C . , 95, 4438 (1973).
Chap. 24 9.
Mechanism-Based I r r e v e r s i b l e Enzyme I n h i b i t o r s
Rando
243
G.A. Hamilton i n P r o g r e s s i n B i o o r g a n i c Chemistry", Vol I. e d . b y E.T. Kaiser and F.J. Kezdy, W i l e y - I n t e r s c i e n c e , New York, 1971, pp. 106-107. 10. C.T. Walsh, A. Schonbrunn and R.H. A b e l e s , J. B i o l . Chem., 246, 6855 (1971). 11. C.T. Walsh, R.H. Abeles and H.R. Kaback, J. B i o l . Chem., 247, 7858 (1972). 1 2 . R.R. Rando and J. D e M a i r e n a , Biochem. Pharmacol., 23, 463 (1974). 13. H. Yamada and K.T. Yasunobu, J. B i o l . Chem., 237, 3077 (1962). 1 4 . K. Watanabe, R.A. Smith, M. Inamasu, and K.T. Yasunobu i n Adv. Biochem. Psychopharm., 5, 107 ( 1 9 7 2 ) , e d . by E. C o s t a and M . S a n d l e r , Raven P r e s s , New York, 1972. 1 5 . J.E. Hucko-Hass and D . J . Reed, Biochem. Biophys. R e s . Corn., 39, 396 (1970). 1 6 . W.A. Wood and I . C . G u n s a l u s , J . B i o l . Chem., 181,1 7 1 (1949). 1 7 . A.T. P h i l l i p s , Biochem. Biophys. Acta, 151,526 (1968). 18. P. F a s e l l a and R. J o h n , Proc. I n t . Cong. Pharm. 5, 1 8 4 ( 1 9 6 9 ) , e d . by R. Ergenmann. 19. Y . Morino and M. Okamoto, Biochem. Biophys. R e s . Comm., 50, 1061 (1973). 20. E.W. Miles and A. Meister, B i o c h e m i s t r y , 5, 1735 (1967). 2 1 . R.R. Rando ( u n p u b l i s h e d e x p e r i m e n t s ) 22. R.H. Abeles and C.T. Walsh, J. h e r . Chem. SOC., 95, 6124 (1973). 23. I. G i o v a n e l l i , L.D. Owens and S. Harvey Mudd, Biochim. Biophys. A c t a , 227, 6 7 1 (1971). 24. L.D. Owens, J.F. Thompson, R.G. P i t c h e r and T. W i l l i a m s , Chem. Corn., 714 (1972). 25. J . P . S c a n n e l l , D.L. P r e u s s , T.L. Demny, L.H. S e l l o , T . Williams and A . S t e m p e l , J. A n t i b i o t i c s , 25, 1 2 2 (1972). 26. R.R. Rando, N a t u r e ( i n p r e s s ) 27. F.C. Neuhaus, Antimicrob. Agents. Chemother., 304-314 (1968). 28. J.L. Lynch and F.C. Neuhaus, J. Bact., 91, 449-460 (1966). 29. E.F. Gale, E. C u n d l i f f e , P.E. Reynolds, M.B. Richmond and M.J. Waring, The M o l e c u l a r Basis of A n t i b i o t i c A c t i o n , p. 6 5 , J. Wiley and Sons, New York, 1972. 30. F.C. Neuhaus, i n A n t i b i o t i c s - Mechanism o f A c t i o n , Vol 1, ed. by G o t t l i e b , D . , and Shaw, P.D., p. 40-83, S p r i n g e r - V e r l a y I n c . , New York, 1967. 31. R.B. L i v i n g s t o n , J . M . V e n d i t t i , D.A. Cooney and S.K. Carter i n Adv. Pharm. and Chemotherapy, 8, 57-113 (1970). 32. H. Z o l l i n g e r , Azo and Diazo Chemistry, Chap. 6 , I n t e r s c i e n c e P u b l i s h e r s , I n c . , New York, 1961. 33. T.C. French, I . B . Dawid, R.A. Day and J . M . Buchanan, J . B i o l . Chem. 238, 2171-2177 (1963) 34. I . B . Dawid, T.C. French and J . M . Buchanan, J . B i o l Chem., 238, 2186-2193 (1963).
Chapter 25.
Adjuvants t o t h e Immune System
Arthur G. Johnson, U n i v e r s i t y of Michigan Medical School, Ann Arbor
-The Immune
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System Immune a d j u v a n t s (L., F r . , a d j u v a r e , t o a s s i s t ) a r e d e f i n e d a s s u b s t a n c e s o r procedures which a i d i n t h e development o r manif e s t a t i o n s of t h e immune system. The p r o t e c t i o n c o n f e r r e d by t h i s system a g a i n s t material f o r e i g n t o t h e h o s t ( a n t i g e n s ) ranges from microorganisms t o tumors, and i t s complexity i s now beginning t o be a p p r e c i a t e d .
Two major limbs of t h e immune response, termed humoral and c e l l med i a t e d , a r e d e f i n e a b l e w i t h p r e s e n t day knowledge'. E i t h e r can b e enhanced by a d j u v a n t s . The two limbs are d i f f e r e n t i a t e d p r i m a r i l y on t h e b a s i s of humoral immunity (HI) being t r a n s f e r r a b l e w i t h serum o r plasma from a n immune donor t o a non-immune r e c i p i e n t , w i t h t h e a c t i v e f a c t o r ( s ) being one o r s e v e r a l of t h e f i v e d e f i n e d immunoglobulin molecules. On t h e o t h e r hand, c e l l mediated immunity (CMI), f u n c t i o n a l p r i m a r i l y a g a i n s t g r a f t s , tumors and i n t r a c e l l u l a r dwelling microorganisms, is p o s t u l a t e d t o occur v i a s o l u b l e mediators, termed lymphokines, s e c r e t e d over a s h o r t range by thymus i n f l u e n c e d lymphocytes. The l a t t e r a r e found mainly i n p e r i p h e r a l blood l e u c o c y t e , spleen, lymph node, o r g u t a s s o c i a t e d populat i o n s . Contrarywise, i n each of t h e s e two limbs s p e c i f i c unresponsiveness o r t o l e r a n c e t o a n t i g e n s , such as w e e x h i b i t t o our own s e l f molecules (and would l i k e t o a c h i e v e w i t h r e s p e c t t o a n t i g e n s on organ t r a n s p l a n t s ) can be induced experimentally. These i n h i b i t o r y procedures a l s o can be r e v e r s e d by several a d j u v a n t s 2 , 3 , providing a model whereby t h e b a s i c biol o g i c p r i n c i p l e s involved i n r e c o g n i t i o n of s e l f o r non-self by t h e immune system, might be uncovered. I n a d d i t i o n , a b n o r m a l i t i e s i n b o t h limbs a r e being recognized i n t h e human b e i n g , and p r o v i d e t h e e t i o l o g i c b a s i s f o r a number of important c l i n i c a l c o n d i t i o n s 4 . Recently, t h e development and m a n i f e s t a t i o n s of both humoral and c e l l mediated immunity have been shown t o r e s u l t from an as y e t undefined i n t e r a c t i o n amongst s e v e r a l d i f f e r e n t c e l l types.596. I m p l i c a t e d a r e a c e l l i n t h e monocyte-macrophage series (MB), which i s thought t o p r e p a r e o r c o n c e n t r a t e t h e f o r e i g n a n t i g e n ; a thymus i n f l u e n c e d lymphocyte (T c e l l ) , which i s n e c e s s a r y f o r antibody s y n t h e s i s but does n o t i t s e l f produce a n t i b o d y , a c c o r d i n g l y termed a "helper" c e l l ; and a bone marrow der i v e d lymphocyte (B c e l l ) which has been shown i n chickens t o be under t h e c o n t r o l of a c e n t r a l lymphoid organ, t h e b u r s a of F a b r i c i u s . The mammal i a n e q u i v a l e n t of t h e l a t t e r i s as y e t unknown. Following union of a n t i gen w i t h s p e c i f i c immunoglobulin r e c e p t o r s on t h e s u r f a c e membrane, t h e B c e l l t r a n s f o r m s i n t o a l a r g e b l a s t c e l l and s y n t h e s i z e s a n t i b o d y d u r i n g m a t u r a t i o n i n t o t h e plasma c e l l series. D i f f e r e n t i a t i o n and d i v i s i o n a r e c h a r a c t e r i s t i c of t h e response of T and B cells and a r e t h e f o c u s of i n t e r f e r e n c e by immunosuppressive drugs i n t r a n s p l a n t a t i o n . Two t y p e s of a n t i g e n s have been recognized, thymus dependent (TD) a n t i g e n s which req u i r e t h e h e l p e r e f f e c t of t h e T c e l l , and thymus independent (TI) a n t i gens, which s t i m u l a t e t h e B c e l l d i r e c t l y . A proposed scheme f o r t h e immune system i s diagrammed as f o l l o w s :
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Although n o t d e p i c t e d i n t h e scheme, t h e whole complex p r o c e s s of inflammation i n many i n s t a n c e s i s i n t e g r a t e d i n t o t h e immune system, i n a s much as t h e union of a n t i g e n s w i t h several a n t i b o d i e s a d s o r b s , and t h e r e b y a c t i v a t e s a p r o e s t e r a s e , t h e f i r s t of a series of n i n e s u b s t a n c e s known as t h e complement system7. A s e q u e n t i a l c h a i n of e v e n t s i s set i n motion, r e s u l t i n g i n e n z y m a t i c a l l y cleaved s p l i t p r o d u c t s which a c t i n one s e n s e as a d j u v a n t s by being s e l e c t i v e l y chemotactic f o r polymorphonuclear leucoc y t e s , monocytes, lymphocytes o r e o s i n o p h i l s . By t h i s means, d e f e n s i v e c e l l s are mobilized i n t h e a r e a of a n t i g e n (microorganism) d e p o s i t i o n , each b r i n g i n g i n t o t h e a r e n a i t s own complex a r r a y of p h y s i o l o g i c a l mediator s u b s t a n c e s .
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The v e r y complexity of t h e immune system i n t r o d u c e s t h e p o s s i Adjuvants b i l i t y of m u l t i p l e ways and t a r g e t s by which i t s a c t i v i t i e s might be a s s i s t e d . The f a c t t h a t i t can be enhanced r e a d i l y i n d i c a t e s t h a t under normal c o n d i t i o n s immunity o p e r a t e s s u b o p t i m a l l y . Areas of immediate conc e r n a r e t h e p o t e n t i a l f o r e l e v a t i n g t h e weak immunity engendered by tumor a n t i g e n s and t h e u t i l i z a t i o n of lesser amounts of v i r u s e s d i f f i c u l t t o i s o l a t e i n q u a n t i t i e s s u f f i c i e n t f o r mass v a c c i n a t i o n . P r i o r t o t h e rec e n t e x p l o s i o n of knowledge i n Immunology, t h r e e major c l a s s e s of p r a c t i c a l a d j u v a n t s were i n use: (a) w a t e r - i n - o i l emulsions, (b) aluminum s a l t c a r r i e r s , and ( c ) mixed v a c c i n e s c o n t a i n i n g G r a m n e g a t i v e organisms. Det a i l e d information a v a i l a b l e t o 1966 on t h e a c t i o n of each of t h e s e has been summarized i n one of t h e few symposia h e l d on t h i s s u b j e c t 8 . Water i n ---
O i l Emulsions
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Freund has documented i n d e t a i l t h e marked
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Topics i n Biology
Wechter, Ed.
i n c r e a s e i n H I and C M I o c c u r r i n g when a n t i g e n s a r e i n c o r p o r a t e d i n t o wat e r - i n - o i l emulsionsg. Complete and incomplete Freund Adjuvants have been d e s i g n a t e d , w i t h t h e former c o n t a i n i n g any of s e v e r a l s p e c i e s of k i l l e d Mycobacteria i n t h e o i l y phase. Antigen i s suspended i n water o r s a l i n e , and t h e d r o p l e t s d i s p e r s e d uniformly throughout t h e o i l phase by e m u l s i f i c a t i o n . The r e s u l t i n g a n t i b o d y r e s p o n s e a f t e r one o r two i n j e c t i o n s i n t r a m u s c u l a r l y o r subcutaneously, i s e l e v a t e d and h i g h l e v e l s p e r s i s t o v e r many months. The mode of a c t i o n of w a t e r - i n - o i l emulsions a p p e a r s t o i n v o l v e t h e r e t a r d a t i o n of d e s t r u c t i o n and e l i m i n a t i o n of t h e a n t i g e n r e s u l t i n g i n a slow, prolonged s t i m u l u s l O . T h i s i s supported by t h e f i n d i n g t h a t minute amounts of a n t i g e n a l o n e g i v e n d a i l y f o r 350 da s, mimic t h e h i g h and prolonged a n t i b o d y t i t e r s s e e n w i t h t h i s a d j u v a n t The a d d i t i o n of t u b e r c l e b a c i l l i i n t o t h e o i l y phase of t h e m i x t u r e (Freund's complete a d j u v a n t ) p o t e n t i a t e s f u r t h e r t h e s e e f f e c t s a s w e l l a s C M I . Res p o n s i b l e f o r t h e p o t e n t i a t i o n e x e r t e d by t h e k i l l e d Mycobacteria i s a wax f r a c t i o n , termed Wax D , which has been i s o l a t e d and shown t o c o n t a i n a peptido-glycan l i n k e d t o a mycolate of a n a r a b i n o - g a l a c t a n . White, e t a l . have documented a s t r i k i n g a c t i o n of t h e wax i n s t i m u l a t i n g macrophage p r o l i f e r a t i o n a t t h e s i t e of i n j e c t i o n 1 2 . The p a r t i c u l a t e d r o p l e t s are phagocytized by wandering polyrnorphonuclear l e u c o c y t e s and macrophages and a r e t r a n s p o r t e d t o o t h e r immunocompetent lymphoid t i s s u e s of t h e body remote from t h o s e d r a i n i n g t h e s i t e of i n j e c t i o n . U n f o r t u n a t e l y , a b s c e s s f o r m a t i o n is f r e q u e n t l y a s s o c i a t e d (up t o 75% i n one s t u d y ) w i t h t h i s t y p e of a d j u v a n t , r e s t r i c t i n g s e v e r e l y i t s usage i n man13. The t o x i c i t y of F r e u n d ' s a d j u v a n t may r e s i d e i n p a r t w i t h f r e e f a t t y a c i d s and d e g r a d a t i o n p r o d u c t s of t h e e m u l s i f i e r 1 4 . I n a d d i t i o n , delayed h y p e r s e n s i t i v i t y , a s w e l l as auto-immune c o n d i t i o n s may be induced r e a d i l y i n e x p e r i m e n t a l a n i m a l s by t h i s a d j u v a n t 1 5 . S u b s t i t u t i o n of v e g e t a b l e o r o t h e r more metabol i z a b l e o i l s may circumvent t h i s c o n t r a i n d i c a t i o n . Their e f f i c a c y with i n f l u e n z a v i r u s h a s a l r e a d y been demonstrated16.
11.
R e c e n t l y , a non-toxic water s o l u b l e a d j u v a n t f r a c t i o n (WSA) w i t h a n approximate m o l e c u l a r weight of 20,000 h a s been i s o l a t e d f o l l o w i n g l y s o zyme, t r y p s i n , and chymotrypsin t r e a t m e n t of d e l i p i d a t e d c e l l w a l l s of Mycobacteria s m e g a t i s . S i m i l a r f i n d i n g s w e r e made w i t h t h e a t t e n u a t e d form of M. b o v i s , B a c i l l u s Calmette Guerin (BCG)I7. T h i s material had a s t r o n g e r a d j u v a n t a c t i v i t y t h a n e q u a l amounts of whole b a c t e r i a , c e l l w a l l s , o r t h e Wax D f r a c t i o n when added t o a w a t e r - i n - o i l emulsion18. Ant i b o d y t i t e r s t o PR 8 i n f l u e n z a v i r u s , Columbia SK v i r u s and p u r i f i e d egg albumin were e l e v a t e d . Furthermore, i n t i a l s t u d i e s showed t h i s product t o b e non-toxic, non-pyrogenic and u n a b l e t o s e n s i t i z e animals t o tubercul i n o r produce a l l e r g i c a r t h r i t i s i n r a t s , a l l of which were f a c e t s of t h e r e a c t i o n t o t h e i n t a c t Mycobacteria. P r e l i m i n a r y evidence i n d i c a t e s WSA i s e f f e c t i v e i n s a l i n e , independent of w a t e r - i n - o i l e m u l s i f i c a t i o n , i n c r e a s i n g i t s p o t e n t i a l f o r human u s a g e l g . Adsorption onto Aluminum S a l t s - The powerful a d s o r p t i v e q u a l i t i e s of t h e aluminum i o n have been u t i l i z e d w i t h a n t i g e n s as a second p h y s i c a l proced u r e which enhances t h e immune system2(). Antigens are adsorbed f i r m l y d u r i n g o r a f t e r t h e f o r m a t i o n of aluminum hydroxide g e l f o l l o w i n g treatment of aluminum s a l t s w i t h a l k a l i . A s w i t h w a t e r - i n - o i l emulsions,
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r e t e n t i o n and wider d i s s e m i n a t i o n of a n t i g e n appear t o b e i m p o r t a n t funct i o n a l a s p e c t s of t h i s a d j u v a n t when d e p o s i t e d s u b c u t a n e o u s l y o r i n t r a m u s c u l a r l y . However, i t i s n o t as e f f e c t i v e as Freund's a d j u v a n t i n t h i s r e g a r d . White, e t a l . have demonstrated a n t i b o d y p r o d u c t i o n i n t h e l o c a l granuloma developed a t t h e s i t e of i n j e c t i o n , as w e l l as w i t h i n t h e reg i o n a l lymphatic g1ands2l. T o x i c i t y and s i d e r e a c t i o n s of aluminum hyd r o x i d e o r phosphate g e l s a r e low, and t h e s e m i n e r a l s a l t carriers have proven u s e f u l w i t h d i p h t h e r i a and t e t a n u s t o x o i d s i n p e d i a t r i c human and animal v a c c i n a t i o n 2 2 . Combined Vaccines - Combined v a c c i n e s , employing k i l l e d Salmonella typhosa (typhoid f e v e r ) o r Hemophilus p e r t u s s i s (whooping cough) a s one of t h e components t o g e t h e r w i t h d i p h t h e r i a and t e t a n u s t o x o i d s , g i v e r i s e t o h i g h e r a n t i t o x i n t i t e r s t h a n when f l u i d t o x o i d s are g i v e n s i n g l y 2 3 . The a c t i v e a d j u v a n t h a s been shown t o b e a l i p o p o l y s a c c h a r i d e component of t h e b a c t e r i a l c e l l w a l l , termed LPS o r e n d ~ t o x i n ~ It ~ . is a p o l y - d i s p e r s e molecule w i t h an a v e r a g e molecular weight g e n e r a l l y over a m i l l i o n . Its a c t i o n i s d r a m a t i c , 1-5 ug s h o r t e n i n g t h e i n d u c t i o n p e r i o d and e l e v a t i n g peak a n t i b o d y t i t e r s , b u t i t s t o x i c i t y i s w e l l known, w i t h p y r o g e n i c i t y , r e n a l damage and shock among i t s c h a r a c t e r i s t i c s 2 5 . I n c o n t r a s t t o Freund's a d j u v a n t o r aluminum c a r r i e r s , l e n g t h y p r o l o n g a t i o n of a n t i b o d y t i t e r s i s n o t a f e a t u r e of t h i s a d j u v a n t . A s t r o n g s t i m u l u s of memory c e l l s d o e s o c c u r , however, and on re-exposure t o a n t i g e n a l o n e , r a p i d and e l e v a t e d r e s p o n s e s are observed.
- Following d e f i n i t i o n of macrophage-T-B c e l l i n t e r a c t i o n s , several s u b s t a n c e s were d e s c r i b e d which s t i m u l a t e d s e l e c t i v e l y e i t h e r T o r B c e l l s t o t r a n s f o r m i n t o l a r g e b l a s t c e l l s and d i v i d e 2 6 . The l i p i d moiety of t h e LPS d i s c u s s e d above, termed L i p i d A, c o n s i s t i n g of phosphor y l a t e d glucosamine d i s s a c h a r i d e u n i t s w i t h ester and amide l i n k e d f a t t y a c i d s , when p r o p e r l y d i s p e r s e d was shown t o b e one of t h e most powerful mitogens f o r a n t i b o d y s y n t h e s i z i n g B lymphocytes i n v i t r o 2 7 , 28, Associa t e d w i t h t h i s p r o p e r t y was t h e s e c r e t i o n by t h e B c e l l s of p o l y c l o n a l a n t i b o d i e s d i r e c t l y w i t h o u t t h e i n j e c t i o n of a n t i g e n s . No m i t o g e n i c e f f e c t w a s e x e r t e d on T c e l l s , a l t h o u g h t h e h e l p e r e f f e c t of T c e l l s on B c e l l s was n e c e s s a r y f o r LPS t r e a t e d a n i a l s t o e x h i b i t t h e i r more profound I n s e r t i o n of t h e l i p i d A i n t o a d j u v a n t a c t i o n on TD a n t i g e n s i n v i v o t h e l i p i d b i l a y e r of t h e B c e l l membrane h a s been p o s t u l a t e d t o t r i g g e r these r e s p o n s e s , and t h e a d j u v a n t p r o p e r t y of l i p i d A i s l o s t f o l l o w i n g removal by a l k a l i t r e a t m e n t of t h e ester l i n k e d f a t t y acids3O. Covalent l i n k a g e of several TD a n t i g e n s t o t h e LPS e l i m i n a t e d t h e i r thymus dependency, and when a t t a c h e d t o t h i s mitogen t h e y became c a p a b l e of a c t i v a t i n g t h e B c e l l d i r e c t 1 t o s y n t h e s i z e s p e c i f i c a n t i b o d y , independent of t h e T It i s surmised t h e r e f o r e , t h a t t h e normal f u n c t i o n cell helper e f f e c t of t h e T c e l l t h a t i s r e p l a c e d by t h e m i t o g e n i c a c t i o n of t h e LPS i s i t s e l f a m i t o g e n i c p r i n c i p l e , and t h a t t h e p r o p e r t y of m i t o g e n i c i t y f o r B c e l l s may be a mandatory c h a r a c t e r i s t i c of thymus independent a n t i g e n s . Thus, 7 d i f f e r e n t T I a n t i g e n s ( l i p o p o l y s a c c h a r i d e s from G r a m n e g a t i v e b a c t e r i a , pneumococcal p o l y s a c c h a r i d e s , b a c t e r i a l f l a g e l l i n , l e v a n , polyV i n y l p y r r o l i d o n e , d e x t r a n , d e x t r a n s u l f a t e ) were found t o s t i m u l a t e t h e u p t a k e of DNA s y n t h e s i s by B lymphocytes and i n d u c e p o l y c l o n a l a n t i b o d y
-B C e l l Mitogens
28 .
31.
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synthesis to several unrelated antigens32. As a result of these findings, it has been postulated that all TI antigens possess 2 different reactive sites on the same molecule. One is an antigenic determinant group which focuses the molecule preferentially on the specific clone of antibody forming B cells reacting only to that antigen via the cell's immunoglobulin membrane receptor, and the second is the mitogen (adjuvant) which gives the actual signal to the cell to divide and synthesize antibody33. Although binding of TD antigens or haptens directly to B cells does occur via the specific Ig receptor, this binding event alone is incapable of triggering the cell to make antibody until the signal from the mitogen is received. For these thymus dependent antigens, the mitogenic signal can come from the T cell, released in some as yet unknown fashion by specific antigen or by non-specific T cell adjuvants as discussed below, o r by concomitant mitogenic stimulation of B cells with TI antigens such as LPS.
- Two plant substances selectively mitogenic over a narrow dose range for T cells are (a) phytohemagglutinin (PHA), isolated from the bean Phaseolus vulgaris, having an affinity for N-acetyl-D-galactosamine34, and (b) Concanavalin A , obtained from the jack bean, Canavalia ensiformis, which binds to a-D-mannopyranosyl, c1 and 6-D-glucopyranosyl and 8-0fructofuranosyl groups35. Responsiveness to these mitogens is a selective marker for T ce s although subpopulations within the T cell family are becoming evident". When added in doses of less than 1 ug with antigen to spleen cell cultures containing T cells, Con A is weakly stimulatory of antibody synthesis. In high concentrations, it is inhibitory-37. On the other hand, the helper effect of T cells is increased strongly following incubation of T cells for 24 hr with Con A. The mechanism of activation is not known, but may be related to a Con A induced release of the mitogenic factor endogenous to T cells. The appearance of a mitogenic factor in the supernatant fluids of cultures of T cells has been described under a number of conditions besides the addition of antigen to sensitized T cells5. Thus, mixtures of immunocompetent lymphoid cells from genetically different individuals, termed allogeneic effect, or mixed lymphocyte reaction, releases a mitogenic factor which acts non-specifically on B cells to stimulate them to antibody synthesis. This factor appears to substitute for the T cell helper effect required by TD antigens and permits direct triggering of B cells by antigens which were previously thymus dependent. Thus, adjuvants exerting an effect on the T cell may function by stimulating the secretion of the same mitogenic factor which acts nonspecifically on B cells signalling them to initiate antibody synthesis. -T Cell Mitogens
Nucleic Acids - The use of nucleic acids as adjuvants to the imune response was first indicated in the demonstration cf a restorative capacity of nucleic acid-rich tissues in animals rendered immunodeficient following x-irradiation38. Their ability to act a s adjuvants to the normal im'une response to unrelated antigens has also been documented and shown to be attributable to low molecular weight oligonucleotides39. Extended study over the past 5 years has shown the more well-defined polynucleotide complexes (poly A:U and poly 1:C) to be powerful adjuvants to many antigens in several animal species4'. Each of the polynucleotides alone were ineffective and complexing was required. Quantities of antigen as low as 1
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nanogram were r e n d e r e d immunogenic by p o l y A:U4I. The p o l y n u c l e o t i d e act i o n w a s e x e r t e d on s e v e r a l c e l l t y p e s ( e . g . , macrophage, memory c e l l and i n p a r t i c u l a r , as a r e s t o r a t i v e a g e n t i n a n i m a l s rendered d e f i c i e n t i n T ~ e l l s ) 4 ~ T. c e l l c y t o t o x i c i t y f o r tumor c e l l s w a s a l s o enhanced43, as w a s p r o t e c t i o n of mice a g a i n s t r a b i e s v i r u s c h a l l e n g e 4 4 . Although amplif i c a t i o n of T c e l l a c t i v i t y i s one way i n which p o l y A:U and o t h e r a d j u v a n t s appear t o a c t i v a t e t h e immune system45, i t remains t o b e d e l i n e a t e d whether t h i s o c c u r s by a mitogenic a c t i v i t y i n c r e a s i n g T c e l l numbers o r an enhancement of i n d i v i d u a l T c e l l e f f e c t o r a c t i o n . Conversion of normal macrophages i n t o c y t o t o x i c c e l l s c a p a b l e of k i l l i n g c u l t u r e d lymphoma c e l l s h a s a l s o been demonstrated t o b e a p r o p e r t y of t h e p o l y n u c l e o t i d e s as w e l l as L P S ~ ~ . A r o l e f o r n u c l e i c a c i d s as a d j u v a n t s i s a l s o i n d i c a t e d by many s t u d i e s documenting a d r a m a t i c i n c r e a s e i n b o t h limbs of t h e immune response by n u c l e i c a c i d - a n t i g e n complexes i s o l a t e d from macrophages6 , 47. I n t h e s e i n s t a n c e s , a n t i g e n was exposed i n v i t r o t o t h i o g l y c o l l a t e induced p e r i t o n e a l macrophages from normal a n i m a l s . Following i n c u b a t i o n and washing, RNA was e x t r a c t e d and i n j e c t e d i n t o normal animals p r e v i o u s l y unexposed t o t h e a n t i g e n . Remarkably h i g h t i t e r s of s p e c i f i c a n t i b o d y have been induced t o many a n t i g e n s u s i n g t h i s t e c h n i q u e . The n a t u r e of t h e unique RNA i s under i n v e s t i g a t i o n . I t s e f f e c t i v e n e s s a s a n a d j u v a n t has p o t e n t i a l f o r t h e weakly f o r e i g n tumor a n t i g e n s o r a n t i g e n s such as v i r a l components which are d i f f i c u l t t o i s o l a t e i n l a r g e q u a n t i t i e s .
The s t r i k i n g potency of ribosomal v a c c i n e s p r o t e c t i v e a g a i n s t t h e b a c t e r i a from which t h e y have been i s o l a t e d u n d e r s c o r e s w i t h a d i f f e r e n t system t h e a d j u v a n t a c t i o n of n u c l e i c a c i d s . Thus, i n 1965, Youmans and Youmans demonstrated t h a t ribosome-rich material i s o l a t e d from Mycobact e r i u m t u b e r c u l o s i s p r o t e c t e d mice a g a i n s t t h i s organism as e f f i c i e n t l y as t h e whole organism vaccines48. The e f f e c t i v e material was h e a t l a b i l e , r i b o n u c l e a s e s e n s i t i v e and had t h e c h a r a c t e r i s t i c s of double-stranded RNA49. I n a d d i t i o n , t h e ribosomal v a c c i n e a c t e d a s an a d j u v a n t t o unrel a t e d a n t i g e n s , as w e l l as i n t h e e x p e r i m e n t a l i n d u c t i o n of auto-immune d i s e a s e 5 0 . These f i n d i n g s have been extended t o o t h e r o r anisms such a s Salmonella t y p h i m ~ r i u m ~P~n ,e u ~ o c o c c u s 5 2 ,Staphylococcus53, and Pseudomonas54. A p l a u s i b l e e x p l a n a t i o n f o r t h e e f f i c a c y of such p r e p a r a t i o n s l i e s i n t h e r e t e n t i o n of minute q u a n t i t i e s of c e l l w a l l a n t i g e n s a t t a c h e d t o t h e RNA, w i t h t h e l a t t e r r e s p o n s i b l e f o r s t i m u l a t i n g t h e immunogenicity of such low amounts of a n t i g e n . Whether i t is due t o a m i t o g e n i c o r a n o t h e r form of s t i m u l u s i s a s y e t a n unanswered q u e s t i o n . I n t h i s c o n t e x t , subp o p u l a t i o n s of T c e l l s have been demonstrated which s u p p r e s s r a t h e r t h a n h e l p immmological responses55, 56. h%ether o r n o t some a d j u v a n t s might f u n c t i o n by removal o f , o r i n t e r f e r e n c e w i t h t h i s s u p p r e s s o r p o p u l a t i o n , i s a n important a s p e c t of t h i s q u e s t i o n .
Non-specific
Tumor Regression Induced by Adjuvants - R e c e n t l y , non-specifi c r e g r e s s i o n of tumors h a s been accomplished by a d j u v a n t procedures i n v o l v i n g t h e inducement of s p e c i f i c c e l l mediated immunity a g a i n s t nontumor a s s o c i a t e d a g e n t s which are h i g h l y e f f e c t i v e a t promoting t h i s limb of t h e immune system57. For example, when human b e i n g s b e a r i n g t u n o r s
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were s e n s i t i z e d w i t h e i t h e r BCG, p u r i f i e d p r o t e i n d e r i v a t i v e of t u b e r c l e b a c i l l i , o r d i n i t r o c h l o r o b e n z e n e , a l l p o t e n t a n t i g e n i c i n d u c e r s of C M I , and s u b s e q u e n t l y provoked i n t o e x p r e s s i o n of c e l l mediated immunity by homologous a n t i g e n c h a l l e n g e , tumor r e g r e s s i o n was observed. T h i s w a s h e i g h t e n e d by d e p o s i t i o n of t h e provoking dose of a n t i g e n d i r e c t l y i n t o t h e tumor, b u t s e n s i t i z a t i o n and c h a l l e n g e a t s i t e s d i s t a n t from t h e tumor a l s o e f f e c t e d r e g r e s s i o n . M e c h a n i s t i c a l l y , t h e c h a l l e n g e d o s e of ant i g e n i s p o s t u l a t e d t o provoke t h e l i b e r a t i o n of lymphokines f o l l o w i n g s p e c i f i c i n t e r a c t i o n w i t h lymphocytes s e n s i t i z e d by t h e f i r s t d o s e of ant i g e n . Once r e l e a s e d , t h e s e e f f e c t o r molecules a c t n o n - s p e c i f i c a l l y res u l t i n g i n t h e d e s t r u c t i o n of t h e tumor t i s s u e a s "innocent b y s t a n d e r . " Conclusion - I n t e l l i g e n t , c o n t r o l l e d i n t e r f e r e n c e w i t h t h e immune r e s p o n s e i s a f o r e s e e a b l e g o a l w i t h i n t h e n e x t few decades. P e r t i n e n t t o t h i s achievement w i l l be chemical d e f i n i t i o n of t h e a d j u v a n t induced molecular s i g n a l s which a m p l i f y M@, T and B c e l l a c t i v i t y . I n t h i s r e g a r d , chemic a l l y d e f i n e d molecular a d j u v a n t s a r e beginning t o be i d e n t i f i e d . Thus, t h e p h e n y l i m i d o t h i a z o l e s a l t , l e v a m i s o l e , h a s been shown r e c e n t l y t o be e f f e c t i v e a g a i n s t a syngeneic mouse sarcoma58, and a i d a n t i - b a c t e r i a l i m m ~ n i t y ~ C~h.a r a c t e r i z a t i o n of c e l l r e c e p t o r s f o r a d j u v a n t s should prove t o be p a r t i c u l a r l y r e v e a l i n g , as should t h e i n t r a c e l l u l a r s i g n a l s a c t i v a t i n g t h e c e l l . The c y c l i c AMP system h a s a l r e a d y been i m p l i c a t e d i n t h e latter60,61. References 1. R.A. Good i n Immunobiology, R.A. Good and D.W. F i s h e r ( e d i t o r s ) , S i n a u e r A s s o c i a t e s , I n c . , Stamford, Conn. (1971) p.3. 2 . Y. Kong and S. Capanna, C e l l . Immunol., 11,488 (1974). 1481 3. J . A . L o u i s , J . M . C h i l l e r and W.O. Weigle, J. Exp. Med., (1973). 4. Freedman, S.O., C l i n i c a l Immunology, Harper & Row, I n c . , N . Y (1971). 5. D.H. Katz and B. Benacerraf i n Adv. Immunol., F.J. Dixon and H.G. Kunkel ( e d i t o r s ) , Academic Press, N . Y . , 15, (1972) p.1. 6. E.R. Unanue i n Adv. Immunol., F.J. Dixon and H.G. Kunkel (ed t o r s ) , Academic Press, N . Y . , 15, (1972) p. 95. 7. H.R. C o l t e n i n Annual R e p o r t s i n Medicinal Chemistry, R.V. Heinzelman ( e d i t o r ) , Academic Press, N . Y . , 7, ( 1 9 7 2 ) - p . 228. 8 . Symp. S e r i e s i n Immunobiol. Stand. 6 . I n t . Symp. on Adjuvants of Immunity, S. Karger, B a s l e , (1967). 9. J . Freund i n Ann. Rev. M i c r o b i o l . , C.E. C l i f t o n ( e d i t o r ) , G . Banta Publ. Co., S t a n f o r d , C a l i f . , 1,p. 291. 10. R.G. White i n Symp. S e r i e s i n Immunobiol. Stand., R.H. Regamey, W. Hennessen, D . I k i c , and J . Ungar ( e d i t o r s ) , S. Karger, Basle, 6 , (1967) p. 3. 11. W. H e r b e r t i n Symp. S e r i e s i n Immunobiol S t a n d . , R.H. Regamey, W. Hennessen, D. I k i c , and J. Ungar ( e d i t o r s ) , S. Karger, Basle, 6, (1967) p. 213. 1 2 . R.G. White, A.H. Coons, and J . M . Connolly, J . Exp. Med., 102, 8 3 (1955).
138,
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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.
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P. Alexander and R. Evans, N a t . New B i o l . , 232, 76 (1971). A.A. G o t t l e i b and R. Schwartz, C e l l . Immunol., 5, 341 (1972). A . S . Youmans and G.P. Youmans, J. B a c t e r i o l , 89, 1291 (1965). A . S . Youmans and G.P. Youmans, J. B a c t e r i o l , 9, 42 (1969). P.Y. P a t e r s o n , J . R e t i c u l o e n d o t h e l i a l SOC., 426 (1973). M.R. Venneman, N . J . B i g l e y and L . J . B e r r y , I n f . Immun., 1,574 (1970). H.C.W. Thompson and I . S . Snyder, I n f . Immun., 3, 1 6 (1971). S.H. Winston and L . J . B e r r y , J . R e t i c u l o e n d o t h e l i a l S O C . , 8, 66 (1970). S.H. Winston and L . J . B e r r y , J. R e t i c u l o e n d o t h e l i a l SOC., 8, 13 (1970). P . J . Baker, P.W. S t a s h a k , D.F. Amsbaugh, B. P r e s c o t t , and R.F. B a r t h , J . Immunol., 105,1 5 8 1 (1970). T-Y. H a , B.H. Waksman, and H.P. T r e f f e r s , J. Exp. Med. , 13 (1974). Conference on Immunology of C a r c i n o g e n e s i s , N a t i o n a l Cancer I n s t . , Monograph 35, (Supt. of Documents, DHEW P u b l i c a t i o n s No.(NIH) 72-334), Dec. 1972. G . Renoux and M. Renoux, N a t . New B i o l . , 240, 217 (1972). G . Renoux and M. Renoux, I n f . & Immun., S, 544 (1973). W. Braun, J . I n f . D i s . , ( S u p p l . ) , 1 8 0 (1973). H.R. Bourne, L.M. L i c h t e n s t e i n , K.L. Melmon, C . S . Henney, Y. W e i n s t e i n , and G.M. S h e a r e r , S c i e n c e , 1 9 (1974).
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Chapter 26. The Cannabinoids: Therapeutic Potentials Robert A. Archer, The Lilly Research Laboratories, Indianapolis, Indiana
This review will survey the literature of the past year with particular reference to those articles which indicate the therapeutic potentials for cannabinoids’. For a comprehensive, up-to-date survey of cannabis chemistry, pharmacology and clinical studies, the reader is referred to a new book’ edited by R. Mechoulam. In regard to marijuana or its active component, AS-THC, the following potential medical applications have been listed3 as areas still t o be explored: analgesia, antihypertensive activity, treatment of migraine, management of dying patients and sexual stimulation. Considering the large number of publications in the marijuana area, few of them examine structure-activity relationships. To date, very little has appeared to show whether the A’-THC molecule can be chemically modified to achieve an enhancement of one particular activity present in A’THC itself. Thus, any therapeutic possibility for the cannabinoids rests mainly on pharmacological and clinical evidence gathered on A’-THC or marijuana itself. New activities were discovered last year. It is still too early to decide which of the following areas represents the best possibility for a useful drug in the cannabinoid area. Anti-Edema, Analgesic, Antipyretic and Anti-Inflammatory Effects - A’-THC inhibits the in v i t r o biosynthesis of staglandin E2 (PGE’) from arachidonic acidfroand the biosynthesis of PGEl from 8,11,14-eicosaH trienic acid’. The inhibition of PGE‘ biosynthesis has been postulated as a mechanism of CH3 . / MH action for such non-steroidal anti-inflammatory agents as aspirin and indomethacin6. Some in CH3 V ~ V Odata’,’ appear to correlate. In the carrageenan-induced rat paw edema, a dose of 10 mg/ A -THC kg AS-THC caused a 40% inhibition of paw swelling. Daily administration of 20 mg/kg A’-THC inhibited the developing adjuvant-induced polyarthritis in rats. In several tests of analgesic activity (acetic acid-induced abdominal constriction, Haffner’s tail pinch, hot plate test and Randall-Selitto test) A’THC showed effectiveness with ED50 of 0.9 to 11.6 mg/kg. At doses up to 20 mg/kg Ag-THC did not affect yeast-induced pyrexis in rats.
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In another study’ of the effects of A’-THC on yeast-induced pyrexis, carrageenan-induced edema and yeast-induced rat paw hyperesthesia, the following results were obtained: (1) The oral antipyretic totency of A’THC is approximately 2 times that of phenylbutazone, (2) A -THC was essentially devoid of antinociceptive activity except at elevated doses which produced a marked catatonic-like state, and (3) A’-THC at doses up to and including 100 mg/kg was totally ineffective in reducing or preventing the edematous response. Anti-Fertility Activity - At a dose of 2 mg in rats, A’-THC suppressed the cyclic surge of luteinizing hormone secretion and also su pressed ovulation (a characteristic shared by other CNS-active drugs)”. However , mating and fertility indices were similar for control and all treatment groups in a study of the effect of Ag-THC on reproduction in rats”. At doses of 2 and 3 mg/kg, A9-THC caused a deterioration in the sexual performance of male rats (interpreted as reflecting a decreased motivation to copdate) 2. Anti-Epileptic, Anticonvulsant Action - The anticonvulsant properties of marijuana and Ay-THC have been recognized for several years. Now cannabidiol and cannabinol have also been found to be effective anticonvulsants using a maximal electroshock test in mice13. (EDSO’S: A’-THC, 80 mg/kg: CBD, 105 mg/kg and CBN, 230 mg/kg).
CH3 Cannabidiol (CBD)
Cannabinol (CBN)
Treatment of freely moving cats with A’-THC (0.25-0.5 mg/kg) temporarily reduced seizure activity induced by electrical stimulation of subOne study concluded that THC closely resembles cortical structures14 acetazolamide rather than diphenylhydantoin’6 .
.
Antihypertensive , Cardiotonic Effects - A previous review” suggested that “the tetrahydrocannabinols warrant evaluation in the treatment of essential hypertension”. A’-THC has since been found to significantly lower the blood pressure of rats and block the appearance of hypertension in immobilized rats”. At 20 mg/kg s.c., the hy-potensive effect persisted over a 96 hr. period and completely counteracted the pressor effects of repeated immobilization stress. In female rats showing adrenal regeneration hypertension, a dose of 3 mg/kg of As-THC was capable of lowering blood pressure” (this moderate dose for a rat does not produce somatic side effects). One study2’ draws attention to the fact that doses of marijuana
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which produce marked psychological alterations in man, do not alter blood pressure in a systematic fashion. The same paper notes that tolerance to the hypotensive effects of A’-THC (5-25 mg/kg/day) develops rapidly in the spontaneously hypertensive rat. The mechanism of these blood pressure and other cardiovascular effects has been the subject for much study. Reduction in blood pressure in anesthesized dogs caused by administration of 2.5 mg/kg i .v. of A’-THC is accompanied by a decrease in cardiac output and an increase in local vascular resistance”. The fractional blood flow to the vital coronary, cerebral and renal beds was unchanged“. The reduction in cardiac output appears to result from the action of A9-THC on the heart rate (bradycardia) as well as venous r e t ~ r n ~ ~ , ~A’maximal . degree of bradycardia is produced only when both sympathetic and parasympathetic innervation of the heart is intactz4. A’-THC appears to be devoid of any ganglionic or 6adrenergic blocking properties“. Interestingly, A9-THC produces a doserelated tachycardia in manz5. While both A’-THC and propanolol delay the onset of ventricular ectopic extrasystoles A’-THC does not delay the onset of ouabain-induced arrhythmias’6 .
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Pulmonary Effects Asthma Three groups Yublished last year on the pulmonary effects of marijuana. In one study ‘ Y “ of nine, normal volunteers with previous marijuana smoking experience, airway resistance (measured in a body plethysmograph) fell 38% and specific airway conductance increased 44% at a dose of 84 pg/kg. This effect was accompanied by a 28% increase in heart rate. At a dose of 32 pg/kg, there was no increase in heart rate but there were significant changes in airway dynamics. In another paper‘ thirty-two experienced male marijuana smokers were given A9-THC by smoking or orally (20 mg dose). Again specific airway conductance increased immediately (48% at 1 5 min. after smoking marijuana assayed at 2% A’-THC; 45% at 180 min. after ingestion of 20 mg A’TKC). A study” suggested that these effects operated via a mechanism other than deep breathing or 6-adrenergic stimulation, the dilation being due to, most probably, relaxation of the smooth muscle of the tracheobronchial tree. No data were available on isolated bronchial smooth muscle. These data strongly suggest a therapeutic potential for AS-THC in the treatment of pulmonary congestion; e.g. asthma3’. Nevertheless , one paper32 has pointed out that these pulmonary effects are not reflections of changes in the clinically important peripheral airways (<2 mm diameter). Potentiation of Barbiturates and Anesthetics: - Pretreatment of mice for six successive days at 20 mg/kg of Ay-THC, caused a decrease in the duration of sleeping time after dosing with zoxazolamine and hexobarbital’ . However, under the same experimental conditions, the duration of barbital sleeping time was enhanced. The authors suggest an induction by AS-THC of the hepatic enzyme systems involved in the metabolism of zoxazolamine and hexobarbital, but no metabolism studies are offered in support of this
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possibility. Another paper3‘ from the same laboratory reports that pretreatment of mice with doses of A’-THC (0.625 to 40 mg/kg) caused a significant reduction in the onset to and duration of sleeping time of most of the sedative-hypnotic drugs tested. These studies cast some degree of doubt on the therapeutic potential of A’-THC to potentiate barbiturate sleeping time, unless it is done on an acute dosing basis rather than after chronic treatment. report on the interaction of A’-THC with anestheTwo studies3” tics. The minimum alveolar anesthetic (MAC) requirements for cyclopropane in rats3= and halothane in dogs36 were significantly decreased by pretreatment with A’-THC. Analgesia, sedation and prolonged barbiturate sleeping time after acute A’-THC injection may reflect the potential additive anesthetic-like action of the drug. Alternatively, drugs that deplete norepinephrine in the CNS decrease halothane MAC36. A’-THC and Sleep - A’-THC continues to be studied as a sedative-hypnotic. One study” of the sleep-wakefulness cycle in rabbits was stimulated by the effects of A’-THC on 5HT metabolism in the brain, an effect hypothesized to play an important role in the production of slow wave sleep. No evidence was found that A’-THC in doses of 0.5 and 1.0 mg/kg increased total REM sleep time or prolonged the duration of‘ single episodes3’. In another study3’ in cats, slow wave sleep was significantly decreased.
A clinical study3’ of A’-THC as a hypnotic found that: (1)A’-THC significantly decreases the time it takes to fall asleep in physically healthy insomniacs. ( 2 ) The most significant effect of an oral dose of 30 mg was a “hangover” phenomena or continued ”high” the next day. (3) This “hangover” seemed severe enough to eliminate the 30 mg dose range for clinical use as a hypnotic. A comparison study of A’-THC at doses around 20 mg with a standard hypnotic is in progress. AO-THC when administered to cats at doses of 1-10 mg/kg, i.p. or i.v. , induced sedation and caused a trend toward fewer and lon er REM sleep episodes following initial suppression of paradoxical sleep“. In rats tolerance developed to both the suppression of paradoxical sleep and to behavioral effects (principally excitation)‘l. CH3
CH 3 AO-THC
-
Other CNS Effects: Biogenic Amines With several standard procedures commonly used to detect antidepressant activity, A’-THC was virtually inactive by i.p. administration at doses up to 20 mg/kg42. Clinically, A’-THC (0.3
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mg/kg, p.0. twice a day for 7 days) in 8 hospitalized depressed patients failed to produce significant euphoria or antidepressant response43 . The mechanism of the central effects of cannabinoids continues to be investigated. A'-THC, hA-THC and DMHP all increased the amounts of noradrenaline and dopamine accumulated from tyrosine in rat whole brain44. The hypothalmus region was most susceptible to these changes in catecholm i n e biosynthesis. In spite of the observed increase in catecholamine biosynthesis in the brain and adrenals, the endogenous content of NA and DA in the brain and DA and NA in the adrenals remained unchanged45.
DMHP Repeated administration of A'-THC to rats leads to an increase in activity of tyrosine hydroxylase4 ; tryptophane hydroxylase and DOPA decarboxylase were not significantly affected. Because of the possible implication of PEA as one of the adrenergic ergotropic modulators, alterations in its disposition may be responsible for the eu horic effects of A'-THC in man4'. Acute administration of 3 mg/kg of A -THC increased 4-fold the brain levels of PEA and daily administration of 0.3 mg/kg for 8 days doubles PEA brain levels. In contrast A'-THC induces only relatively small changes in the brain levels of serotonin, catecholamines and acetylcholine.
8
Confirming earlier work on ingested marijuana, smoked cannabis has been found to produce little changes in the urinary excretion of epinephrine and NE but decreases VMA excretion by 25% from baseline value at 2 hrs. in humans4'.
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Summary Even a cursory look at the literature on cannabis would lead one to the conclusion that marijuana is "a drug for all reasons". Perhaps the many biological activities of marijuana impeded its therapeutic use during the past 40 years. A s recently as 1971 the natural material or THC appeared to offer little advantage over currently used medications used as sedatives, analgesics, antidepressants and antihypertensive drugs4'. With the identification in 1964 of A'-THC as the "active" ingredient in marijuana, a new chapter in marijuana research was opened. The possibility of structural modification to enhance activity was again explored. Undoubtedly during the past decade many investigators (especially those in the pharmaceutical industry) have pursued the possibility of modification of the A'-THC structure to delineate activities.
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An indication of the efforts along these lines has been reported5' by Abbott Laboratories. Compound, ABBOTT 40656 , ~ ~ 1 ,0a6water-soluble benzopyran derivative is presently under Phase I clinical evaluation as a sedative-hypnotic. Time will tell whether the pursuit of structure-activity relationships will lead to therapeutically useful drugs in any or all of the disease areas 'HC1 mentioned in this review. Also new activities might be discovered.
ABBOTT 40656 ( ~ ~ 1 0 6 ) References 1. The term "cannabinoids" has been defined as the group of C21 compounds typical of and present in Cannabis s a t i v a , their carboxylic acids, analogs, and transformation products. 2. R. Mechoulam (Ed.) , "Marijuana" , Academic Press , Inc , New York and London, 1973. 128 (1972). 3. L. Hollister, Psychopharmacologia, 4 . S. Burstein and A. Raz, Prostaglandins, 2, 369 (1972). 5. S. Burstein, E. Levin and C. Varanelli, Biochem. Pharmacol., 22, 2905
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J . R. Vane, Nature New Biol. , 232 (1971). R. D. Sofia, L. C. Kuobloch and H. B. Vassar, Res. Comun. Chem.
Pathol. Pharmacol., 6, 909 (1973). R. D. Sofia, S. D. Nalepa, J. J. Harakal and H. B. Vassar, J. Pharmacol. Exp. Therapeutics, 646 (1973). 9. D. S. Kosersky, W. L. Dewey and L. S. Harris, Eur. J. Pharmacol., 24, 1 (1973). . ~. 10. I. Nir, D. Agalon, A. Tsafriri, T. Cordova and H. R. Linder, Nature,
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L. Wright, S. L. Haley, J. B. Plank, M. C. Brande, and J. C. Calandra, Toxicol. Appl. Pharmacol. , 25, 449 (1973). 1 2 . A. Merari, A. Barak and M. Plaves, Psychopharmacologia, 28, 243 (1973). 13. R. Karler, W. Cely and S. A. Turkanis, Life Sci., 13,1527 (1973). 1 4 . J. A. Wada, M. Sato and M. E. Corcoran, Experimental Neurology, 2, 11. T L . Keplinger, P.
157 (1973). 15. J. A. Wada, M. Corcoran, M. Sat0 and J. McCaughran, Epilepsia, 14,101 (1973). 16. R. Karter,'Fed. Proc., 32, 756 (1973). 17. H. F. Hardman, E. F. Domino and M. H. Seevers, Pharmacol. Rev., 23, 295 (1971). 18. R. B. Williams, L. K. Y. Ng, F. Lamprecht, K. Roth and I. J. Kopin, Psychopharmacologia, 28, 269 (1973). 19. M. K. Birmingham, Br. J. Pharmacol., 48, 169 (1973). 20. G . G . Nahas, I. W. Schwartz, J. Adamee and W. M. Manger, Proc. SOC. Exp. Biol. Med., 142, 58 (1973).
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I. Cavero, R. Ertel, J. P. Buckley and B. S. Jandhyala, Eur. J. Pharmacol. , 20, 373 (1972). 22. I. Cavero, J. P. Buckley and B. S. Jandhyala, Eur. J. Pharmacol., 24, 243 (1973). 23. I. Cavero and B. S. Jandhyala, Fed. Proc., 32, 755 (1973). 24. I. Cavero, T. Solomon, J. P. Buckley, B. S. Jandhyala, Eur. J. Pharmacol. , 22, 263 (1973). 25 B. R. Manno and J. E. Manno, Toxicol. Appl. Pharmacol. , ;15, 451 (1973). 26. J . F. Schaffer Jr., R. Loetzer and R. D. Sofia, Arch. Int. Pharmacodyn. 205, 5 (1973). 27. L. Vachon, M. X. FitzGerald, N. H. Solliday, I. A. Gould and E. A. Gaensler, N. Engl. J. Med., 288, 985 (1973). 28. L. Vachon, M. X. FitzGerald, I. A. Gould, E. A. Gaensler and N. H. Solliday, Am. Rev. Respir. Dis., 3, 1099 (1973). 29. D. P. Tashkin, B. J. Shapiro and I. M. Frank, N. Engl. J. Med., 2&, 336 (1973). 30. B. J. Shapiro, D. P. Tashkin and I. M. Frank, Ann. Intern. Med., 78, 832 (1973). 31. P. Beaconsfield, J. Ginsburg and R. Rainsbury, N. Engl. J. Med. 389, 1315 (1973). 32. T. P. Bright, M. A . Evans, R. Martz, D. J. BroTj, and R. B. Forney, Pharmacologist , 15,238 (1973 ) 33. R. D. Sofia, Res. Commun. Chem. Pathol. Pharmacol., 5, 91 (1973). 34. R. D. Sofia and L. C. Kuobloch, Psychopharmacologia, 2,185 (1973). 35. T. S. Vitez, W. L. Way, R. D. Miller and E. I. Eger, Anesthesiology, 38, 525 (1973). 36. R. K. Stoelting, R. C. Martz, J. Gartner, C. Creasser, D. J. Brown and R. B. Forney, Anesthesiology, 3 8 , 521 (1973). 37. M. Fujiwasi and H. E. Himurch, Physiol. Behav., 11,291 (1973). 38. E. S . Barrati and P. M. Adam, Pharmacol. Psychiatry, 6, 207 (1973). 39. K. Consens and A. DiMascio, Psychopharmacologia, 33, 375 (1973). 40. M. B. Wallach and S. Gershan, Eur. J. Pharmacol.,?4, 172 (1973). 41. J. E. Moreton and W. M. Davis, Neuropharmacology, l2, 897 (1973). 42. R. D. Sofia, R. K. Kubena and H. Barry, Psychopharmacologia, 2,1 2 1 (1973). 43. J. Kotin, R. M. Post and F. K. Goodwin, Arch., Gen. Psychiatr., 28, 345 (1973). 44. L. Maitre, P. C. Waldmeier and P. A. Baumann, Life Sci., l3, R 1 1 2 (1973). 45. I. M. Mazurkiewica-Kwilecki and M. Filczewski, Psychopharmacologia, -333 7 1 (1973). 46. B. T. Ho, D. Taylor and L. F. Englert, Res. Commun. Chem. Pathol. Pharmacol. , 5, 851 (1973). 47. H. C . Sabelli, W. A. Pedemonte, C. Whalley, A. D. Mosnaim and A. J. Vazquez, Life Sci., 149 (1974). 48. F. S. Messiha and R. A. Soskin, Res. Commun. Chem. Pathol. Pharmacol 6 , 325 (1973). 49. L. E. Hollister, Science, 172, 2 1 (1971). 50 * W. G. Jochimsen and A. T. Dren, E d . Proc. , 33, 527 (1974). 21.
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Section V I Editor:
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i n Chemistry
Robert A. Wiley, C o l l e g e of Pharmacy, U n i v e r s i t y of Kansas Lawrence, Kansas
Chapter 27 : Reactions of I n t e r e s t i n Medicinal Chemistry Dwight S. F u l l e r t o n t , George L. Kenyon+', and Dolan H. E a r g l e , Jr.?* Department of Medicinal Chemistry, College of Pharmac U n i v e r s i t y of Minnesota, Minneapolis, Minnesota 55455 U n i v e r s i t y of C a l i f o r n i a , San F r a n c i s c o , C a l i f o r n i a 94122++
v
S e v e r a l u s e f u l r e f e r e n c e books of g e n e r a l i n t e r e s t were published i n 1973 i n c l u d i n g : Annual Reports & Organic Synthesis-=, t h e t h i r d of t h e annual series by J . McMurry and R. B. Miller (covering t h e l i t e r a t u r e t o March, 1973); P r o t e c t i v e Groups i n Organic Chemistry by J . F. W. McOmie; and Volume 27 of S y n t h e t i c Methods of Organic Chemistry by William Theilheimer. I n T h e i l h e i m e r ' s p r e f a c e is i n c l u d e d an i n t e r e s t i n g summary of "Trends i n S y n t h e t i c Organic Chemistry" c i t i n g eighty-one r e f e r e n c e s t o t h e s y n t h e t i c l i t e r a t u r e 1971-1973. Computers - The p o s s i b i l i t i e s and l i m i t a t i o n s of u s i n g computer literature searches t o f i n d p a r t i c u l a r k i n d s of s y n t h e t i c r e a c t i o n s has been reviewed. Formation - T e r t i a r y phosphines have been shown t o b e good catalysts for MichueZ reactions, o f f e r i n g a u s e f u l a l t e r n a t i v e t o a l k o x i d e o r hydroxide c a t a l y s i s . 2 Lithium "mnides" have been used t o form e n o l a t e s
-C-C Bond
and c a r b a n i o n s a t -78' f o r a v a r i e t y of s y n t h e t i c purposes c i t e d througho u t t h i s c h a p t e r , i n c l u d i n g f o r C-C bond formation. The a l k y l a t i o n of c y c l o h e ~ e n o n e s(~o r a-diketones4) and of l a c t o n e s 5 i s i l l u s t r a t i v e :
L
Acetylene i s normally a v e r y s l u g g i s h d i e n o p h i l e i n Diels-Alder rea c t i o n s , b u t two acetylene synthons have been r e p o r t e d t o b e v e r y reactive -- v i n y l e n e t h i o n o c a r b o n a t e and 2-phenyl-1 ,3-dioxol-4-ene.
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Chemical Reactions
Fullerton, Kenyon, Eargle
261
E d n e s t o be used i n C-C bond formation may be prepared under very mild conditions by treatment of the aldehyde or ketone with the trimethylsilyl derivative of the appropriate amine. Organocopper Reagents - Many new organocopper reagents and uses of these reagents have been reported in 1973--including one report of lithium din-butyl cuprate acting as a reducing agent in certain situations.8 The following examples illustrate recent advances. The eZectronic and s t m e turaZ requirements for addition of organocopper reagents to a,B-unsaturated carbonyl compounds have been studied in detail.9 Epoxides treated with organocopper reagents form simple alcohols' and even a-hydroxyallenes.l 1 On the other hand, treatment of lithium dimethyl copper with some a,B-epoxy ketones failed to yield any alkylated product. l 2 Li (CH3) 2Cu /O\ CHz-CH2
63..
*
YH (-70%) CH3CH2CHR
+o&
&:2)3Hc(iL
O
dH
0
5%
85%
Lithiwn diaZkyZ cuprates react with enol acetates of B-diketones to give the corresponding B-alkyl-a,B-unsaturated ketone. (CH3)2CuLi
AcO 0
AA.
w
91%
A new class of mixed cuprate (I) reagents, Het(R)CuLi, allows seleet i v e aZkyZ group transfer. l 4 Lithium diorganocuprates add t o p r i m a r y tosylates i n high yieZds (70-loo%), but secondary tosylates give somewhat a-AZkyZ ketones have been prepared by treatment lower yields (10-75%). of dimethylcuprate (I) on a,a'-dibromoketones. Yields are good, and the reaction appears to proceed via a cyclopropanone intermediate.l6 Reaction of a,B-unsaturated sulfones with lithium dialkylcuprates has been used to prepare gem-dialkyl products.l7 R CI-!?
A
bx2Ar R2CuLi
-
x
h ~ 2 s o ~ ~ ~ a / H g 15-89%
Preparation of C=C and CEC Epoxides have been used as starting materials for several olefin syntheses. Olefins may be prepared by addition of trifluoroacetic acid to a solution of the epoxide and triphenyzphosphine seZenide. Trisubstituted aZZy Zic aZcohoZs may be stereoselectively prepared in good yield by the acid catalyzed ring-opening o f cyclopro yloxiranes19 and by conjugate reduction of a , B-unsaturated epoxides.g 0
Sect. VI
262
-
Topics in Chemistry
+ iBu2AlH
-F-
+ Ca/NH3
Wiley, Ed.
95%
6
HO
5%
h 8%
b
92%
IntramoZecuZar W i t t i g reactions have been used to form new C=C bonds in high yields.21 Disubstituted acetylenes may be obtained in high yields by treating lithioalkynes with alkyl halides ;2 2 by treating carbonyl compounds in a one-step, base-induced reaction with trimethylsilyldiazomethane or dimethylphosphonodiazomethane;2 3 or by treatment of lithium 1alkynyltriorganoborates.24 R C3 - Li+
R2X
+
RiB-[R1CX-iR2]Li-R1CECR2 3
R1CXR2(70-90%)
+ I2
R;BI
+ LII
(90-100%)
Unsaturated Carbonyl Compounds - A two carbon-homologation of-ketones, aldehydes , or halides to a ,B-unsaturated aldehydes has been achieved using 2,3,4,6-tetramethy1-4H-l,3-0xazine.~~ Ketones have been brominated selectively on one side of the carbonyl group with specifically generated enolates . Dehydrobromination then yields position-speci f i e a, &unsaturated ketones. 26
&-€ 87%
a,f~;llnsaturatedketones and e s t e r s may be obtained from the corresponding saturated compounds by treatment with PhSe-Na', PhSeCl , or PhSeBr followed by oxidation with H202 or sodium p e r i ~ d a t e . New ~ ~ syntheses o f a-methylene Zactones continue to be reported. For example, lactones may be treated with a lithium anride a t -78' and carbon dioxide to form the a-carboxy-lactone, and then with formaldehyde2* (or in a longer sequence with iodomethyl methyl sulfide2') to form the a-methylene product. Alkylation of a ,6-unsaturated carbonyl compounds usually occurs in the a-position, but it has been shown that deconjugative a l k y l a t i o n can occur in the presence of hexamethylphosphoramide (HMPA) 30
.
Chap. 27
Chemical Reactions
Fullerton, Kenyon, Eargle
COOEt l)LINRz,HMPA -78" Z)RX(l.lEq)
COOEt
263
COOEt (repeat
* rl\R 88-98%
steps)-R+R 88-98%
-
Protective Groups and Their Removal Lactones and e s t e r s may be protected The ester or lactone is treated with bis (dimethylas 2,3-dithioZanes. aluminum) 1,2-ethanedithiolate, and deprotection is achieved with mercuric oxide and BF3-Et20 in 15% aqueous THF. The dithiolanes were shown to be stable to acetic acid, to KOH-aqueous methanol, and to LiAlH4 or methyllithium in ether. 3 1 Another report appeared describing the use of the t-butyzdimethyls i l y z protecting group f o r aZcohoZs,32 and it was also found to be useful for protecting deoxynuczeosides--especially the 5 ' hydroxyl. 3 3 YZids containing s i z y z protective groups have also been successfully employed in Wittig reactions to synthesize complicated olefins under very mild conditions. However, the ylid must be pure and salt free, and a precise stoichometry must be used. 34 A r e v i m of nezJ amino function protecting groups was published3= and the 4-~sopropylo;cycarbonyZo~benzy~oxy carbonyz group has appeared as a new alkaZi-labile m i n o protecting group. 36
S-substituted mercaptoethanols can serve as phosphate protective groups in nucleotide synthesis,3 7 and 1-oxopyridin-2-yldiazomethane has been reported to be a water soluble phosphate blocking group.38 Tetrahydropyranyl ethers and 4-methoxy-THP ethers may be prepared i n only f i v e minutes by treatment of the parent alcohol with dihydropyran in anydrous dioxane, using p-toluenesulfonic acid as a catalyst. Yields are 80-90%. 39 BromoketaZs have been investigated to meet the requirements of protective group removal under neutral conditions. Ketalization is carried out by treatment of the carbonyl compound with bromoglycol and p-toluenesulfonic acid as a catalyst. Deketalization is accomplished by refluxing the bromoketal in methanol containing zinc dust. 4 0 Saturated Aldehydes Ketones - Both primary and secondary alcohols can be oxidized in 94-98% yield to their corresponding carbon 1 compounds with dimethy7 suZfoxide-chlorine complex at -45' to -10' ,81 a procedure which is reportedly superior to previous oxidation methods. Terminal alkynes are converted t o ketones by alkylation o r protonation of their lithium alkynyltrialkylborate salts, followed by H202 oxidation.4 2
264
Sect. VI
-
Topics in Chemistry
Wiley, Ed.
AcetonyZ functions can be prepared from the readily available lithium salt of acetylmethylene triphenylphosphorane by reaction with alkyl halides.
9
Rx
LiCH2CCH=P(Ph)
b
i? RCH2CCH3
39-93% 8-Ketoesters are produced in 40-90% yield by condensation of 0silylketone acetals with acid chlorides followed by hydrolysis .44 yKetoaZdehydes result from alkylation of the lithium salt of 2-ethoxyallyl vinyl sulfide, followed by thio-CZaisen rearrangement and hydrolysis.4 5
+ Li EtO
xs] R
H~O/DME
+ RBr
OHDC&'
reflux 56-70% 12 hr High yields of tY.isubstituted enoZ ethers, readily convertible to methyl ketones, result when ketones are treated with dilithium 0-methyl lactate followed by decarboxylation.46 EtO
R1
\C=O
R2'
+
I;i CH3CC02Li bCH3
1)THF 2) PhS02C1 88-90%
3 ) K CO
Olefins may be converted in high2 i&d to a-chloroketones by treatment with chromyl chloride in acetone.47 a-Carboqketones, generally relatively inaccessible, can be prepared by treatment of ketones with lithium 4-methyl-2,6-di-tert-butylphenoxide and carbon dioxide.48 CycZic ketones result from ring expansion of methy Zene qcZoaZkanes with cyanogen azide followed by hydrolysis, a procedure which complements the standard diazomethane-ketone approach . 4 9 Functionalized cis-bicycZic ketones can be synthesized by haloketal cyclization followed by hydrolysis. 5 0 Epoxides - A modified method for the conversion of ketones t o epoxides via 8-hydroxysulfides has been developed. 51
Acid-sensitive oZef i n s may be epoxidized i n high yield using a twophase system of dichloromethane and aqueous sodium bicarbonate t o which is added m-chloroperbenzoic acid.52
v' n l
0 7
*
80-85%
Two new routes to arene oxides of carcinogenic aromatic hydrocarbons have been reported, which employ 0sO4 or NBS sequences to prepare the required 1,2-diol or a-bromo alcohol intermediates for epoxidation. 5 3 y 54
Chap. 27
Chemical Reactions
Fullerton, Kenyon, Eargle
265
-Amines and
Nitriles - Primary and secondary amines may be methylated in high yield by treatment with formaldehyde in methanol followed by reduction with sodium borohydride. 5 5 Similarly, aromatic primary amines may be monomethylated by sodium borohydride reduction of their N-aryl-amino methylsuccinimides, generated by treatment of the amine with aqueous alcoholic formaldehyde and succinimide.56 A general, stereospec; f i e synthesis of secondary a&nes can be achieved in high yield by treatment of alkyl or aryl azides with alkyl- or aryl-dichloroborane (RBC12) followed by base hydrolysis. The reaation proceeds with retention of configuration of the alkyl group in the alkyldichloroborane.57
GpticaZZy a c t i v e a&nes can be prepared in 80-90% yield (90% optical purity) by reaction of imines or nitriles with optically active p-tolylsulfinyl carbanion followed by reduction with Raney nickel.5 a
C@PhCH=NPh i+L2HCH3
\ /
..
THF
,
$H3 Ph-C-NHPh Raney Ni HI 81% c
-10
A new B-ary Zeth Za&ne synthesis by aryl aldehyde homologation has also been de~eloped.~' A procedure has been found for the specific ortho aZkyZation of aromatic amines.60 A review of the use of quaternary ammonium compounds i n organic synthesis has recently appeared.6 1 Mannich bases may be prepared regiospecifically using the reaction of enol borates with dimethyl-(methylene) -ammonium iodide.6 2 A very complete review of RIB
+ R~CONH~
v2
+
R;BOC=CHR~
-N2
3
(CH3) 2NzCH2I8 R~-c-~HR~ 80-100% CH2N (CH3) 2
synthetic uses of Mannich bases has been published.63
It is now possible to convert ketones i n t o n i t r i l e s directly using tosylmethylisocyanide.64 N i t r i l e s may also be prepared using the action of in situ generated dichlorocarbene on amides, thioamides or aldoximes . 6 4 Under these same conditions ureas yield N-cyano&nes. Carboxylic Acids and Derivatives - A convenient one-flask method for e s t e r aZky Zation also uses lithium diisopropylamide at low temperature.6 5 i)Lim2, -780 repeat R3 R~~HCOOCH~ * R1-b-CO0CH3 R~CH~COOCH~ A2 2)R2X in HMPA R2 88-98% with R3X
-
88-98%
DecarbaZkoxy Zations of geminal diesters can be effected by treatment with sodium chloride in hot, wet dimethylsulfoxide.66 Similar yields are obtained in the decarbethoxylation of both 6-keto and a-cynanoesters. NaC 1
* R1R2CCOOEt
R1R2C(COOEt) DMSO-H-0 140-186'
85-95%
+ c02
+ EtOH
266
Sect. V I
-
Wiley, Ed.
Topics i n Chemistry
A l k y Z halides (R’X) may b e c o n v e r t e d t o t h e c o r r e s onding a l i p h a t i c , esters (R1COOR2) o r amides (R CONR2R3) u s i n g disodium tetracarbony Zferrate followed by o x i d a t i o n w i t h O2 o r N a O C 1 , o r
P
c a r b o x y l i c a c i d s (R’COOH)
h a l o g e n a t i o n on t h e p r e s e n c e of t h e a p p r o p r i a t e a l c o h o l o r amine.67 P r i mary and secondary alcohols (R1R2CHOH) may b e c o n v e r t e d i n good y i e l d s t o arrtides (R1R2CHNHC0R) u s i n g chlorodipheny h e thy l i u m h e x a c h l o r o a n t imonat e i n a-A&no acid derivatives may b e p r e p a r e d from malonic n i t r i l e solvents. a c i d h a l f - e s t e r s u s i n g diphenylphosphorylazide. S u b s t i t u t e d g l y c i n e , v a l i n e , g h e n y l a l a n i n e and t r y p t o p h a n were g e n e r a t e d , among o t h e r amino acids. F i n a l l y , t h e r e i s a n e x c e l l e n t review of modern approaches t o 6Zactam s y n t h e s e s . 70
*
H e t e r o c y c l e s - A l k y l o r a r y l d i c h l o r o b o r a n e s react w i t h 2 - i o d o a l k y l a z i d e s t o g i v e s t e r e o s p e c i f i c a l l y N-alkyl and N-aryZ aziridines i n y i e l d s of 7394%.71 The r i n g c l o s u r e of a n i l i n e s t o 2-substituted indozes may b e accomplished i n t h e f o l l o w i n g manner,72 and oxindoles may b e o b t a i n e d w i t h a change i n t h e s u l f i d e r e a g e n t of s t e p 2 t o CH3SCH2C(=O)OCZH5.73
@
1)tBuOCl
Raney N i
mR2 2) CH3SCH2 3)Et3N
R1
A d i r e c t s y n t h e s i s of substituted pyridines i n y i e l d s of a b o u t 50% is found i n t h e t r e a t m e n t of a c e t y l e n e and a n a l k y l n i t r i l e i n t h e p r e s e n c e of a c o b a l t c a t a l y s t . 7 4 A g e n e r a l method f o r t h e s y n t h e s i s of 3- and 3 , 4 - s u b s t i t u t e d furans from ketones h a s been developed.75 A new r o u t e t o v a r i o u s h e t e r o c y c l e s w i t h 2 or more heteroatoms h a s been p r e s e n t e d , as w e l l as a p r o c e d u r e i n v o l v i n g 1 , 3 - o r 3 , 3 - c y c l i z a t i o n on d i f u n c t i o n a l n u c l e o p h i l e s such as h y d r a z i n e , hydroxylamine, a l k y l and a r y l diamines 76 ’ 37 AgCN /CH2\ RCN + H2N(CH2)nQH W N Q +RNH2 %H d Q=O ,NH9 S
.
S u l f u r Chemistry - Two f a c i l e methods of t h e h e r e t o f o r e d i f f i c u l t sulfoxi d e t o su-lfide reduction have been accomplished w i t h d i i s o b u t y l aluminum h ~ d r i d eand ~ ~d i c h l o r o b o r a n e i n THF a t Oo.79 With t h e l a t t e r r e a g e n t , ket o n e s , esters, and amides remain u n a f f e c t e d . A review on sulfoximes and d e r i v a t i v e s as s y n t h e t i c r e a g e n t s p r e s e n t s some new methods f o r t h e prep a r a t i o n of v a r i o u s oxiranes, a z i r i d i n e s , alcohols, eyclopropmes, and alkenes. 8o A l l y l i e suzfoxide anions have proven u s e f u l f o r t h e synthesis of a l l y Zic alcohols, i n c l u d i n g t r i s u b s t i t u t e d o l e f i n i c a l l y l i c a l c o h o l s . T r a n s e s t e r i f i c a t i o n between a d i a l k y l a c y l p h o s p h o n a t e and a s u l f o n i c a c i d y i e l d s sulfonate e s t e r s . 82 The o x i d a t i o n of a l i p h a t i c mercaptans t o sulf i n i c acids w i t h t h e u s e of m-chloroperbenzoic a c i d i s e s p e c i a l l y u s e f u l i n t h a t t h e e x c e s s p e r b e n z o i c a c i d i s removed by p r e c i p i t a t i o n a t -80°.83 Reduction
-
Ether soluble AZH, may b e made by t r e a t m e n t of L i A 1 H 4 w i t h
Chap. 27
Chemical Reactions
Fullerton, Kenyon, Eargle
267
BeC12 or ZnC1, (with concomitant precipitation of LiBeH2C12 or LiC1). 8 4 The Birch reduction has now been applied to Ar heterocycle^^^ yielding, for example, N-substituted 1,4-dihydroquinolines from quinoline. A variety of reductive uses for LiEt3BH have been reported.8 6 9 8 7 LiEt3BH RBr
RH 90-100%
99% Lithium-ethylamine reduction of l,&dialkylbenzenes gives the corresponding 1,4-dialkylcyclohexa-1,4-diene.8 8 Among the many new applications of metal carbonyls are to be found the reductions of enol acetates, vinyl chlorides, and a,@-unsaturated aldehydes to olefins; and a-acetoxyketones to ketones.8 9
O H 40-70%
X=OAc,Cl,CHO Removal of aromatic phenoZic groups is accomplished in 65-90% ield by conversion to the sulfonate, followed by catalytic hydrogenation.Bo Aromatic &nes are deaminated by aprotic diazotization with pentyl nitrate in yields up to 89%.91
-
Useful Reactions (not elsewhere classified) FZuovination of 12 types of compounds has been simplified and improved by the use of diaZkyZanrino t r i f l u o r i d e s , resulting in faster reaction times (15 to 30 min.), lower temperatures (0 to 80°), and better yields (55-89%) than with SF4. 9 2
+R~CO~H RiNSF3
+ R2CH0
+ R~COR
-
R~COF eR2CF2H tR ~ C F ~ R
The difficult task of selective a-bromination of a,B-unsaturated ketones may be achieved without affecting the double bond.93
3 PhCH=CHCCH3
i-
Br)i$ -I: Br 0
PhCH=CHljCH2Br 0 91%
’‘
Reviews (not mentioned elsewhere) - Or anoboron chemistry, MetaZr-aZZyl comp Zexes,= organic eZectrochemistry, % free-radicai! preparative organic chenristry, 9 7 and asymmetric syntheses (utilizing n-ally1 nickel halides , Lewis acids , and phosphoranes to introduce chiral centers,98 have been reviewed.
Sect. VI
-
Topics in Chemistry
Wiley, Ed.
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7. 8.
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34. 35. 36. 37. 38. 39. 40.
41. 42. 43. 44. 45. 46. 47,
48. 49. 50.
m.,
Chap. 27 51. 52. 53. 54. 55. 56. 57. 58. 59. 60. 61. 62. 63. 64. 65.
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Chemical R e a c t i o n s
F u l l e r t o n , Kenyon, E a r g l e
269
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u.,
w.,
-
e., m.,
w.,
u.,
m.,
=.,
2 70
Chapter 28.
The Intramolecular Diels-Alder Reaction in Organic Synthesis
Robert G. Carlson, Department of Chemistry U n i v e r s i t y of Kansas, Lawrence, Kansas
The [4+2]cycloaddition r e a c t i o n l o n g known t o o r g a n i c chemists a s t h e Diels-Alder r e a c t i o n i s among t h e most powerful of t h e many s y n t h e t i c t o o l s a v a i l a b l e t o chemists i n t e r e s t e d i n t h e s y n t h e s i s of complex o r g a n i c I t s a b i l i t y t o produce w i t h high s t e r e o s e l e c t i v i t y , good y i e l d molecules. and i n a p r e d i c t a b l e manner a s i n g l e s t e r e o i s o m e r c o n t a i n i n g s e v e r a l c h i r a l c e n t e r s i s almost unique among o r g a n i c r e a c t i o n s . For exaxple t h e a d d i t i o n of m a l e i c anhydride t o trans, trans-2,4-hexadiene g i v e s s t e r e o s e l e c t i v e l y t h e adduct 1 c o n t a i n i n g f o u r asymmetric c e n t e r s .
Because of t h e s e remarkable a s s e t s , as w e l l a s a tremendous v e r s a t i l i t y , t h e Diels-Alder r e a c t i o n has been u t i l i z e d i n a l a r g e number of t o t a l s y n t h e s e s of complex molecules. Although t h e e a r l y l i t e r a t u r e r e c o r d s a number of r e a c t i o n s which can be i n t e r p r e t e d a s i n v o l v i n g i n t r a m o l e c u l a r Diels-Alder r e a c t i o n s , it is only i n t h e l a s t decade t h a t o r g a n i c chemists have s t u d i e d t h e i n t r a m o l e c u l a r Diels-Alder r e a c t i o n i n terms of i t s scope and l i m i t a t i o n s and i n t e n t i o n a l l y a p p l i e d t h e i n t r a m o l e c u l a r c y c l i z a t i o n i n t h e s y n t h e s i s of comp l e x molecules. This c h a p t e r w i l l review some of t h e more i n t e r e s t i n g s t u d i e s of t h e i n t r a m o l e c u l a r Diels-Alder r e a c t i o n s and i t s a p p l i c a t i o n t o t h e p r e p a r a t i o n of a v a r i e t y of classes of n a t u r a l p r o d u c t s . The i n t r a molecular Diels-Alder r e a c t i o n i s of g r e a t p o t e n t i a l u t i l i t y because it may be used t o s o l v e o r i e n t a t i o n problems t h a t might be encountered i n t h e corresponding i n t e r m o l e c u l a r r e a c t i o n . I t is a l s o l i k e l y t h a t t h e r e w i l l be enhanced r e a c t i v i t y i n some systems because t h e e n t r o p y of a c t i v a t i o n w i l l be a p p r e c i a b l y lower t h a n t h a t of t h e analogous b i m o l e c u l a r r e a c t i o n s . House3 r e p o r t e d i n 1 9 6 5 t h e f i r s t s y s t e m a t i c s t u d y of t h e stereochemi s t r y and s t r u c t u r a l l i m i t a t i o n s of t h e i n t r a m o l e c u l a r Diels-Alder r e a c t i o n . I n t h i s s t u d y t h e t h e r m a l r e a c t i o n s of t h e i s o m e r i c t r i e n e e s t e r s 1 and 2 were examined. With both and 3 t h e i n t r a m o l e c u l a r Diels-Alder r e a c t i o n f a i l e d , b u t with t h r e e carbon atoms s e p a r a t i n g t h e d i e n e and d i e n o p h i l e & c and t r a n s - i s o m e r s (2 and underp o r t i o n s of t h e molecule both t h e went s t e r e o s e l e c t i v e c y c l i z a t i o n t o produce t h e hydrindane d e r i v a t i v e s 2 and 5.
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n=2 n=3
"" 4 A
d
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O f p a r t i c u l a r i n t e r e s t was t h e f i n d i n g t h a t t h e *-1,3-pentadiene deriv a t i v e 2 underwent ready c y c l i z a t i o n because systems of t h i s t y p e o f t e n f a i l t o undergo b i m o l e c u l a r Diels-Alder r e a c t i o n s . Secondly, t h e i n t r a molecular r e a c t i o n d i c t a t e s a s p e c i f i c o r i e n t a t i o n of t h e d i e n e and dienop h i l e which i s d i f f e r e n t from t h a t observed i n t h e analogous i n t e r m o l e c u l a r r e a c t i o n . I t i s t h e s e two f e a t u r e s which make t h e i n t r a m o l e c u l a r DielsAlder r e a c t i o n p a r t i c u l a r l y a t t r a c t i v e as a s y n t h e t i c method.
-
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One of t h e e a r l i e s t a t t e m p t s t o use t h e i n t r a m o l e c u l a r Diels-Alder r e a c t i o n f o r t h e s y n t h e s i s of a n a t u r a l p r o d u c t was r e p o r t e d i n 1963 by B r i e g e r who a t t e m p t e d t o p r e p a r e l o n i f o l e n e ( 5 ) by a r o u t e i n v o l v i n g an i n t r a m o l e c u l a r Diels-Alder r e a c t i o n . The s y n t h e t i c s t r a t e g y was based upon t h e c o n s i d e r a t i o n t h a t a compound such as % c o u l d be r e a d i l y c o n v e r t e d t o l o n g i f o l e n e ( 5 ) by r e d u c t i o n of t h e double bond followed by dehydrat i o n and t h a t 6 could p o t e n t i a l l y be p r e p a r e d by an i n t r a m o l e c u l a r c y c l i z a t i o n of L. The r e q u i r e d i n t e r m e d i a t e 2 was p r e p a r e d as a mixture of isomers by t h e c o u p l i n g of c y c l o p e n t a d i e n y l magnesium bromide w i t h t h e hydrogen c h l o r i d e a d d i t i o n p r o d u c t of g e r a n y l acetate. U n f o r t u n a t e l y ,
272 -
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++@+ 170°
CH 20H
-6
-7 d i d
n o t c y c l i z e t o g i v e t h e product w i t h t h e l o n g i f o l e n e s k e l e t o n b u t a s t h e p r o d u c t of t h e i n t r a m o l e c u l a r i n s t e a d gave t h e t r i c y c l i c isomer Diels-Alder r e a c t i o n .
a
A similar c y c l i z a t i o n has been e x p l o i t e d by Corey f o r t h e p r e p a r a t i o n of t h e t r i c y c l i c system
z.5
K l e m m has p u b l i s h e d an e x t e n s i v e s e r i e s of papers d e s c r i b i n g h i s work which has taken advantage of t h e i n t r a m o l e c u l a r Diels-Alder r e a c t i o n f o r t h e p r e p a r a t i o n of a series of l i g n a n l a c t o n e s , podophyllotoxins and p i c r o p o d o p h y l l i n s m 6 I n t h e s e r e a c t i o n s an a r o m a t i c r i n g c o n t r i b u t e s one of t h e double bonds of t h e d i e n e p o r t i o n o f t h e molecule. A t y p i c a l example i s given below.6b Although t h e y i e l d s i n t h e s e c y c l i z a t i o n s a r e o f t e n low t h e i n t r a m o l e c u l a r Diels-Alder r o u t e p r o v i d e s a remarkably s h o r t s y n t h e t i c pathway t o t h e s e systems from r e a d i l y a v a i l a b l e i n t e r m e d i a t e s . Two novel s y n t h e t i c approaches t o t h e s e s q u i t e r p e n e s e y c h e l l e n e (lo) have been based upon t h e u t i l i z a t i o n o f t h e i n t r a m o l e c u l a r Diels-Alder r e a c t i o n as t h e key s t e p i n t h e g e n e r a t i o n of t h e t r i c y c l i c carbon s k e l e t o n . I n t h e f i r s t approach7 t h e s t r a t e g y involved t h e p r e p a r a t i o n o f t h e t r i c y c l i c i n t e r m e d i a t e 2 by t h e i n t r a m o l e c u l a r c y c l i z a t i o n of t h e cyclohexa-
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The Intramolecular Diels-Alder Reaction
2 73 -
Carlson
I
Meoq +
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dienone 2. The r e q u i r e d cyclohexadienone 2 was prepared i n s e v e r a l s t e p s a s a mixture of d i a s t e r e o m e r s from 2,3-dimethylcyclohexenone and g e n e r a t e d i-n s i t u by an amine oxide p y r o l y s i s . Only a l o w y i e l d o f t h e i n t e r n a l add u c t & w a s o b t a i n e d and it was e a s i l y converted i n t o s e y c h e l l e n e .
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Very r e c e n t l y an a l t e r n a t i v e u t i l i z a t i o n of t h e i n t r a m o l e c u l a r D i e l s Alder r e a c t i o n for t h e s y n t h e s i s of s e y c h e l l e n e (2) was r e p o r t e d . a I n t h i s case an i s o m e r i c t r i c y c l i c i n t e r m e d i a t e was prepared and s u b j e c t e d t o a Wagner-Meerwein rearrangement t o g i v e s e y c h e l l e n e . A s o u t l i n e d below, t h e i n t r a m o l e c u l a r Diels-Alder r e a c t i o n u t i l i z e d t h e cyclohexadienone port i o n of t h e molecule a s t h e d i e n o p h i l e r a t h e r t h a n t h e d i e n e a s had been done i n Fukamiya' s approach.
t
isomer Wenkert' has developed an i n t e r e s t i n g s y n t h e s i s of t h e a- and Bhimalchenes (L3 and 14) which uses an i n t r a m o l e c u l a r Diels-Alder r e a c t i o n as t h e key s t e p i n e s t a b l i s h i n g t h e p r o p e r carbon s k e l e t o n . The b i c y c l i c u n s a t u r a t e d ketone 15 s e r v e d as an a p p r o p r i a t e p r e c u r s o r which could be converted r e a d i l y t o e i t h e r of t h e two himalchenes 13 and 2 and was its e l f produced by t h e c y c l i z a t i o n o f t h e t r i e n e &J i n u n s p e c i f i e d y i e l d .
13
14
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The I n t r a m o l e c u l a r Diels-Alder
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An e l e g a n t example of t h e tremendous p o t e n t i a l of t h e i n t r a m o l e c u l a r Diels-Alder r e a c t i o n f o r s o l v i n s t e r e o c h e m i c a l and o r i e n t a t i o n problems was r e c e n t l y provided by Coreyl' i n some model s t u d i e s d i r e c t e d toward a t o t a l s y n t h e s i s of g i b b e r e l l i c a c i d (17).A p r e v i o u s s t u d y l l had e s t a b l i s h e d t h a t t h e f u n c t i o n a l i t y and s t e r e o c h e m i s t r y of r i n g A could be i n t r o duced i n a s e r i e s of s e v e r a l s t e p s from 1 8 . A p o s s i b l e r o u t e t o 1 8 would i n v o l v e t h e p r e p a r a t i o n and a l k y l a t i o n of a c y c l o h e x a d i e n i c e s t e r o f t h e 2 ) . The i n t e r m o l e c u l a r r e a c t i o n t y p e 1 9 by a Diels-Alder r e a c t i o n would l i k e l y g i v e p r o d u c t s a r i s i n g from both p o s s i b l e o r i e n t a t i o n s a s w e l l as p r o d u c t s r e s u l t i n g from approach of t h e p r o p a r g y l e s t e r t o b o t h s i d e s of t h e d i e n e molecule. However, by c o n s t r u c t i n g t h e system i n such a way
(cf.,
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17
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t h a t t h e A r i n g i s i n t r o d u c e d by an i n t r a m o l e c u l a r Diels-Alder r e a c t i o n b o t h of t h e s e problems can be s o l v e d . I n o r d e r t o t e s t t h e f e a s i b i l i t y of t h i s p r o c e s s t h e model system 22 was p r e p a r e d . On h e a t i n g a t t e m p e r a t u r e s above 135O, 22 c y c l i z e d t o g i v e t h e t r i c y c l i c l a c t o n e 23 i n y i e l d s of up t o 70%. The s t e r e o c h e m i s t r y can be unambiguously a s s i g n e d as t h e only pos-
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s i b l e s t e r e o c h e m i s t r y for t h e product of t h i s i n t r a m o l e c u l a r Diels-Alder reaction.
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C02H
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.-24
An added advantage of t h i s approach i s t h a t l a c t o n e 23 can be a l k y l a t e d s t e r e o s e l e c t i v e l y w i t h l i t h i u m i s o p r o p y l c y c l o h e x y l amide and methyl i o d i d e t o g i v e 2,which was converted i n s e v e r a l s t e p s t o containing stereochemistry analogous t o t h a t p r e s e n t i n g i b b e r e l l i c a c i d . S e v e r a l examples have r e c e n t l y appeared which demonstrate t h e u t i l i t y o f t h e i n t r a m o l e c u l a r Diels-Alder r e a c t i o n for t h e c o n s t r u c t i o n of h e t e r o c y c l i c systems. Oppolzerl’ has e s t a b l i s h e d t h a t t h e r m a l l y g e n e r a t e d oquinodimethanes w i l l undergo i n t r a m o l e c u l a r Diels-Alder r e a c t i o n s t o produce p o l y c y c l i c amides (%, 2 6 ’+ 2 7 ) . The r e q u i r e d amides of t h e t y p e 26 a r e r e a d i l y o b t a i n a b l e from benzocyclobutanecarboxylic a c i d . This method h a s been a p p l i e d t o t h e t o t a l s y n t h e s i s of t h e a l k a l o i d d l - c h e l i d o n i n e . 12b
@p%[&--q4 $yH 26 -
-
27, -
85%
Gschwend has examined t h e i n t r a m o l e c u l a r c y c l i z a t i o n of p e n t a d i e n y l acrylamides for t h e p r e p a r a t i o n of p e r h y d r o i s ~ i n d o l i n e s ~and ~ a has a p l i e d t h i s r e a c t i o n t o t h e p r e p a r a t i o n o f aza- and d i a z a - s t e r o i d a n a l o g s . b A t y p i c a l example i s shown below. A number of t h e s e c y c l i z a t i o n s t a k e p l a c e under exceedingly mild c o n d i t i o n s .
2
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It
Ph \
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r
. 2c1
0
Yates l 4 h a s a p p l i e d t h e i n t r a m o l e c u l a r D i e l s - A l d e r r e a c t i o n t o t h e s y n t h e s i s of t h e 2,4a-ethano-2,3,4,4a-tetrahydroxanthene s y s t e m which i s p r e s e n t i n t h e n a t u r a l l y o c c u r r i n g compounds m o r e l l i n and gambogic a c i d . The d i e n o n e 29 was g e n e r a t e d by o x i d a t i o n of t h e xanthene 28 w i t h l e a d t e t r a a c e t a t e i n a c r y l i c a c i d t o g i v e t h e d e s i r e d system 2.
278
S e c t . VL
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F i n a l l y , an i n t r a m o l e c u l a r c y c l i z a t i o n of a bis-o-quinonemethide (32) was employed by Chapman15 i n t h e t o t a l s y n t h e s i s of t h e l i g n a n c a r p a n o n e (2). I n t h i s r e m a r k a b l y f a c i l e s y n t h e s i s t h e r e q u i r e d 2-quinonemethi.de was g e n e r a t e d -i n s i t u by p h e n o l i c c o u p l i n g o f two m o l e c u l e s of 2 - ( t r a n s with palladium d i c h l o r i d e . This l - p r o p e n y l ) - 4 , 5 methylenedioxyphenol i n 46% y i e l d and i n v o l v e s t h e p r o c e d u r e l e d d i r e c t l y t o carpanone i n t r o d u c t i o n of f i v e asymmetric c e n t e r s i n a s i n g l e s t e p .
(a) (a)
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f
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32
I n a d d i t i o n t o t h e f o r e g o i n g examples, t h e l i t e r a t u r e r e c o r d s a numb e r of o t h e r examples of i n t r a m o l e c u l a r D i e l s - A l d e r r e a c t i o n s . The examp l e s c i t e d , however, g i v e a n i n d i c a t i o n o f t h e ways i n which t h i s method can b e u s e d i n o r g a n i c s y n t h e s i s . References
1. For r e c e n t r e v i e w s of t h e D i e l s - A l d e r r e a c t i o n and i t s mechanism s e e ( a ) J . G . M a r t i n and R.K. H i l l , Chem. Rev., 61, 537 ( 1 9 6 1 ) ; (b) J. S a u e r , Angew. Chem. I n t e r n . E d . , 5, 2 1 1 ( 1 9 6 6 ) ; ( c ) J . S a u e r , 6 , 1 6 ( 1 9 6 7 ) ; ( d ) A.S. Onishckenku, Diene S v n t h e s i s , D a n i e l Davey, New York, 1 9 6 4 ; ( e ) J . H a m e r , e d . , l.4-C~, Academic P r e s s , New York, 1967; ( f ) Y . A . T i t o v , Russ. Chem. Rev., 2, 267 ( 1 9 6 2 ) ; ( g ) S . S e l t z e r , i n 1 > HHart and G . J . K a r a b a t s o s , E d . , Vol. 2 , p . 1, Academic Press, N e w York, 1968. 2 . K . A l d e r and W . Vogt, J u s t u s L i e b i g s Ann. Chem. , 571, 137 ( 1 9 5 1 ) . 3. H . O . House and T.H. C r o n i n , J . Org. Chem., 30, 1 0 6 1 ( 1 9 6 5 ) . 4, G. B r i e g e r , J . A m e r . Chem. S o c , , 85, 3783 ( 1 9 6 3 ) . 5. E . J . Corey and R.S. Glass, J. A m e r . Chem. S O C . , 89, 2600 ( 1 9 6 7 ) . 6. ( a ) L . H . K l e m m , J. H e t e r o c y c l i c Chem., 2, 1215 ( 1 9 7 2 ) ; ( b ) L. H . K l e m m , D.R. Olson and D.V. W h i t e , J . Org. Chem., 36, 3740 ( 1 9 7 1 ) and e a r l i e r papers i n t h i s series. 7. N . Fukamiya, M . Kato and A . Y o s h i k o s h i , J. Chem. S O C . , P e r k i n T r a n s . I , 1843 (1973). 8. G . F r a t e r , Helv. Chim. Acta, 5 7 , 172 ( 1 9 7 4 ) . 9. E . Wenkert and K. Naemura, S y z h e t i c Commun., 3, 45 ( 1 9 7 3 ) . 1 0 . E . J . Corey and R . L . D a n h e i s e r , T e t r a h e d r o n L e t t . , 4477 ( 1 9 7 3 ) . 11. E . J . Corey, T.M. Brennan and R . L . Carney , J . A m e r . Chem. SOC., 93, 7316 ( 1 9 7 1 ) . 1 2 . ( a ) W. O p p o l z e r , J . A m e r . Chem. SOC., 93, 3833, 3834 ( 1 9 7 1 ) ; ( b ) W . Oppolzer and K. Keller, i b i d . , 93, 3 8 3 r ( 1 9 7 1 ) .
G.,
..
Chap. 28
The Intramolecular Diels-Alder Reaction
Carlson
__ 2 79
( a ) H.W. Gschwend, A.O. Lee a n d H.-P. Meier, J . Org. Chem., 38, 2169 (1973); ( b ) H.W. Gschwend, Helv. Chim. Acta, 56, 1763 (1973): 14. D . J . B i c h a n and P . Yates, J . A m e r . Chem. S O C . , 94, 4773 (1972). 15: O.L. Chapman, M.R. E n g e l , J . P . S p r i n g e r and J . C T C l a r d y , i b i d ., 93, 6696 (1971).
13.
Chapter29-
Drug B i n d i n g and Drug A c t i o n
C o l i n F. C h i g n e l l N a t i o n a l H e a r t and Lung I n s t i t u t e , B e t h e s d a , Maryland
-
The i n t e r a c t i o n s of a drug w i t h a b i o l o g i c a l s y s t e m c a n be Introduction d i v i d e d i n t o two c a t e g o r i e s , namely s p e c i f i c and n o n s p e c i f i c . F o r most d r u g s t h e s p e c i f i c i n t e r a c t i o n i n v o l v e s c o m b i n a t i o n w i t h a t i s s u e compon e n t c a l l e d a r e c e p t o r . Such a n i n t e r a c t i o n u s u a l l y i n i t i a t e s a c h a i n of e v e n t s which f i n a l l y c u l m i n a t e s i n t h e e x p r e s s i o n of t h e p h a r m a c o l o g i c a l a c t i v i t y of t h e d r u g . However, b e f o r e a drug c a n combine w i t h i t s r e c e p t o r , i t w i l l i n t e r a c t w i t h many o t h e r body components. While t h e s e nons p e c i f i c i n t e r a c t i o n s do n o t r e s u l t i n any e x p r e s s i o n of t h e pharmacologi c a l a c t i v i t y , they a r e n e v e r t h e l e s s important, s i n c e they modulate t h e amounts of drug which f i n a l l y a r r i v e a t i t s r e c e p t o r s i t e . It i s t h e p u r p o s e of t h i s a r t i c l e t o examine t h e ways i n which b i n d i n g t o plasma and t i s s u e p r o t e i n s c a n a l t e r t h e d i s t r i b u t i o n , metabolism and e x c r e t i o n of a d r u g . The b i n d i n g of d r u g s t o plasma p r o t e i n s . The i n t e r a c t i o n of a d r u g (D) w i t h a p r o t e i n (P) may b e d e s c r i b e d i n terms of t h e Mass-Action Law by t h e following expression where k l and k-1 a r e t h e r a t e c o n s t a n t s f o r a s s o c i a t i o n and d i s s o c i a t i o n r e s p e c k-1 tively. I f t h i s p r o t e i n h a s m u l t i p l e , mut u a l l y i n d e p e n d e n t , b i n d i n g s i t e s and i f a l l t h e s i t e s have t h e same a f f i n i t y f o r t h e d r u g , t h e n i t may be shown that
kl
D + P
'
(1)
DP
CDB1 l ~ (~W-LDB!) l
where [ D F ~and [DR] a r e t h e molar conc e n t r a t i o n s of f r e e and bound d r u g , res p e c t i v e l y , K is t h e a s s o c i a t i o n cons t a n t f o r t h e i n t e r a c t i o n , n i s t h e number of d r u g b i n d i n g s i t e s p e r mole of p r o t e i n and [PI i s t h e molar c o n c e n t r a t i o n of p r o t e i n . It may r e a d i l y be s e e n from e q u a t i o n 2 t h a t , i n t h e a b s e n c e of c o m p l i c a t i n g f a c t o r s , s u c h a s t h e p r e s e n c e o f o t h e r d r u g s o r endogenous l i g a n d s , t h e d e g r e e of d r u g b i n d i n g u n d e r e q u i l i b r i u m c o n d i t i o n s depends on (i) t h e t o t a l c o n c e n t r a [DF] o f d r u g i n t h e s y s t e m , and (ii) t h e a f f i n i t y t i o n DT ( i . e . [DB] c o n s t a n t (K) € o r t h e i n t e r a c t i o n . S i n c e , f o r any g i v e n s y s t e m , t h e v a l u e s f o r n,[P] and K a r e c o n s t a n t , i t i s o b v i o u s t h a t t h e t o t a l d r u g c o n c e n t r a t i o n i s p r o b a b l y t h e most i m p o r t a n t f a c t o r t h a t d e t e r m i n e s how much of a d r u g i s bound. The e f f e c t o f [D.,.1 on t h e p e r c e n t b i n d f n g o f a h y p o t h e t i c a l d r u g t h a t i s bound s o l e y t o plasma albumin is shown i n F i g . 1.
K
=
+
(*)
Chap. 29
Drug Binding and Drug Action
2 81 -
Chignell
06 \
\
'\.
/
I I
I I
I I I I
l'DF
---. I I 8 / J
* O t
,__-**,
0 0
Fig. 1. The e f f e c t of drug conc e n t r a t i o n , [DT], on p e r c e n t bindi n g of a drug i n plasma. Each curve represents a d i f f e r e n t value of K. C a l c u l a t e d from e q u a t i o n 5 a s s ming t h a t n = 1 and P = 5.8 x Adapted from r e f e r e n c e 6. 10'
x.
.ooos
I
.a01
F i g . 2. The e f f e c t of drug concent r a t i o n [DT] on f r e e [DF] and bound phenylbutazone i n plasma. Calr c u l a t e d from e q u a t i o n 5 assuming n = 1, P = 5.8 x M and K = lo5.* Adapted from r e f e r e n c e 6.
[w]
It may be s e e n from F i g . 1 t h a t a s t h e c o n c e n t r a t i o n of drug inc r e a s e s t h e per c e n t of drug bound d e c r e a s e s . Whjle t h i s e f f e c t is q u i t e small f o r d r u g s t h a t have a weak a f f i n i t y (K-10 ) f o r plasma albumin, i t c a n be q u i t e l a r g e for d r u g s t h a t a r e s t r o n g l y bound. Brodie and Hogbed have shown t h a t t h e f r e e c o n c e n t r a t i o n of phenylbutazone i n plasma i n c r e a s e s d r a m a t i c a l l y when [DTJ exceeds t h e c o n c e n t r a t i o n where t h e plasma p r o t e i n s become s a t u r a t e d . The t h e o r e t i c a l v a l u e s of [ D B ~and [DFl f o r phenylbutazone a r e shown i n F i g . 2.
The e f f e c t s of plasma p r o t e i n b i n d i n g on drug d i s t r i b u t i o n and k i n e t i c s a r e b e s t understood i n terms of a simple two compartment ~ y s t e m . ~ - ~ (i) t h a t t h e drug i s g i v e n T h i s model makes t h e f o l l o w i n g assumptions6: i n t r a v a s c u l a r l y , (ii) t h a t t h e drug is d i s t r i b u t e d i n t o two aqueous comp a r t m e n t s , i . e . plasma water (volume V1) and a second compartment (volume V2) which r e p r e s e n t s t h a t p a r t of t h e r e s i d u a l body water which i s a c c e s s i b l e t o t h e d r u g , ( i i i ) t h a t p r o t e i n b i n d i n g is governed s o l e l y by t h e Mass-Action Law w i t h i n t h e plasma w a t e r compartment, ( i v ) t h a t t h e r a t e of e q u i l i b r a t i o n between t h e tm compartments i s much f a s t e r t h a n t h e r a t e of drug e l i m i n a t i o n , (v) t h a t drug metabolism and e x c r e t i o n a r e f i r s t - o r d e r p r o c e s s e s , ( v i ) t h a t drug metabolism and e x c r e t i o n a r e a f u n c t i o n of t h e c o n c e n t r a t i o n of unbound drug and ( v i i ) t h a t t h e r a t e c o n s t a n t s f o r
282 -
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metabolism and e x c r e t i o n , km and k e , can be combined t o g i v e k e l , the r a t e constant f o r elimination,
I f t h e s e assumptions h o l d , t h e n t h e body c o n t e n t a t any g i v e n time is g i v e n by Body c o n t e n t = V1cDT1 4. V2[DF1
(41
Plasma p r o t e i n b i n d i n g and drug d i s t r i b u t i o n . Martin5 has d e r i v e d t h e f o l l o w i n g e q u a t i o n from t h e r e l a t i o n s h i p g i v e n i n e q u a t i o n 2 .
)
I f i t i s assumed t h a t f o r a 70 kg man, plasma volume is 3 1, e x t r a c e l l u l a r f l u i d volume i s 9 1 and t o t a l body w a t e r i s 39 1, t h e n e q u a t i o n 5 may be used i n c o n j u e t i o n w i t h e q u a t i o n 4 t o p r e d i c t t h e e f f e c t of p r o t e i n b i n d i n g on and [ D F ~ drug d i s t r i b u t i o n . I n Fig. 3 t h e r e l a t i o n s h i p between [DJ and t o t a l body c o n t e n t of a drug i s shown f o r drug A which i s l i p i d i n s o b l e and i s unable t o c r o s s c e l l membranes and t h u s is r e s t r i c t e d t o e x t r a It may be s e e n c e l l u l a r f l u i d and drug B which has a c c e s s t o c e l l water. from F i g . 3 t h a t when t h e t o t a l body c o n t e n t is low (about 1 mmole) t h e r e is l i t t l e d i f f e r e n c e between [QJand [ S ] f o r d r u g s A and B , because b o t h a r e almost completely bound t o plasma albumin and a r e t h e r e b y confined t o t h e plasma compartment. However, a s t h e body load of drug d e c r e a s e s t h e plasma p r o t e i n b i n d i n g s i t e s become s a t u r a t e d and t h e unbound drug p a r t i t i o n s between t h e plasma w a t e r and t h e second compartment. A t h i g h body l o a d s t h e f r e e c o n c e n t r a t i o n of drug B i s about o n e - t h i r d of t h a t f o r drug A , because B h a s a c c e s s t o a l a r g e r e x t r a v a s c u l a r compartment.
[DTl = b F ' I
(1 i- K-l
nP cDF1
-k
.0012.001
-
(5)
F i g . 3. R e l a t i o n s h i p between t o t a l amount of drug i n t h e bod , c o n c e n t r a t i o n of d r u g , [DT5, i n plasma and c o n c e n t r a t i o n of unbound d r u g , ~ D F ], i n t f s s u e f l u i d . C a l c u l a t e d from e q u a t i o n s 4 , 5 and 10 assumi K = lo5, n = 1, P = 5.8 x 10' M, V 1 = 3 l i t e r s . Drug A, (-), V2 = 9 l i t e r s . Drug By (----), V2= 39 l i t e r s . Drug C, 6 -), V2 = 39 l i t e r s , V3 = 100 l i t e r s , Adapted from r e f e r e n c e 6.
2
0
.ooz
.004
-001
Body content (moles)
Chap. 29
Drug B i n d i n g and Drug A c t i o n
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283
T i s s u e b i n d i n g and drug d i s t r i b u t i o n . B e f o r e examining t h e e f f e c t of t i s s u e b i n d i n g on t h e d i s t r i b u t i o n of a d r u g , i t i s n e c e s s a r y t o i n t r o d u c e t h e c o n c e p t of a p p a r e n t volume o f d i s t r i b u t i o n , VA, c a l c u l a t e d from t h e following expression,
vA
=
T o t a l d o s e o f d r u g i n body w a t e r a t a n y time
(6)
[DFI
The a p p a r e n t volume of d i s t r i b u t i o n may b e d e f i n e d a s t h a t volume o f body f l u i d s which would h o l d t h e d r u g i n s o l u t i o n a t t h e same c o n c e n t r a t i o n a s i n body w a t e r . It s h o u l d b e emphasized t h a t VA i s n o t t h e same a s t h e g e n e r a l l y a c c e p t e d d e f i n i t i o n of volume of d i s t r i b u t i o n which i s c a l c u E q u a t i o n s 4 and 6 may be combined t o g i v e l a t e d from [DT] and n o t [DFJ.
It may b e s e e n from e q u a t i o n 8 t h a t i f a d r u g i s bound t o plasma p r o t e i n s VA w i l l b e g r e a t e r t h a n t h e t r u e volume of d i s t r i b u t i o n . The b i n d i n g of d r u g s t o t i s s u e s w i l l a l s o i n c r e a s e t h e volume of d i s t r i b u t i o n . I n t h e o r y i t s h o u l d b e p o s s i b l e t o i n t r o d u c e terms i n t o e q u a t i o n 8 s i m i l a r t o t h a t f o r plasma p r o t e i n b i n d i n g which would a c c u r a t e l y p r e d i c t t h o e f f e c t of I n p r a c t i c e t h i s is i m p o s s i b l e due t o t h e l a r g e t i s s u e b i n d i n g on VA. number of d i f f e r e n t t i s s u e components t h a t c a n i n t e r a c t w i t h d r u g s . I n a d d i t i o n , l i t t l e i s known a b o u t t h e n o n s p e c i f i c t i s s u e b i n d i n g s i t e s , a l though some a t t e m p t s a r e now b e i n g made t o s t u d y t h i s i n t e r a c t i o n (M. B i c k e l , p e r s o n a l communication). I n t h e a b s e n c e of s p e c i f i c b i n d i n g d a t a , t i s s u e b i n d i n g c a n b e e x p r e s s e d a s a t h i r d compartment t h a t i s a c c e s s i b l e t o t h e d r u g . E q u a t i o n s 4 and 8 may t h e n be r e w r i t t e n t o g i v e e q u a t i o n s 9 and 10 r e s p e c t i v e l y , Body c o n t e n t = V1[DT!
+
V2[DF]
+ V3[DF]
(9)
(10)
The e f f e c t of V 3 on t h e c o n c e n t r a t i o n of f r e e and bound d r u g i n plasma is shown i n F i g . 3 (drug C).
Drug d i s p l a c e m e n t and d r u g d i s t r i b u t i o n . It h a s o f t e n b e e n s u g g e s t e d t h a t t h e d i s p l a c e m e n t o f one h i g h l y bound d r u g from i t s plasma b i n d i n g s i t e s b y a n o t h e r d r u g c a n c a u s e a l a r g e i n c r e a s e i n t h e plasma c o n c e n t r a t i o n of f r e e drug. More r e c e n t l y , however, t h i s p o s t u l a t e h a s b e e n c a l l e d i n t o q u e s t i o n . 7-10 The two compartment model c a n be used t o p r e d i c t t h e e f f e c t of s u c h a drug-drug i n t e r a c t i o n (Table 1). The d a t a i n T a b l e 1 i n d i c a t e t h a t , a t t h e lower body l o a d , d r u g A would show t h e l a r g e s t ( 8 - f o l d ) inc r e a s e i n f r e e drug C o n c e n t r a t i o n . I n c o n t r a s t , d r u g B , which h a s a l a r g e r volume of d i s t r i b u t i o n , shows o n l y a t h r e e f o l d i n c r e a s e i n f r e e d r u g c o n c e n t r a t i o n . However, d r u g C , which i s a l s o bound t o t i s s u e p r o t e i n s , would e x h i b i t a s i x f o l d i n c r e a s e i n f r e e c o n c e n t r a t i o n i f d i s p l a c e d from b o t h plasma and t i s s u e b i n d i n g sites. Displacement of C from j u s t t h e plasma b i n d i n g s i t e s would r e s u l t i n a t w o f o l d i n c r e a s e i n t h e plasma
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c o n c e n t r a t i o n of f r e e C , i f t h e d i s p l a c e d drug was t a k e n up by t h e t i s s u e s . A t t h e higher body l o a d , drug C would show a f o u r f o l d i n c r e a s e i n f r e e drug c o n c e n t r a t i o n when d i s p l a c e d from both plasma and t i s s u e b i n d i n g s i t e s . Very l i t t l e change would be observed by t h e displacement of any of t h e drugs s o l e l y from plasma p r o t e i n s . These c a l c u l a t i o n s show t h a t t h e change i n f r e e plasma c o n c e n t r a t i o n of a d i s p l a c e d drug depends upon (i) volume of d i s t r i b u t i o n , ( i i ) whether o r not t h e r e is concomitant d i s placement from t i s s u e b i n d i n g s i t e s and ( i i i ) t h e body load of drug. The most dangerous c l i n i c a l i n t e r a c t i o n would be one i n which displacement occurred from b o t h plasma and t i s s u e b i n d i n g s i t e s . Table 1 E f f e c t of drug displacement on [DFj and [DT] Body c o n t e n t
Drug
IDTI+
1 millimole [DFl' [D1*
A
30.3
1.02
8.33
8.17
109.0
B
24.4
0.69
2.38
3.45
79.6
C
16.1 16.1
0.37 0.37
2.38$ 0.70**
6.43 1.89
52.3 52.3
8 millimoles b F p [DJ*
[D]/cDFJ
1
b]/bF'J
52.4
66.60
1.27
15.2
19.00
1.25
19.004 5.63**
4.13 1.22
4.6 4.6
'Drug concentrations (M x 105) calculated from Fig. 3 Plasma drug concentration (M x 105) assuming complete displacement from binding sites. *Calculated assuming complete displacement from plasma and tissue binding sites. Jrk Calculated assuming complete displacement from plasma binding sites only.
*
The e f f e c t of drug b i n d i n g on k i n e t i c s . I f , a s i s suggested by t h e twocompartment model, drug e l i m i n a t i o n , whether by metabolism o r e x c r e t i o n , is a f i r s t - o r d e r p r o c e s s depending on [DFj and having a r a t e c o n s t a n t k e l , t h e n we c a n w r i t e where [DFjo is the v a l u e of [DF] a t t h e time of a d m i n i s t r a t i o n assuming i n s t a n taneous e q u i l i b r a t i o n . Since e l i m i n a t i o n i s a f i r s t - o r d e r p r o c e s s , i t may be expressed i n terms of c l e a r a n c e where c l e a r a n c e i s t h a t volume of plasma water which i s e f f e c t i v e l y c l e a r e d of a drug i n u n i t time. Furthermore, B u t l e r 1 1 has pointed o u t t h a t [DF]
3
[DF]'
e'kelt
6
(11)
Clearance (ml/min) VA ( m l )
Chap. 29
Drug B i n d i n g and Drug A c t i o n
285
Chignell
From e q u a t i o n 11 i t c a n be shown t h a t tf
-*
= In Ice 1
(13)
When e q u a t i o n s 13 and 14 a r e combined, t h e f o l l o w i n g r e l a t i o n s h i p i s ohtained
VA In
t'
Clearance
(14)
I t h a s been p r e v i o u s l y shown t h a t b i n d i n g t o plasma end t i s s u e p r o t e i n s , by r e d u c i n g [Dp], w i l l i n c r e a s e VA. I f t h e r a t e c o n s t a n t f o r d i s s o c i a t i o n of t h e d r u g p r o t e i n complex (k-1) i s v e r y r a p i d , t h e d r u g w i l l b e e l i m i n a t e d a s though it were a l l f r e e i n one l a r g e component of volume VA. Und e r t h e s e c o n d i t i o n s t h e two-compartment s y s t e m behaves k i n e t i c a l l y a s a pseudo one-compartment model.
F i g . 4. P o t e n t i a l e f f e c t o f unbound d r u g c o n c e n t r a t i o n , in t i s s u e f l u i d w i t h t i m e . Each l i n e r e p r e s e n t s a d i f f e r e n t v a l u e of per c e n t b i n d i n g which is assumed n o t t o change w i t h time. The i n t e r c e p t o n t h e o r d i n a t e , [DF!', was c a l c u l a t e d from e q u a t i o n 4 . S l o p e of l i n e c a l c u l a t e d from e q u a t i o n s 6 and 14. Dose = moles, V 1 = 3 l i t e r s , V2 = 9 lit e r s , c l e a r a n c e = 0.13 l i t e r s / m i n . Adapted from r e f e r e n c e 4.
[%I,
0
100
200
300
400
500
Time (mid
The e f f e c t o f v a r i o u s d e g r e e s of p r o t e i n b i n d i n g on t h e plasma decay c u r v e f o l l o w i n g a s t a n d a r d d o s e of d r u g i s shown i n F i g . 4. It w i l l be s e e n t h a t p r o t e i n b i n d i n g h a s two e f f e c t s : (i) i t r e d u c e s t h e i n i t i a l c o n c e n t r a t i o n of f r e e d r u g , [DF'j0, and (ii) i t i n c r e a s e s tf so t h a t [DF] f a l l s l e s s r a p i d l y . The f a l l i n drug c o n c e n t r a t i o n w i t h t i m e w i l l be l i n e a r (Fig. 4 ) o n l y i f VA has t h e same v a l u e a t a l l d r u g c o n c e n t r a t i o n s . However, a s i s shown i n F i g . 1, t h e b i n d i n g of d r u g s w i t h a h i g h a f f i n i t y f o r plasma albumin changes w i t h [DT] s o t h a t a t h i g h v a l u e s of [DT- t h e v a l u e of V A w i l l d e c r e a s e and e l i m i n a t i o n w i l l b e more r a p i d (Eq. 13).
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Burns and co-workers12 have r e p o r t e d t h a t , when a h i g h d o se of p h e n y l b u t a zone i s g i v e n t o a p a t i e n t who a l r e a d y h a s a " p l a t e a u " blood l e v e l of t h e d r u g , t h e r a t e of e l i m i n a t i o n , which i s i n i t i a l l y v e r y r a p i d , e v e n t u a l l y s low s t o i t s p r e v i o u s v a l u e . ,KrUger-Thiemer3 h a s developed a r a t e equat i o n f o r t h e change of [ D F ~ w i t h t i m e u s i n g t h e pseudo one-compartment model t o g e t h e r w i t h e q u a t i o n 5. T h i s e x p r e s s i o n was s o l v e d w i t h t h e a i d of a d i g i t a l computer, and t h e l i n e s g e n e r a t e d a r e shown i n F i g . 5. The model a c c u r a t e l y p r e d i c t e d t h e k i n e t i c b e h a v i o r of a s i n g l e o r a l d o se of s u l f a o r thod ime t h o x i ne s 4
.
F i g . 5. P o t e n t i a l e f f e c t of p l a s ma b i n d i n g on t h e chanpe of [DF] i n t i s s u e f l u i d w i t h time. Model curves generated according t o the pseudo one-compartment model assuming a n i n i t i a l d r u g d o s e of 5 mmoles, V 1 = 3 l i t e r s , V2 = 40 l i t e r s , n = 2 , P = 10-3 M, k i d n e y c l e a r a n c e = 0.82 l i t e r s / h r . 3 ~ 4
Time (hr)
Some o t h e r f a c t o r s not c o n s i d e r e d i n t h e pseudo one-compartment model. T h i s model cannot p r e d i c t t h e phar maco k i n e t i c s of d r u g s t h a t a r e r a p i d l y me t abol i zed by l i v e r enzymes o r e l i m i n a t e d by k i d n e y t r a n s p o r t sy st e m s. G i l l e t t e h a s r e c e n t l y p o i n t e d out8 t h a t , f o r d r u g s whose c l e a r a n c e by t h e l i v e r o r ki dney appr oaches t h e blood f l o w t h r o u g h t h a t o r g a n , b i n d i n g t o plasma p r o t e i n s may enhance t h e r a t e of e l i m i n a t i o n . T h i s e f f e c t i s i l l u s t r a t e d i n T able 2 , where t h e a p p a r e n t r a t e c o n s t a n t s o f e l i m i n a t i o n ( r a t e / c o n c e n t r a t i o n ) a t v a r i o u s c o n c e n t r a t i o n s of two h y p o t h e t i c a l d r u g s have been c a l c u l a t e d . Cne of t h e d r u g s i s c o m p l e t e l y c l e a r e d from t h e plasma a s i t p a s s e s thr ough t h e l i v e r , w h i l e t h e o t h e r i s e l i m i n a t e d so s l o w l y t h a t o n l y 1%of t h e unbound dr u g i s e x t r a c t e d a s i t p a s s e s t h r o u g h t h e l i v e r . In making t h e s e c a l c u l a t i o n s , i t was a l s o assumed ( i ) t h a t t h e d i s t r i b u t i o n phas es had been com p l e t e d , (ii) t h a t t h e unbound forms of t h e dr ug d i s t r i b u t e d i n body w a t e r , (iii) t h a t t h e k i n e t i c volumes of d i s t r i b u t i o n (VD) were t h e same a s t h e e q u i l i b r i u m a p p a r e n t volumes of d i s t r i b u t i o n (VA), i . e . t h a t t h e r a t e c o n s t a n t s f o r t h e t r a n s f e r of d r u g s from t h e body compartment t o t h e blood were l a r g e compared w i t h t h e
Chap. 29
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Chignell
Drug B inding and Drug A c t i o n
a p p a r e n t r a t e c o n s t a n t s of e l i m i n a t i o n , ( i v ) t h a t t h e r a t i o KnP/(l + KnP) e q u a l s 0.98 and (v) t h a t t h e blood f low r a t e t h r o u g h t h e l i v e r i s 1.6 lit e r s / m i n . 8 When t h e t o t a l c o n c e n t r a t i o n s of t h e d r u g s i n plasma a r e i n f i n i t e l y g r e a t e r t h a n t h e c o n c e n t r a t i o n s of t h e bound f o r m s, VD would b e 50 l i t e r s and k e l f o r t h e r a p i d l y e l i m i n a t e d d r u g would b e 100 times t h a t f o r t h e s l o w l y e l i m i n a t e d drug. However, a s t h e plasma l e v e l s of t h e d r u g s d e c r e a s e , t h e r a t i o of t h e t o t a l amounts of t h e d r u g s i n t h e body t o [DTJ d e c r e a s e s , whereas t h e r a t i o of t h e t o t a l amount of t h e d r u g s i n t h e body t o [DF] i n c r e a s e s . Thus k e l f o r t h e r a p i d l y e l i m i n a t e d d r u g i n c r e a s e s a s t h e plasma l e v e l of t h e dr ug d e c l i n e s ( s e e Eq. 1 2 ) , whereas k e l f o r t h e s l o w l y e l i m i n a t e d drug d e c r e a s e s . T able 2 R e l a t i o n s h i p s between p r o t e i n b i n d i n g o f d r u g s i n plasma, t h e e x t r a c t i o n r a t i o i n l i v e r , and t h e a p p a r e n t r a t e c o n s t a n t of elim i n a t i o n * t t * j r
100% of T o t a l amount i n plasma is extracted Bound i n plasma k (%) (min-1)
Drug concentration
(MI
0 32. 6 82. 0 96.0 97.8 97.98
00 /Ka 100 /Ka lO/Ka 1/Ka O.l/Ka O.Ol/Ka
0.032 0,045 0.218 0. 221 0. 245 0.250
1%of Unbound amount i n plasma i s e x t r a c t e d
k
tk (min)
(min- 1 x 103)
21.6 15.4 5.9 3.14 2.84 2.80
0.320 0.304 0.215 0.0865 0.0543 0.0505
tf (min)
2 160 2280 3220 8000 12,750 13 ,700
( d a y s) 1.5 1.6 2.2 5.5 8.8 9.5
*Unbound drug d i s t r i b u t e d w i t h body w a t e r ; VD = 50 l i t e r s . tKnP/(l KnP) = 0.98. $Blood f l o w r a t e = 1 . 6 l i t e r s / m i n . *Taken from r e f e r e n c e 8 .
+
G i l l e t t e8 h a s a l s o examined t h e e f f e c t o f t i s s u e b i n d i n g on t h e e l i m i n a t i o n of a d r u g t h a t i s r a p i d l y m e t a b o l i z e d by t h e l i v e r . I n T a b l e 3 t h e h a l f - l i v e s of a s e r i e s of h y p o t h e t i c a l d r u g s t h a t have d i f f e r e n t k i n e t i c volumes of d i s t r i b u t i o n (V,) b u t a r e c o m p l e t e l y c l e a r e d by t h e l i v e r a r e shown. The minimal h a l f - l i f e of a drug t h a t i s e l i m i n a t e d by t h e l i v e r and has a V,/kg of 20 l i t e r s / k g i n man would be a b o u t 10 h r . If o n l y 50% of t h e dr ug were c l e a r e d a s i t p a sse d t h r o u g h t h e l i v e r , t h e b i o l o g i c a l h a l f - l i f e would b e 20 h r . Thus i t would b e a m i s t a k e t o assume t h a t t h e c l e a r a n c e of a dr ug is always slow when i t h a s a l o n g b i o l o g i c a l half- l i f e .
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Table 3 R e l a t i o n s h i p s between VD, l i v e r plasma flow, e x t r a c t i o n r a t i o s , and
100% (hr)
t#
5.0
0.071 0.214 0.71 1.0 2.0 4.0 10.0 20.0 40.0
15 50 70 140 280 700 1400 2800
0.036 0.11 0.36 0.5 1.0 2.0 5.0 10.0 20.0
tf*t
50% tf ( h r )
25% tf ( h r )
10% t# ( h r )
0.072 0.22 0.72 1.0 2.0
0.14 0.44 1.4 2.0 4.0 8.0 20.0 40.0 80.0
0.36 1.1 3.6 5 10 20 50 100 200
4.0 10.0 20.0
40.0
*For t h e s e c a l c u l a t i o n s , i t was assumed t h a t t h e blood flow r a t e was 1 . 6 l i t e r s / m i n in a 70-kg man, which is e q u i v a l e n t t o 96.0 l i t e r s l h r and t o 1.3 7 li t e r s / k g / h r ?Taken from r e f e r e n c e 8.
.
In c o n s i d e r i n g t h e p o s s i b l e f a c t o r s t h a t a f f e c t t h e metabolism of h i g h l y bound d r u g s , i t would appear t h a t t h e r a t e of d i s s o c i a t i o n of a d r u g - p r o t e i n complex might l i m i t t h e e l i m i n a t i o n of t h e bound form of t h e drug. However, G i l l e t t e has shown8 t h a t f o r t h i s mechanism t o be impcrtent Table 4 Rate constants f o r t h e formation and d i s s o c i a t i o n of drug-protein complexes 14 Drug
41: (5,7-Disulfonic a c i d na ph- 2- 01)-1 - a z o j benzene sulfonamide (I)
-
-
1 Na ph t h o 1-2 su 1f o n i c acid-4-[4- (4'-azo benzene- azo)l-phenyla r s o n i c a c i d (11)
Protein
kl
k- 1
(~-1 sec-1)
(sec-1)
(M- 1)
Carbonic anhydrase
5.8 x
lo5
0.075
7.7 x
lo6
Plasma albumin
3.6 x
lo5
2.5
1.4
105
t h e h a l f - t i m e f o r d i s s o c i a t i o n of t h e complex would have t o be e q u a l t o o r g r e a t e r than t h e mean t r a n s i t time of t h e blood through t h e s i n u s o i d s of t h e l i v e r . In t h e dog t h i s t r a n s i t t i m e i s about 10 sec13 so t h a t about
Chap. 29
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50% of a d r u g - p r o t e i n complex could d i s s o c i a t e i f k - 1 i s a p p r o x i m a t e l y 0.07 s e c . The r a t e c o n s t a n t s € o r two d r u g s t h a t i n t e r a c t w i t h c a r b o n i c a n hydrase and plasma albumin a r e shown i n T a b l e 4. I f t h e t r a n s i t time of blood t hr ough t h e human l i v e r i s a b o u t 10 s e c , t h e c l e a r a n c e o f d r u g 11, bound t o albumin, would n o t b e l i m i t e d by k - 1 , whereas o n l y a b o u t 50% o f d rug I , bound t o c a r b o n i c a n h y d r a s e , c o u l d b e removed. It sh o u l d b e emp h a s i z e d , however, t h a t i t i s i m p o s s i b l e t o p r e d i c t from t h e a s s o c i a t i o n c o n s t a n t (K) a l o n e t h a t k-1 f o r a g i v e n d r u g - p r o t e i n i n t e r a c t i o n i s r a t e l i m i t i n g . A c omplete k i n e t i c a n a l y s i s o f t h e sy st e m must b e made.
-
Concl usi ons Binding t o plasma p r o t e i n s c a n have a profound e f f e c t on t h e d i s t r i b u t i o n of a drug and on t h e r a t e a t which i t i s e l i m i n a t e d from t h e body. Plasma b i n d i n g h a s a l e s s e r e f f e c t on a drug t h a t i s a b l e t o e n t e r c e l l s t h a n on one t h a t is r e s t r i c t e d t o e x t r a c e l l u l a r f l u i d . Plasma b i n d i n g has a marked e f f e c t on t h e p r o p e r t i e s of a d r u g o n l y i f t h e K f o r t h e Plasma b i n d i n g may i n c r e a s e d r u g - p r o t e i n i n t e r a c t i o n is g r e a t e r t h a n 104 t h e r a t e of e l i m i n a t i o n of d r u g s t h a t a r e c o m p l e t e l y c l e a r e d by o r g a n s s u c h a s t h e l i v e r or kidney. The e f f e c t o f d i s p l a c e m e n t o f one d r u g by a n o t h e r o n t h e f r e e c o n c e n t r a t i o n of f i r s t d r u g is governed by t h e volume of d i s t r i b u t i o n of t h e d r u g s , t h e body load of t h e f i r s t d r u g , w h e t h e r t h e f i r s t dr ug i s a l s o bound t o t i s s u e s and w h e t h e r t h e second d r u g d i s p l a c e s t h e f i r s t from b o t h t i s s u e and plasma b i n d i n g s i t e s .
.
R ef er en c e s
1. 2. 3. 4. 5. 6.
7. 8. 9. 10. 11. 12.
13. 14.
B.B. B r o d i e and C.).. M. Hogben, J.Pharm.Pharmacol., 2, 345 (1957). H.M. Solomon and J. J. S c h r o g i e , BiochemPharmacol. , 1219 (1967). E. KrUger-Thiemer, A r z n e i m i t t e l - F o r s c h . , 1431 (1966). E. KrUger-Thiemer , W. D i l l e r and P. Burger , Antimicrob.AgentsChemother. a 183 (1966). B.K. M a r t i n , N a t u r e , 274 (1965). P.M. Keen, Handb.Exp.Pharmacol., 28, P t . 1, 213 (1971) J.R. G i l l e t t e , Importance of Fundamental P r i n c i p l e s i n Drug Evaluat i o n . D.H. T edes chi and R.E. T ede sc h i ( e d s . ) Raven P r e s s , New York. J . R . G i l l e t t e , Ann. N.Y. Acad. S c i . , 226, 6 (1973). W.M. W ar dell, I n t e r n a t i o n a l Symposium on Drug I n t e r a c t i o n s , M i l a n , 1973, S. G a r a t t i n i ( e d . ) Raven P r e s s , New York. C.F. C h i g n e l l and D.K. S t a r k w e a t h e r , Pharmacology, 2 , 235 (1971). T.C. B u t l e r , Fed.Proc., 1158 (1958). J.J. Burns, R.K. Rose, T. Chenkin, A. Goldman, A. S c h u l e r t , B.B. B r o d i e , J.Pharmacol.Exp.Ther., 109, 346 (1953). C. Goresky, h e r . J . Phys io1. , 3 ,606 (1963). P.W. T a y l o r , MethodsPharmacol. , 2 , 351 (1972).
16,
1965,
207,
17,
16,
C h a p t e r 30.
Cytochrome P-450 Monoxygenases and Drug Metabolism
J.E. Tomaszewski, D.M. J e r i n a , and J.W. Daly N a t i o n a l I n s t i t u t e o f A r t h r i t i s , Metabolism and D i g e s t i v e Diseases, N a t i o n a l I n s t i t u t e s of H e a l t h , Bethesda, Maryland 20014
-
Introduction O x i d a t i v e metabolism of f o r e i g n compounds h a s been known t o o c c u r i n l i v i n g organisms f o r more t h a n one hundred years.1, 2 Howe v e r , i t h a s o n l y been d u r i n g t h e p a s t twenty y e a r s t h a t a group of u n u s u a l hemoproteins d e s i g n a t e d by t h e t e r m cytochrome P-450 have been e s t a b l i s h e d a s t h e t e r m i n a l oxygenases i n t h e r e s p i r a t o r y c h a i n r e s p o n s i b l e f o r many of t h e s e o x i d a t i o n s . One such cytochrome P-450, which c a t a l y s e s t h e o x i d a t i o n of camphor, h a s r e c e n t l y been i s o l a t e d from s o i l b a c t e r i a and ~ r y s t a l l i z e d . ~I n h i g h e r organisms t h e P-450 enzymes a r e membranal p r o t e i n s and d i s p l a y a wide spectrum o f o x i d a t i v e a c t i v i t y towards d r u g s , p e s t i c i d e s and o t h e r x e n o b i o t i c compounds. T h e i r s o l u b i l i z a t i o n and i s o l a t i o n as p u r e enzymes have proven d i f f i c u l t . High levels o f P-450 a r e found i n microsomal f r a c t i o n s from l i v e r and a d r e n a l c o r t e x and i n m i t o c h o n d r i a 1 f r a c t i o n s from t h e l a t t e r t i s s u e . These hemoproteins have a l s o been i m p l i c a t e d i n t h e b i o s y n t h e s i s of c h o l e s t e r o l , s t e r o i d hormones and b i l e a c i d s . 4 The p r e s e n t a r t i c l e h i g h l i g h t s c e r t a i n a s p e c t s of t h e c h e m i s t r y o f mammalian P-450 monoxygenases w i t h emphasis on t h e i r r o l e i n d r u g metabolism.
-
S p e c t r a l P r o p e r t i e s of Cytochrome P-450 Monoxygenases L i v e r microsomal f r a c t i o n s c o n t a i n two m a j o r hemoproteins: cytochrome b and cytochrome P-450. Both e x h i b i t an a b s o r b t i o n maximum n e a r 420nm, z u t t h e l a t t e r hemoprotein i s a t y p i c a l i n t h a t t h e spectrum of t h e reduced c a r b o n monoxide-complexed pigment shows a s t r o n g absorbance band a t 450nm; hence t h e o r i g i n o f t h e d e s i g n a t i o n P-450. The cytochrome monoxygenases p r e s e n t i n microsomes from a n i m a l s p r e t r e a t e d w i t h p o l y c y c l i c hydrocarbons d i f f e r s u b s t a n t i a l l y from t h e P-450 monoxy e n a s e s t h a t a r e p r e s e n t i n u n t r e a t e d o r p h e n o b a r b i t a l - t r e a t e d a n i m a l s m 5 - f 3 S i n c e t h e spectrum of t h e r e d u c e d c a r b o n monoxide complex o f t h i s new c l a s s of cytochromes h a s a maximum a t This 448nm i n s t e a d of 450nm, t h e t e r m cytochrome P-448 was i n t r o d u c e d . new c l a s s o f cytochromes h a s a l s o been t e r m e d p l - 4 5 0 . 6 Distinctive spectral have been n o t e d a f t e r a d d i t i o n of v a r i o u s compounds t o t h e o x i d i z e d form o f cytochrome P-450. The compounds which e l i c i t such s p e c t r a l changes have been p l a c e d i n two c a t e g o r i e s : Type I s u b s t r a t e s s u c h a s h e x o b a r b i t a l , ethylmorphine and t e s t o s t e r o n e which c a u s e l o s s of a b s o r b a n c e a t 420nm w i t h a c o n c o m i t a n t i n c r e a s e a t 385nm, and Type I1 s u b s t r a t e s such as a n i l i n e , p y r i d i n e and a c e t a n i l i d e which c a u s e an i n c r e a s e i n absorbance a t 430nm and a d e c r e a s e a t 400nm. Such a l t e r a t i o n s i n t h e heme chromophore r e f l e c t i n t e r a c t i o n a t o r n e a r t h e a c t i v e s i t e of t h e enzyme. Type I1 s u b s t r a t e s have nonbonding e l e c t r o n p a i r s which a r e t h o u g h t t o i n t e r a c t w i t h t h e heme i r o n . Three t y p e s of EPR s p e c t r a have been s e e n f o r o x i d i z e d cytochrome P-450, two low s p i n forms and one h i g h s p i n form.17 One o f t h e low s p i n forms i s a s s o c i a t e d w i t h s u b s t r a t e - f r e e o x i d i z e d P-450, w h i l e t h e o t h e r i s o b s e r v e d
Chap. 30
Cytochrome P-450 Monoxygenases
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291
o n l y upon i n t e r a c t i o n w i t h a Type I1 s u b s t r a t e . 1 8 When a Type I s u b s t r a t e i s added t o o x i d i z e d P-450, t h e low s p i n form i s c o n v e r t e d t o t h e h i g h Evidence f o r a n o t h e r s p e c t r a l s p e c i e s o f cytochrome P-450, s p i n form.lg presumably a t e r n a r y complex of r e d u c e d P-450,oxy en and s u b s t r a t e h a s been r e p o r t e d f o r b a c t e r i a l and mammalian systemsfot 2 1 ( see however r e f . 22). S p e c t r a l e v i d e n c e f o r P-450-product complexes g e n e r a t e d d u r i n g s u b s t r a t e metabolism h a s a l s o been o b t a i n e d O 2 3
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P r e t r e a t m e n t of C h a r a c t e r i z a t i o n of Cytochrome P-450 Monoxygenases a n i m a l s w i t h b a r b i t u r a t e s o r p o l y c y c l i c a r o m a t i c hydrocarbons r e s u l t s i n s u b s t a n t i a l i n c r e a s e s of t h e l e v e l s of P-450 i n l i v e r . A l t e r a t i o n s i n p r o d u c t d i s t r i b u t i o n a n d / o r changes i n s u b s t r a t e s p e c i f i c i t i e s a f t e r s u c h t r e a t m e n t s p r o v i d e e v i d e n c e f o r more t h a n one form of cytochrome P-450. 5-7, 24-26 Data on c o m p e t i t i v e i n h i b i t i o n s p r o v i d e c o r r o b o r a t i v e e v i d e n c e f o r t h e p r e s e n c e of more t h a n one monoxygenase i n microsomal membranes.27-29 Although a number of cytochrome monoxygenases t h u s a p p e a r t o b e p r e s e n t i n l i v e r , e a c h enzyme p r o b a b l y can a c c e p t a v a r i e t y of s u b s t r a t e s and e a c h i s c a p a b l e of c a t a l y z i n g v a r i o u s o x i d a t i v e t r a n s f o r m a t i o n s i n c l u d i n g e p o x i d a t i o n s , a r y l and a l k y l h y d r o x y l a t i o n s and o x i d a t i o n s a t n i t r o g e n and s u l f u r . E f f o r t s t o s o l u b i l i z e and i s o l a t e t h e P-450 monoxygenases from t h e microsomal membranes have been hampered by t h e f a c i l e c o n v e r s i o n t o a c a t a l y t i c a l l y i n a c t i v e form, cytochrome P-420. I n 1968, however, a s u c c e s s f u l s o l u b i l i z a t i o n of c a t a l y t i c a l l y a c t i v e P-450 from h e p a t i c microsomes was r e p 0 r t e d . 3 ~ ~ 31 The s o l u b i l i z e d system, which e f f e c t e d w-hydroxylation of f a t t y a c i d s , r e q u i r e d t h r e e components f o r c a t a l y t i c a c t i v i t y ; cytochrome P-450, NADPH-cytochrome c r e d u c t a s e , and a h e a t s t a b l e , c h l o r o f o r m - s o l u b l e f a c t o r , t h e a c t i v e component of which was i d e n t i f i e d a s t h e microsomal l i p i d p h o s p h a t i d y l c h o l i n e . 3 2 Further studies w i t h t h i s s o l u b i l i z e d and r e c o n s t i t u t e d system have i n d i c a t e d t h a t t h e c tochrome P-450 and P-448 f r a c t i o n s have d i f f e r e n t c a t a l y t i c a c t i v i t i e s 3T-35 and t h a t t h e t e r m i n a l o x i d a s e a c t i v i t y r e s i d e s i n t h e b-type c y t o chrome (P-450 o r P-448) f r a c t i o n r a t h e r t h a n t h e cytochrome c r e d u c t a s e o r l i p i d f r a c t i o n s . 3 3 , 36, 37 The cytochrome P-450 and P-448 were found t o compete f o r r e d u c t a s e when p r e s e n t t o g e t h e r . 3 8 Cytochrome b5 d i d n o t a p p e a r t o be an o b l i g a t o r y component of t h e r e c o n s t i t u t e d system.39 Cytochrome P-450 and P-448 f r a c t i o n s from r a t l i v e r were found t o c o n t a i n h i g h l e v e l s of an e p o x i d e h y d r a s e , which can c o n v e r t i n t e r m e d i a t e o x i d e s t o v i c i n a l d i 0 l s . ~ 0 Further p u r i f i c a t i o n has afforded f r a c t i o n s r e l a t i v e l y f r e e of c r o s s - c o n t a m i n a t i o n of monoxygenase and h y d r a s e enzymes.41 R e c e n t l y , d i f f e r e n t i a l s o l u b i l i z a t i o n of monoxygenase a c t i v i t y towards a Type I s u b s t r a t e ( n a p h t h a l e n e ) and a Type I1 s u b s t r a t e ( a n i l i n e ) w a s r e p o r t e d -42
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Mechanism of E l e c t r o n T r a n s p o r t and O x i d a t i o n The g e n e r a l l y a c c e p t e d mechanism f o r e l e c t r o n t r a n s p o r t d u r i n g o x i d a t i v e r e a c t i o n s c a t a l y z e d by The o x i d i z e d cytochrome reacts cytochrome P-450 i s shown below.43 r a p i d l y w i t h s u b s t r a t e (SH) t o form a P - 4 5 0 - s u b s t r a t e complex. One
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cytochrome C reductase)
P- 450++-SH
e l e c t r o n r e d u c t i o n o f t h e i r o n t o t h e f e r r o u s s t a t e i s c a t a l y z e d by t h e NADPH-dependent f l a v o p r o t e i n , cytochrome c r e d u c t a s e . T h i s reduced enzyme-substrate complex b i n d s molecular oxygen. The q u e s t i o n a s t o which of t h e two extreme formalisms, Few-02 v e r s u s Fe*-02’, best d e s c r i b e s t h e r e s u l t i n g complex i s u n c l e a r . However, superoxide ( 0 2 ’ ) h a s been i m p l i c a t e d i n t h e reaction.44 T r a n s f e r of a second e l e c t r o n i s followed r a p i d l y by r e l e a s e of p r o d u c t and water without d e t e c t a b l e i n t e r m e d i a t e s . The e x a c t r o l e of cytochrome b5 i n t h i s sequence i s unknown. However, r e d u c t i o n of cytochrome b5 i s an NADH-dependent p r o c e s s and t h e o x i d a t i o n of v a r i o u s s u b s t r a t e s w i t h h e p a t i c microsomes i s known t o be s t i m u l a t e d by t h e p r e s e n c e of NADH.45 A number of s t u d i e s have i n d i c a t e d t h a t t h e r a t e determining s t e p o f t h e above sequence i s t h e r e d u c t i o n of t h e enzyme-substrate complex and t h a t t h e r a t e of t h i s r e d u c t i o n i s s t r o n g l y dependent on t h e n a t u r e of t h e ~ u b s t r a t e . ~ 6 - ~ 8
.
Criteria
f o r t h e involvement of t h e P-450 system i n t h e metabolism i) enhancement o f metabolism a f t e r p r e t r e a t m e n t of animals w i t h p h e n o b a r b i t a l and i n some i n s t a n c e s a f t e r t r e a t m e n t w i t h p o l y c y c l i c aromatic hydrocarbons ii) l o c a l i z a t i o n of enzyme a c t i v i t y i n microsomal f r a c t i o n s w i t h h i g h l e v e l s p r e s e n t i n l i v e r iii) i n h i b i t i o n by carbon monoxide, p i p e r o n y l b u t o x i d e , p - d i e t h y laminoe thyl-3,3-dip heny lpropy l a c e t a t e ( SKF 525a) and c e r t a i n o t h e r compounds i v ) requirement f o r NADPH a s a source of reducing e q u i v a l e n t s . The f o l l o w i n g s e c t i o n s provide r e p r e s e n t a t i v e examples of t y p e s of drug metabolism which appear t o be c a t a l y z e d by P-450 monoxygenases of s p e c i f i c drugs o r x e n o b i o t i c compounds include:
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Aromatic Oxidation Mamnalian metabolism of a r o m a t i c compounds a p p e a r s t o proceed L a an i n i t i a l I?-450-catalyzed formation of r e l a t i v e l y l a b i l e a r e n e o x i d e s which l e a d t o phenols, d i h y d r o d i o l s , c a t e c h o l s and p r e m e r c a p t u r i c a c i d s . The conversion of a t y p i c a l a r e n e oxide, benzene oxide, t o a d i h y d r o d i o l and a p r e m e r c a p t u r i c a c i d w i t h h e p a t i c enzymes
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and t o p h e n o l by i s o m e r i ~ a t i o nwas ~ ~ consonant w i t h t h i s view. However, i t was n o t u n t i l n a p h t h a l e n e 1,2-oxide 2 w a s i d e n t i f i e d as t h e o b l i g a t o r y n a p h t h a l e n e 1 t h a t an a r e n e i n t e r m e d i a t e i n the h e p a t i c metabolism o x i d e w a s i s o l a t e d i n a b i o l o g i c a l system.50, 51 Subsequently, t e n t a t i v e e v i d e n c e h a s been p r e s e n t e d f o r t h e f o r m a t i o n of a r e n e o x i d e s from a number of o t h e r p o l y c y c l i c a r o m a t i c hydrocarbons (see r e f e r e n c e 52).
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Isomerization of appropriately deuterated arene oxides t o phenols i s accompanied by m i g r a t i o n and r e t e n t i o n of t h e d e u t e r i u m i n a n a d j a c e n t p o s i t i o n , a s i n t h e i s o m e r i z a t i o n of 1 - d e u t e r o - n a p h t h a l e n e 1,2-oxide t o 2 - d e u t e r o - l - n a p h t h 0 1 . ~ ~ S i m i l a r m i g r a t i o n s of r i n g s u b s t i t u e n t s o c c u r d u r i n g a r o m a t i c l ~ h y d r o x y l a t i o n s l l and have been c a l l e d t h e " N I H S h i f t . " Because of i t s widespread and c h a r a c t e r i s t i c n a t u r e , t h e " N I H S h i f t " i s now used as a c r i t e r i o n f o r monoxygenase-catalyzed p h e n o l formation.54, 55 The o n l y a p p a r e n t e x c e p t i o n s are d u r i n g lthydroxylationsI1 o f p h e n o l s and a n i l i n e i n which r e t e n t i o n s a r e v e r y low p r o b a b l y due t o t h e reactive n a t u r e of t h e r i n g . The proposed mechanism f o r t h e " N I H S h i f t " i s as shown. The s u b s t r a t e 5 i s c o n v e r t e d t o t h e a r e n e o x i d e 7, which t h e n
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undergoes a s p o n t a n e o u s h e t e r o l y t i c opening of t h e o x i r a n e r i n g ( p a t h a). The r e s u l t a n t z w i t t e r i o n i c s p e c i e s 2 can e i t h e r l o s e t h e d e u t e r i u m s u b s t i t u e n t d i r e c t l y ( p a t h i ) o r form t h e k e t o n e w i t h concomitant migration of deuterium ( p a t h s ) . The k e t o n e t h e n e n o l i z e s t o t h e p h e n o l w i t h p r e f e r e n t i a l r e t e n t i o n of d e u t e r i u m ( p a t h $ ) . The e x t e n t of o v e r a l l d e u t e r i u m r e t e n t i o n w i l l depend on t h e r e l a t i v e importance of p a t h s 2 and 5 and t h e i s o t o p e e f f e c t i n t h e e n o l i z a t i o n ( p a t h A more v s d'). complete d i s c u s s i o n of a r e n e o x i d e s and t h e " N I H S h i f t " c a n b e found i n r e c e n t reviews.55~ 56 The magnitude o f t h e " N I H S h i f t " o b s e r v e d d u r i n g f o r m a t i o n of
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4-hydroxyacetanilide from 4-deuteroacetanilide was significantly less with cytochrome P-448 as compared to that with P-450.57 This provided evidence for differences in the active site of these two classes of monoxygenases. The extent of retention of deuterium in ttcatecholict' metabolites from 4-deuterochlorobenzene was explicable in terms of intermediate dihydrodiols, rather than with pathways involving conversion o f 4-chlorophenol to the catechol by a second tthydroxylation.tt58Phenols in certain instances also appear to be formed by a direct oxygen-insertion pathway similar to that involved in aliphatic hydroxylation.59 An example is the formation of 3-nitrophenol from nitrobenzene.
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Aliphatic Oxidation and 0-Dealkylation Cytochrome P-450 monoxygenases appear to be involved in a variety of aliphatic hydroxylations. One common pathway involves an initial w-hydroxylation as with pentobarbital, 6o followed by further metabolism to the aldehyde and acid by dehydrogenases. Hydroxylation also frequently occurs at the penultimate carbon as in amobarbitalbl, at benzylic and allylic positions and at carbons bearing a heteroatom. Many examples of such oxidations are cited in a recent review. 62 Hydroxylation at carbon bearing a heteroatom such as oxygen, nitrogen or sulfur leads to the so-called dealkylation reactions so common in drug metabolism. The mechanism of aliphatic hydroxylation by cytochrome monoxygenases would appear to involve a direct oxygen-insertion with retention of configuration. For example, when S-(+)-l-2H-ethylbenzene was incubated with liver microsomes, the 1-phenylethanol produced in the reaction consisted of 92% of the ,R isomer and only 8% of the isomer.63 phenobarbital pretreatment diminished the stereoselectivity of this reaction. 639 64 The reaction of the ara-dideutero-derivative exhibited an isotope effect o f 1.8 when compared to the undeuterated substrate, consistant with an oxygen-insertion mechanism. In certain examples o f aliphatic hydroxylation, however, it appears from the absence of an isotope effect with labeled substrates that the insertion reaction is not the rate-limiting step.65-67 Another class of aliphatic oxidations catalyzed by microsomal monoxygenases is 0-dealkylation. The reaction involves cleavage of the alkyl-oxygen bond as shown by the lack o f incorporation of oxygen-18 into the p henolic product, 4- hydroxyacetani1ide, when 4-methox acetani1ide was treated with microsomes in the presence of H2180 and 1802,'8 The mechanism of such 0-dealkylations appears to involve hydroxylation of the alkyl moiety to form a hemiacetal or hemiketal which decomposes to form the 0-dealkylated product and, respectively, either an aldehyde or a ketone*69 An isotope effect of 2.0 for the demethylation of 4-nitroanisole containing a trideutero-methyl group70 is consonant with a mechanism in which oxygen-insertion is rate-limiting.
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N-Dealkylation and Oxidative Deamination Formally, N-dealkylation, a common metabolic pathway for xenobiotic compounds, would appear quite
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a n a l o g o u s t o 0 - d e a l k y l a t i o n r e a c t i o n s . However, t h e N - d e a l k y l a t i o n r e a c t i o n can p r o c e e d v i a e i t h e r o f two mechanisms3 d i r e c t f o r m a t i o n of an u n s t a b l e c a r b i n o l a m i n e 6 9 , 7 1 o r t h e f o r m a t i o n of an N-oxide which can t h e n r e a r r a n g e t o t h e ~ a r b i n o l a m i n e . 7 ~The r e s u l t s f o r t h e m a j o r i t y of t h e compounds s t u d i e d f a v o r t h e f i r s t mechanism, b u t t h e y do n o t r u l e o u t t h e second e n t i r e l y . Formation o f t h e c a r b i n o l a m i n e would a p p e a r t o be c a t a l y z e d by P-450, w h i l e N-oxide f o r m a t i o n i s c a t a l y z e d by h e p a t i c flavoproteins. There a r e now many examples where c a r b i n o l a m i n e s , c a r b i n o l a m i d e s and c a r b i n o l i m i n e s have been i m l i c a t e d o r i s o l a t e d a s i n t e r m e d i a t e s i n N - d e a l k y l a t i o n r e a c t i o n s . 62, 73-76 O x i d a t i v e deamina t i o n , a s f o r example i n t h e c o n v e r s i o n o f amphetamine t o p h e n y l a c e t o n e 7 7 , may be c o n s i d e r e d a s a v a r i a n t of t h e N - d e a l k y l a t i o n r e a c t i o n , where t h e s u b s t r a t e i s a p r i m a r y amine. Carbinolamines, oximes, i m i n e s , and hydroxylamines have a l l been i m p l i c a t e d i n t h e metabolism of amphetamine. 78-80
R e a c t i o n s a t S u l f u r - Methylmercapto-compounds undergo m e t a b o l i c demethyla t i o n presumably s i m i l a r i n mechanism t o t h a t of 0 - d e a l k y l a t i o n s . 8 1 O x i d a t i o n a t s u l f u r r e p r e s e n t s a n o t h e r major pathway of metabolism, as i n t h e c o n v e r s i o n of c h l o r p r o m a z i n e t o t h e s u l f o x i d e 8 2 and i n t h e c o n v e r s i o n o f p h e n y l m e t h y l s u l f i d e , a m e t a b o l i t e o f t h e p e s t i c i d e Dyfonate, t o b o t h t h e s u l f o x i d e and t h e sulfone.83
Reductions -
Cytochrome P-450 monoxygenases have been i m p l i c a t e d i n t h e r e d u c t i o n of n i t r o - and a z o - f u n c t i o n s under a n a e r o b i c c o n d i t i o n s . 8 4 An example i s t h e c o n v e r s i o n of 4 - n i t r o b e n z o i c a c i d t o 4-aminobenzoic * such r e d u c t i o n s a r e p r o b a b l y c a t a l y z e d by o t h e r enzymes.85 acid. 2 i
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O x i d a t i v e Model Systems The c h e m i c a l n a t u r e o f t h e o x i d i z i n g s p e c i e s which i s g e n e r a t e d from m o l e c u l a r oxygen by cytochrome P-450 i s unknown. C u r r e n t r e v i e w s on t h i s problem are a v a i l a b l e . 8 6 , 87 I n g e n e r a l most a u t h o r s a g r e e t h a t an lloxenell o r floxenoid" mechanism i s o p e r a t i v e , whereby t h e n e t e q u i v a l e n t of an oxygen atom t r a n s f e r o c c u r s d u r i n g t h e oxidation. S i m i l a r i t y between t h e r e a c t i o n s of c a r b e n e s and n i t r e n e s ( a d d i t i o n t o d o u b l e bonds, C-H bond i n s e r t i o n s ) w i t h t h e o x i d a t i o n s c a t a l y z e d by cytochrome P-450 ( e p o x i d a t i o n , a r e n e o x i d e f o r m a t i o n , a l i p h a t i c h y d r o x y l a t i o n ) have l e d t o t h e I1oxene1l terminology. T h r e e g e n e r a l t y p e s of o x i d a n t s 8 6 a r e c a p a b l e of e f f e c t i n g t h e Molecular oxygen i n r a n g e of r e a c t i o n s c a t a l y z e d by cytochrome P-450. t h e p r e s e n c e o f a metal i o n and a s o u r c e of r e d u c i n g e q u i v a l e n t s , i n some i n s t a n c e s t h e reduced m e t a l i o n , i s one of t h e s e . Such r e a c t i o n s are t y p i f i e d by t h e U d e n f r i e n d system88, 89 which employs F e ( I I ) , O2 and a s c o r b i c a c i d . A second c l a s s c o n s i s t s of t h e r e a c t i o n s of H O2 and Examples from t h i s c l a s s i n c l u d e t h e m e t a l - c a t a l y z e d o r g a n i c peroxides. decomposition of H202905 9 1 o r o r g a n i c p e r o x i d e 9 2 and t h e r e a c t i o n s of Reducing e q u i v a l e n t s a r e n o t consumed i n t h e s e peroxy-acids.93-95 r e a c t i o n s . F i n a l l y , t h e r e a r e n o n - p e r o x i d i c o x i d a n t s which t r a n s f e r These i n c l u d e p ho t o e x c i t e d azaromat ic-N-oxide s92 and i n o r g a n i c oxygen.
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r e a g e n t s s u c h a s chromyl a c e t a t e . 9 6 The most s e r i o u s problems e n c o u n t e r e d w i t h such model systems have been t h e complexity of t h e r e a c t i o n s and t h e q u e s t i o n of what c r i t e r i a s h o u l d be a p p l i e d when comparing models t o t h e enzyme-catalyzed r e a c t i o n s . Advent of t h e " N I H S h i f t " h a s p r o v i d e d an i n v a l u a b l e c r i t e r i o n f o r such comparisons. Models which do n o t d i s p l a y t h i s phenomenon must be c o n s i d e r e d i n a d e q u a t e , d e s p i t e how a t t r a c t i v e t h e y m i g h t a p p e a r from o t h e r s t a n d p o i n t s . A t p r e s e n t a s u i t a b l e model which i n v o l v e s t h e r e d u c t i o n of m o l e c u l a r oxygen i s , t h e r e f o r e , u n a v a i l a b l e . 8 6 H y d r o x y l a t i o n s by o r g a n i c p e r o x i d e s 9 2 , peroxy a c i d s g 3 3 97 p h o t o a c t i v a t e d N-oxidesg2, and chromyl a c e t a t e 9 do show t h e r e q u i s i t e m i g r a t i o n s . These o x i d a n t s i n d i c a t e t h e t y p e of oxygen atom t r a n s f e r which i s n e c e s s a r y , b u t a f f o r d no e v i d e n c e f o r how such a s p e c i e s could be g e n e r a t e d from molec u l a r oxygen.
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Summary Cytochrome P-450 monoxygenases have now been i m p l i c a t e d i n m a j o r Such metao x i d a t i v e metabolisms f o r most f o r e i g n compounds i n mammals. b o l i s m s u s u a l l y r e s u l t i n t h e f o r m a t i o n of p o l a r p r o d u c t s ( a l c o h o l s , p h e n o l s , secondary amines, e t c ) which a r e more r e a d i l y c o n j u g a t e d and e x c r e t e d . These metabolisms, t h e r e b y , appear t o accomplish t h e p r i m a r y f u n c t i o n of such monoxygenases, i - e . , d e t o x i f i c a t i o n via enhanced r a t e s of e l i m i n a t i o n . I n c e r t a i n i n s t a n c e s , however, s u c h a s i n t h e c o n v e r s i o n of imipramine by N-demethylation t o desmethylirnipramine,98 t h e m e t a b o l i s m may r e s u l t i n t h e f o r m a t i o n of a compound w i t h h i g h e r b i o l o g i c a l a c t i v i t y . I n o t h e r c a s e s , metabolism may r e s u l t i n t h e f o r m a t i o n of l t b i o a c t i v a t e d l l m e t a b o l i t e s which upon r e a c t i o n w i t h t i s s u e c o n s t i t u e n t s e l i c i t c y t o t o x i c o r carcinogenic sequelae. Examples o f such b i o a c t i v a t i o n s i n c l u d e t h e c o n v e r s i o n of h a l o b e n z e n e s and p o l y c y c l i c hydrocarbons t o r e a c t i v e a r e n e o x i d e s ( f o r a c u r r e n t r e v i e w s e e r e f . 52). I t i s t h u s clear t h a t a complete u n d e r s t a n d i n g o f t h e P-450 monoxygenase system assumes p r i m e importance w i t h r e s p e c t t o b o t h p h a r m a c o k i n e t i c s and t o x i c i t y of m e d i c i n a l agents
.
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Cytochrome P-450 Monoxygenases
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248,
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57. 58. 59. 60 61. 62 63. 64 65
Sect. V I
- Topics i n Chemistry
Wiley, Ed.
A.Y.H. Lu, R. Kuntzman, S. West, M. Jacobson, and,A.H. Conney, J. B i o l . Chem., 1727 (1972). A.Y.H. Lu and S.B. West, Mol. Pharmacol., 2, 490 (1972) S.B. West and A.Y.H. Lu, Arch. Biochem. Biophyso 298 ( 1 9 7 2 ) . W. Levin, DO Ryan, S.B. West, and A.Y.H. Lu, J. B i o l . Chem. i n p r e s s . F O Oesch, D.M. J e r i n a , J.W. Daly, A.Y.H. Lu, R. Kuntzman, and A.H. Conney, Arch. Biochem. Biophys., 153, 62 (1972). Daly, W. Levin, A.Y.H. Lu, R. Kuntzman P.M. D a n s e t t e , D.M. J e r i n a , J.W. and A.H. Conney, Arch. Biochem. Biophys. i n p r e s s . W. B l e e c k e r , J. C a p d e v i l a , and M. Agosin, J. Biol. Chem. 248, 8474 ( 19 73). R.W. Estabrook, i n "Handbook of Experimental Pharmacology,tl eds. B.B. B r o d i e and J.R. G i l l e t t e , S p r i n g e r - V e r l a g , B e r l i n , 1971, V o l . 2 8 / 2 p. 264. H.W. S t r o b e l and M.J. Coon, J. B i o l . Chem., 246, 7826 ( 1 9 7 1 ) . A. H i l d e b r a n d t and R.W. Estabrook, Arch. Biochem. Biophys., 143, 66 (1971). P.L. Gigon, T.E. Gram, and J.R. G i l l e t t e , Biochem. Biophys. R e s . Commun., 2, 558 (1968). J.B. Schenkman, and D.L. C i n t i , Biochem. Pharmacol., 2, 2396 (1970). He D i e h l , J e S c h a d e l i n , and V. U l l r i c h , Hoppe-Seyler's 2. P h y s i o l . Chem., 351, 1359 (1970). D.M. J e r i n a , J.W. Daly, B. Witkop, P. Zaltzman-Nirenberg, and S. U d e n f r i e n d , Arch. Biochem. Biophys., 176 ( 1968) D.M. J e r i n a , J.W. Daly, B e Witkop., P. Zaltzman-Nirenberg, and S. U d e n f r i e n d , J. Am. Chem. Soc., 90, 6525 (1968) D.M. J e r i n a , J.W. Daly, B. Witkop, Pe Zaltzman-Nirenberg, and S. U d e n f r i e n d , B i o c h e m i s t r y , 2, 147 (1970). J.W. Daly and D.M. J e r i n a , S c i e n c e , i n p r e s s . D.R. Boyd, J.W. Daly, and D.M. J e r i n a , B i o c h e m i s t r y 2, 1961 (1972). G. G u r o f f , J.W. Daly, G.M. J e r i n a , J. Renson, S. Udenfriend, and B. Witkop, S c i e n c e , 1524 (1967). J.W. Daly, D.M. J e r i n a , and B. Witkop, E x p e r i e n t i a , 28, 1129 (1972) D.M. J e r i n a and J.W. Daly, i n "Oxidases and R e l a t e d Redox Systems,tt eds. T.E. King, H.S. Mason, and M. Morrison, U n i v e r s i t y P a r k Press, B a l t i m o r e , 1973, Vole 2, p. 143. J.W. Daly, D.M. J e r i n a , J. Farnsworth, and G. G u r o f f , Arch. Biochem. 238 (1969) Biophys., D.M. J e r i n a , J.W. Daly, and B. Witkop, J. Am. Chem. SOC., 89, 5488, ( 1967) J.E. Tomaszewski, D.M. J e r i n a , and J.W. Daly, B i o c h e m i s t r y , s u b m i t t e d . J.R. Cooper and B.B. B r o d i e , J. Pharmacol. Exp. Ther., 75 (1955). E.W. Maynert, J. Biol. Chem., 397 (1952) J.W. Daly i n IIHandbook of E x p e r i m e n t a l Pharmacology, eds. B.B. B r o d i e and J.R. G i l l e t t e , S p r i n g e r - V e r l a g , B e r l i n , 1971, Vol. 28/2 p. 285. R.E. McMahon, H.R. S u l l i v a n , J.C. C r a i g , and W.E. P e r e i r a , Jr., Arch. Biochem. Biophys., 575 (1969). R.E. McMahon and H.R. S u l l i v a n , L i f e Sci., 5 , 9 2 1 (1966). R.U. Lemieux, K.F. Sporek, I . O I R e i l l y , and E. Nelson, Biochem. 31 (1961) Pharmacol.,
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1,
173,
2, 150,
+,
-
.
Chapter 31. Use of Chemical Relationships in Design of DDT-Type Insecticides Robert L. Metcalf, Department of Entomology, University of Illinois, Urbana-Champaign, Illinois
-
General The pharmacology of insecticides is of broad general interest in that it involves details of action and detoxication both in target insects, in man and domestic animals, and in the thousands of non-target species in the environment. Thus comparative pharmacology is rapidly becoming a recognized area of study. For the past 25 years new candidate insecticides were selected largely on the basis of insecficidal effectiveness and longterm persistence. Those most successful in meeting these requirements are now being phased out1 Many of the less persistent organophosphate and carbamate replacements are extremely toxic to man and possess little or no intrinsic selectivity. There is an urgent need to develop biodegradable and pharmacologically selective insecticides which are also relatively persistent on surfaces. Restudy of the DDT-type molecule provides an avenue for meeting these objectives.
.
Mode of Action of DDT - Despite thirty years of intensive study and the ----utilization of more than 4 x lo9 lb. of DDT, there is little definitive knowledge about the critical lesion produced in DDT poisoning. , I n both insects and mammals, DDT produces hyperexcitability, violent tremors, convulsions, prostration, and death. Physiologically, the tremors result from initiation of repetetive after-discharge in the nerve axon, so that a single efferent nerve impulse produces a prolonged volley of afferent impulses. These may have the effect of literally stimulating the insect to death that may result from metabolic exhaustion, or from endogenously produced neurotoxin2. This axonal effect is associated with an increase in negative after-potential and results in prolongation of the action potenThe moletial of the nerve3 presumably through inhibition of @efflux. cular mechanisms for this action are the subject of substantial conjecture. DDT has a high electron affinity and Matsumura and O'Brien4 suggested charge -transfer complex formation between DDT and nerve membrane and have isolated a DDT protein combination that appears to have these properties. The well known negative temperature coefficient of DDT intoxication5supports this idea. Gunther et a16 suggested that DDT must bond with a cavity in the protein matrix, and Mullins7 proposed a generalized receptor site in the regular interstices of cylindrical lipoprotein strands forming the membrane lattices of the nerve axon. The lipoprotein strands were estimated to be about 40 A 0 in diameter and when packed in hexagonal array with a 2 A0 separation, provide hypothetical pores into which the DDT molecule should fitsnuggly inend-on position,distorting the membrane and presumably producing ion-leaks causing excitation. HolanS interwove these ideas into the concept of the DDT molecule acting as a molecular wedge, entering into the lipoprotein of the nerve membrane so that the dichlorodiphenylmethane moiety is locked into the overlying protein-layer through molecular complex formation, and the trichloromethyl group fits into a
Chap. 31
Design of DDT-Type Insecticides
Metcalf
301
pore in the membrane, the "sodium gate", keeping it open to Na+ ions and delaying the falling phase of Na+ ion potential. Weiss' has explained the implied paradox of DDT blockage o f the membrane pore as leading to Na+ leakage, by proposing ''molecular springs" of helical phospholipids which when expanded prefer hydrated Na+ to K+ and when contracted prefer unhydrated K+ to Na+. Absorption o f DDT molecules between the expanded springs would prevent their closure and facilitate abnormal Na+ permeability. From parallel beam projections of a number of toxic DDT analogues, Holan5concluded that the van der Waals' diameter o f the aliphatic apex of the active molecular wedge should be 60.t 0.5 Ao, or close to that o f the hydrated sodium ion; and that the maximum allowable distance between the 2, p' substituents of the aryl rings is about 14.0 Ao. More recent data described later, suggest that the latter measurement will need revision. Matsumura and Narahashi' found DDT to strongly inhibit Na+, K+, and ATP'ase of rat brain and have suggested a correlation between ATPIase inhibition and the electrophysiological symptoms of DDT poisoning. The strong ATP'ase inhibition by the insecticidally and electrophysiologically inert DDE or 2,2-bis-(~-chlorophenyl)-l,l-dichloroethylene remains difficult to explain. Mg*
-
Critical Fit at Site of Action The present views of the action of DDT strongly support a precise physical interaction between a macro-molecular element of the nerve axon, the "DDT-receptor", and the DDT-type molecule 5s6s7 . Analogues isosteric with DDT (9 and with appropriate lipophilicity produce typical DDT-like axonal instability. This phenomenon can be best observed by prior inhibition of mixed function oxidase (MFO) detoxication by administration of a synergist such as 3,4-methylenedioxy6-propylbenzyl z-butyl diethyleneglycol ether (piperonyl butoxide) "or, Under 2- (3,4-methylenedioxyphenoxy) -3,6,9 - trioxaundecane (sesamex) these conditions, the completely methylated DDT-isostere 1,l-bis-(E-toly1)approaches the toxicity neopentane (2) (synergized LD50 Musca 35 pg/g)
'.
cla HDC1 C I
C k E;c1
CH3
QyIH3 C I
C H j+CH3 CH3
LD50 values for Musca domestica in pg per g. or Culex pipiens fatigans larva i pp will be given subsequently without prefix and are taken from f.0'p2s T3 unless otherwise indicated.
Sect. VI
302 -
- Topics in Chemistry
Wiley, Ed.
of DDT (LD50 Musca 5.5 ) I g / g ) 12. In general typical DDT-like activity is found in all the isostere combinations of C1 (Van der Waals' radius 1.75 There is, however, clearly an optimum Ao) and CH3 (radius 1.97 A O ) . 1 3 size both to the constituent parts and to the molecule as a whole. A series of silicon analogues of DDT including bis-(e-chloropheny1)-tertbutylsilane bis-(E-chloropheny1)-dichloromethylsilane (k) and bis(E-chloropheny1)-trimethylsilylmethane (2) proved to be nearly inactive as toxicants to Musca and Culex 1 4and it was suggested that the nearly
(z),
-4
-3
-5
50% larger size of silicon (Si-C bond 1.86 AO) compared to carbon (C-C bond 1.48 AO) caused an increase in size of the molecule in all tetrahedral dimensions so that interaction with the DDT receptor site was prevented, e.g. the va der Waals' radius of -C(CH3)3 is 2.79 A 0 vs. 4 5 3 . 3 A0 for -Si(CH3)3
.
This narrow range of restriction in the physical size of the active DDT-type molecule is supported by the relative inactivity of 2,2-bis-(~chloropheny1)-l,l,l-tribromomethane (Musca 500, Culex 0.55) where the van der Waals' radius of -CBr3 is 3.21 Ao; and by comparison of 2,2-bis(~-chlorophenyl)-2,1,l,l-tetrachloroethane (completely inactive) vs. 2,2-bi~~(~-chlorophenyl)-2-fluoro-l,l,l-trichloroethane (Musca 125, Culex where the atomic radius of F is 1.47 A0 compared to 1.75 Ao for O . 9 W With C1 decreased crowding around the t-carbon, using 2,2-bis-(~chloropheny1)-1,l-dichloroethane as a model (h) (Musca 19.7, Culex 0.038), insertion of 4 - F (7J substantially improved toxicity (Musca 4.1, Culex 0.024), insertion ofcT-Cl (8) is acceptable (Musca 18.5, Culex 0.24, and toxicity is further decreased with#-Br (2,2-bis-~-chlorophenyl)-2-bromo-
.
Cl+l
C l ' $
c
Chap. 31
Design of DDT-Type Insecticides
1,l-dichloroethane)
Metcalf
303 -
(Musca 55,.Culex 0.19).
Other Aliphatic Moieties - Other DDT-like compounds include 1,l-bis-(Echlorophenyl)-2-nitropropane or Prola@. lThe analogue 1,l-bis-(e-ethoxy(Musca 5.0, Culex 0.045). phenyl)-2-nitropropane (2) is very toxic Replacement of t h e C-H with CH3 in the corresponding 2-nitroisobutane (10)sharply reduced toxicity (Musca 80, Culex 0.2) probably because of the increased Van der Waals’ radius (CH(CH3)N02 ca 3.23 AO, C(CH3)2 NO2 ca 3.00 Ao) .
-3
10 -
11 -
Introduction of the cyclopropane group into DDT chemistry was first is essentially tried in 1944 l6 but 1,1-bis-@-chloropheny1)-cyclopropane inactive (Musca 7500, Culex 1.0). However, the corresponding 1,l-di-(kchlorophenyl)-2,2-dichloro-cyclopropane (z)5(Musca 12.0, Culex 0.042) is apqpximately as toxic as DDD (&), whose configuration it approximates . The corresponding l,l-di-(~-chlorophenyl)-2-chlorocyclopropane was about one-tenth as active and in agreement with the trihaloethanes; the 2,2-difluorocyclopropane (Musca 415) was of low activity, the 2,2dibromocyclopropane (Musca 3 6 5 ) only slightly more active, while the 2chloro-2-fluoro (Musca 60) and 2-chloro-2-bromocyclopropane were intermediate 5
.
Essential Configuration - The biological action of DDT).I( and analogues depends upon optimum configuration of the phenyl rings and trichloromethylgroup about the centralw-carbon. For maximum tivic action the rings must The essentiality of assume the butterfly or trihedral configuration this configuration is demonstrated by the low insecticidal activity of 2,7- dichloro-9-trichloromethyl-9,lO-dihydroanthracene (12)(Musca)500, Culex 0.35), in which the aryl rings are planar19*20 The diphenylmethane moiety is not essential for DDT-like activity and an atom of N, 0, or S can be interposed between the two aryl moieties to produce compounds that approach DDT in toxicity to insects21 These compounds such as e - t r i c h l o r o m e t h y l - ~ - e t h o x y b e n z y l N-(E-ethoxyaniline) (2)(Musca 15.5, Culex 0.19) and H-trichloromethyl-2-ethoxybenzyl E-ethoxyphenyl ether (2)(Musca 27 Culex 0.1l2l , produce characteristic DDT-like symptoms of intoxication 22’ .Observations of Fischer-Hirschfelder-Taylor molecular models of these compounds show that the staggered alignment of the groups aryl-X-C-aryl (13, 14) permits the p, 2’-substituents to assume configurations very close to those of DDT. The importance of this staggered align-
.
.
.
-
304 -
Sect. VI
12 -
- Topics in Chemistry
13 -
Wiley, Ed.
14 -
ment in permitting the q-trichloromethylbenzylphenyl ethers andw-trichloromethylbenzyl anilines to fit the DDT receptor is shown in Holan's 5 results with substituted diphenyldichlorocyclopropanes. The l,l-di-(~chlorophenyl)-2,2-dichlorocyclopropane (11) (Musca 12) was highly toxic while its conformer trans-1,3-di-(~-chlorophenyl)-2,2-dichlorocyclop--Lopane (Musca >10,000) was inactive. The rigid cyclopropane ring in the last clearly prevents assumption of the staggered configuration found DDT and in (11). X-ray diffraction determinations of the crystalline structures of DDT, l,l-bis-(g-ethoxyphenyl)-2,2-dimethylpropane and l,l-di-(g-chlorophenyl)-2,2-dichlorocyclopropane have recently been completed , and it was found that these three compounds were extremely similar in stereochemistry. The studies showed that there is a lack of a mirror plane running through the alkyl chain so that the two phenyls are unevenly twisted.
-
Asynnnetrical Analogues The earlier literature of DDT toxicology contains many statements that symmetry of structure is an essential feature of all good DDT analogues 2 4 . A few attempts were made to investigate asymmetrically substituted E,g'-diaryltrichloroethanes with substituents such as Musca 16, Culex 0.039), Cl-CH3 (Musca 62.5, Culex 0.032), CH'jO-OC2H5 Recent study of such asymmetric and F-OCH3 (Musca 47, Culex 0.47). compounds l o shows that insecticidal activity is associated with 3 substantial degree of asymetry and that the permissible limits for high toxicity vary with the nature of the aliphatic moiety. Thus representative LD50 values for E,E'-dialkoxyphenyl-l,l,l-trichloroethanes were: CH30-OC3 H7 (Musca 24, Cd@x 0.18), CH30-OC4Hg (Musca 21, Culex 0.18), CH30-OCgH13 (Musca 500, Culex 1). However for kIE'-dia1koxyphenyl2-nitropropanes the LD50 values were: CH30-OC6H7 (Musca 42, Culex O.ll), CH30-OCqHg (Musca 21, Culex 0.062), CH3O-OC6H13 Musca 37, Culex 18), and CH30-OCgH17 (Musca 75, Culex>l)
.
These several sets of data demonstrate conclusively that the overall size and shape of the DDT-type molecule is the critical factor in deter-
Chap. 31
Design of DDT-Type Insecticides
305
Metcalf
mining the efficiency of interaction with the macromolecular axcjnic receptor. The efficiency of interaction is determined by optimum combination of the substituents on both aromatic rings or in theH-(C~) and @-(C1) carbons. Marked asymmetry of structure, e.g. as great as ~ ~ 3 0 OC8H17, can be associated with high toxicity. In this regard it is interesting that the most toxic diaryltrichloroethane when given orally to the white mouse contained the E,E'- substituents f$H50-OC3H7 (LD50 75), vs. C Z H ~ O - O C ~(300) H ~ and Cl-CI (DDT) (LD50 200)
.
-
Multiple Regression Analysis Quantitative biological data for several hundred DDT analogues supports the simple concept that the biological effectresults from maximum complementarity between the DDT-type molecule and the DDT receptofi and the view that there is a critical optimum for summation of the van der Waals' forces of the four key substituents x, Y, L, and Z l 4 :
Toxic molecules result when these substituents vary over a wide range (e.g. F to C8H170 for X and Y) implying that the receptor is by no means a rigid structure 5 * 7 but must: have a considerable degree of flexibility or elasticity. Therefore as the summation of size of X, Y, L, and 2 'increases, interaction with the cavity or pouch of the DDT receptor increases and eventually reaches a maximum beyond which increases in the sizes of the substituents result in decreased complementarity and loss of insecticidal activity 1 4 . Free energy parameters - the Taft steric substituent constants Es, have been employed to quantify this idea 14. Es values represent sole1 steric
IP.
parameters and are directly proportional to van der Waals' radii A parabolic function was developed to express the relationship of LD50 to Es :
where i = substituents X, Y, or Z
306 -
Sect. VI
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Using LD50 values to Musca for 25 symmetrical and unsymmetrical DDT analogues with variations in X and Y, in multiple regression analyses using Es, 'II , F, and R values, it was shown that the correlation coefficient was 0.74 for LD50 alone and was increased to 0.87 using synergized LD50 values where MFO detoxication was inhibited by prior application of piperonyl butoxide. Es was the only free energy parameter of significance in the regression equations indicating that van der Waals' interaction forces Similar are primarily responsible for the DDT-receptor interaction6 multiple regression analysis of LD50 values to Musca for 9 DDT analogues where X and Y remained constant as C1 and L and Z were varied using Es, , and6*, gave r = 0.86 with the combination of Es andG* as highly significant. Therefore a combination of steric and polar effects contributed to the interaction of L and 2 with the receptor14
.
.
This sort of multiple regression analysis has opened the way to the rational design of effective new DDT analogues based on optimizing the van der Waals' radii of groups X, Y, L, and Z. Several effective new compounds have been designed from these correlations, e.g. 1-(2-fluorophenyl)-l-(~-buto~y)-nitropropane~~ Using data from several new sets of compounds under evaluation it appears feasible to develop a unified mathematical description of the combined effects of groups X, Y, L, and Z upon toxicity of the DD'f type compound, and this is presently under study.
.
-
Comparative Toxicology A substantial amount of precise LD50 data to the house fly Musca domestica, to the black blowfly, Phormia regina, to the house mosquito larva, Culex fatigans, and to the malaria mosquito larva, Anopheles albimanus enables us to take a quantitative look at the dimensions of the macromolecular target site of the DDT-type molecule in several species. Additionally, using the synergized LD50 to virtually eliminate the effects of in vivo detoxication it is feasible to examine intrinsically the most active. which sort of DDT-type configuration is Surprisingly, DDT itself appears to have the best fit for only Anopheles larvae of the species examined. Based on synergized LD50 values, the order of LD50 values to Musca for the g,E'-disubstituted aryl trichloroethanes with the indicated substituents was C 2 ~ 5 0(1.75) cH30 (3.5) C1 To Phormia the order was CH3O ( 4 . 6 ) C2H5O ( 7 . 4 ) Cl (8.25). To (5.5). Culex the order was Q H 5 0 (0.04) C ~ 3 0(0.067) C1 0 07 , and to Anopheles C1 (0.05) C3H70 (0.077) C2H50 ( 0.086)16 * . 'For the E,E'disubstituted-diphenyl-2-nitropropanes,the synergized LD50 values to Musca were in the order C2H5O (1.25) C3H70 (2.3) c ~ 3 0(2.75) C1 7.0; To Culex and to Phormia C2H5O (1.35) CH3 (6.0) CH3O (7.75) C1 (11,O). and Anopheles larvae C2H50 (0.045-0.048) C1 (0.06&-0.066)
-
.
These data show rather clearly that the optimal size of the aryl substituents is related to the size of the aliphatic portion, e.g. -CC1 vs. -CH(CH3) NO2, thus leading back to the hypothesis of Gunther et a1 63
.
Both methoxychlor and methylchlor show decreased insecticidal activity over DDT in some insects especially lepidopterous larvae due to & vivo degradation in the insect body. However, they may be more toxic to other insects, e.g. methoxychlor to the Mexican bean beetle, Epilachne
-
Chap. 31
Design of DDT-Type Insecticides
Metcalf
307
varivestis and to Phormia regina, and their activity can be mterially improved by synergism with piperonyl butoxide l o . Asymmetrical analogues such as 2-(~-methylphen l)-Z-(E-ethoxyphenyl) l,l,l-trichloroethane (15) (Musca 9, Culex 0.13) are insecticidally effective, of low toxicity to fish and nlammals, and readily biodegradable 282 29
''
.
cnoE
l
\
C 1 ' c,Yl c1
CH3 0
\ 1 'CH3
H
H
Alterations of the aliphatic moiety of the DDT molecule to introduce degradophores (easily degraded moieties) have produced compounds such as 1,l-bis-(p-ethoxyphenyl)-3,3-dimethyloxetane (g), (Musca 26) l 1 , 3 0 which is synergized 50-fold by sesamex to the housefly and is readily degradable. References 1.
2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20.
Environmental Protection Agency, The Federal Environmental Pesticide Control Act of 1972, Wash. D. C. Jan. (1973). J.Sternburg, Ann. Rev. Entomology g , 19 (1963). T. Narahashi and H. G. Haas, J. Gen. Physiol. 2, 177(1968). F. Matsumura and R. D. O'Brien, J. Agr. Food Chem. l4, 36 (1966). G. Holan, Nature (London) 221, 1025 (1969). F. A. Gunther, R. C. Blinn, G. E. Carman, and R. L. Metcalf, Arch. Biochem. Biophysics 50, 504 (1954). L. J. Mullins, h e r . Inst. Biol. Sci. Publ. 1, 123 (1956). D. E. Weiss, Austral. J. Biol. Sci. 22, 1355 (1969). F. Matsumura and T. Narahashi, Biochem. Pharmacol. 20, 825 (1971). R. L. Metcalf, I. P. Kapoor, and A. S . Hirwe, Bull. W.H.O. 44, 363 (1971). G. Holan, Nature (London) 232, 644 (1971). R. L. Metcalf, I. P. Kapoor, and A. S . Hirwe, p. 244 in "Degradation of Synthetic Organic Molecules in the Biosphere". National Academy of Sciences, Wash. D. C. (1972). R . L. Metcalf and T. R. Fukuto, Bull. W.H.O. 38, 633 (1968). M.A.H. Fahmy, T. R. Fukuto, R. L. Metcalf, and R. L. Holmstead, J. Agr. Food Chem. 2 l , 585 (1973). M. Charton, J. Amer. Chem. SOC. 91, 615 (1969). uP. Lauger, H. Martin, and P. MuL.ler, Helv Chim. Acta 27, 892 (1944). T. P. DeLacy, C.H.L. Dennard, and G. Holan, Chem. Commun. 930, 1971. E. F. Rogers, H. D. Brown, I. M. Rasmussen, and R. E. Heal, J. Amer. Chem. SOC. 75, 2991 (1953). F. A. Vingiello and P. E. Newallis, J. Org. Chem. 25, 905 (1960). R. L. Metcalf, J. Agr. Food Chem. 2, 511 (1973).
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21. A. S. Hirwe, R. L. Metcalf, and I. P. Kapoor, J. Agr. Food Chem. 2 0 , 818 (1972). 22. T. A. Miller and J. M. Kennedy, Pestic. Biochem. Physiol. 2, 206 (1972). 23. P. MGller, Helv. Chim. Acta 3, 1560 (1946). 24. R. D. O'Brien, "Insecticides, Action and Metabolism" Academic Press. 25. E. A. Prill, M. E. Synerholm, and A. Hartzell, Contrib. Boyce Thompson Inst. l4, 341 (1946). 26. E. Balaban and F. K. Sutcliffe, British Pat. 5 9 7 , 091, Jan. 19 (1948). 27. R. L. Metcalf, A. S. Hirwe, and J. Williams, Unpublished data. 28. I. P. Kapoor, R. L. Metcalf, A. S. Hirwe, J. R. Coats, and M. S. Khalsa, 3 . Agr. Food Chem. 2 l , 310 (1973). 29. R. L. Metcalf, I. P. Kapoor, and A. S . Hirwe, U. S . Pat. 3,787,505, Jan. 22, (1974). 30. G. Holan, Bull. W.H.O. 44, 355 (1971).
COMPOUND NAME AND CODE NUMBER INDEX A9145, 110 ABB (aminobenzylbenzimidazole), 134 Abbott 29590, 199 Abbott 40656 (SP 1061, 258 Abbott 41596, 63 acetaminophen (paracetamol) , 168 acetylcholine, 229 acetylcystein, 85 acetylsalicylic acid (aspirin), 75, 87, 168, 197, 199 actinomycin D, 139, 144 actinomycin D lactam, 144 adamantine derivatives, 134 adenine arabinoside (ara-A), 132 adenosine, 76 adenosine diphosphate (ADP), 226 ADP (adenosine diphosphate) , 226 adrenochrome, 27 Agkistrodon acutus venom, 79 AH 6556, 91 AH 7079, 91 AH 7725, 91 AH 7921, 15 AHR-2277 (lenperone), 3 AHR-3018 (azapropazone dihydrate), 81 AL-1021, 3 alanine-B-sulfonate, 237 albuterol (salbutamol), 80, 86 alkanoic acids, 110 2-alkenoic acids, 110 N-alkyl-norketobemidones, 13 alprazolam (U-31,889) , 5 alprenolol (Aptin@), 59, 68 amantadine, 21 amikacin (BB-K a), 98, 100 aminobenzylbenzimidazole (ABB) , 134
L-2-amino-5-bromo-4-oxopentanoic acid, 141 2-amino-3-butenoic acid, 238 (as, 5S)-a-amino-3-chloro-4,5-dihydro5-isoxazoleacetic acid, 141 (1 -t 4)-2-amino-2-deoxy-B-~-glucopyranuronan, 143 2-amino-7-hydroxytetralin, 32
L-2-amino-4-methoxy-trans-3-butenoic acid, 239 aminophylline, 143 aminotetralins, 29, 32 amodiaquin, 115 amoxicillin, 95, 96
309
amphetamine, 32 amphetamine abuse, 38 amphotericin B, 107, 109, 110, 111 analgetics, 43 angiotensin, 51 angiotensin 11, 228 angiotensin, mustard derivatives, 228 anileridine, 229 annulene, 134 ansomycin, 131 antiestrogens, 226 antitumor lipids, 143 antitumor plant extracts, 144 antitumor tissue extracts, 145 antitussives, 43 apomorphine, 22 aprindine, 69 ara-A (adenine arabinoside), 132 arachidonic acid, 60, 87 arabinofuranosylcytosine-5’-adaman-
toate, 133 ara-C (cytosine arabinoside, cytarubine), 132, 139, 141, 158 aryl acid, 197 arylalkanoic acid, 197 arylhydroxytriazene, 196 ascorbate oxidase, 144 ascorbic acid (vitamin C ) , 83, 128 L-asparaginase, 141, 143 L-asparaginase derivatives, 144 aspirin (acetylsalicylic acid), 75, 87, 168, 197, 199 ~ ~ - 3 0 8 177 , atropine, 32, 89 axenomycin D, 124 AY-16,804, 77 AY-22,284, 186 AY-23,028 (butaclamol), 3 8-azainosine, 142 azanator maleate (Sch15,280), 91, 92 azapropazone dihydrate (AHR-3018), 81 azapropazone, trifluromethyl analogs, 81 azaserine, 141, 241 azathioprine (ImuranB), 196, 219 azidocodeine, 12 azidomorphine, 12
310
COMPOUND NAME, AND CODE NUMBER INDEX
N-0-(pazidopheny1)ethyl phanol, 229 aziridines, 134
norlevor-
bacterial endotoxins, 144 barbiturate abuse, 38 Bay b 5097 (clotrimazole), 108, 109 BB-K 8 (amikacin), 98, 100 BC-2605 series, 12 (-)-BC-2605(oxilorphan), 11, 41, 42 BC-2910, 41, 42 BCNU, 111, 146 beclomethasone-17,21 dipropionate, 90 bee venom, 200 benapryzine (BRL 1288), 23 benorylate, 198 benserazide (Ro 4-4602), 20 benzomorphan, 229 benzothiazolacetic acid, 198 benzylamines, 83 bephenium, 121 BFE-60, 68 BHT(buty1ated hydroxytoluene) , 218 bis (1-aziridinyl)phosphinate derivatives, 145 bisbenzimidazoles, 134 1,3-bis (2-chloroethyl-l-nitrosourea, BCNU), 111, 146 p-(N,N-bis (2-chloroethyl)amino) phenylacetic acid esters, 227 bis (B-dibutylaminopropyl)-9-oxofluorene-2,7-dicarboxylate dihydrocloride, 132 bisobrin (EN-1661), 81 bithionol, 122 BL-3676, 70 BL-3677, 70 BL-20803, 132 bleomycin 2 , 110 boron compounds, 145 bovine serum, 200 BR 750 (guanabenz, FLA 137, WY-8678), 58 bradykinin, 228 bretylium tosylate, 69 Brinerdin@, 59 BRL 1288 (benapryzine), 23 BRL 2288 (ticarcillin), 97 6~-bromoacetoxyprogesterone, 225
lla-bromoacetoxyprogesterone, 225 16a-bromoacetoxyprogesterone, 225 bromoacetyloxytocin, 227 2-bromoalkanoic acids, 110 3-bromo-4-t-butyl benzoic acid, 82 8-bromocyclic GMP, 88 bromoperidol (R 11,333), 2 6a-bromoprogesterone, 227 6@-bromoprogesterone, 225 68-bromotestosterone acetate, 224 bufotenin, 31 bunalol, 67 bunamidine, 124 bunitrolol (Koe 1366), 68 buquinolate, 118 butaclamol (AY-23,028), 3 butorphanol, 43 butylated hydroxytoluene (BHT) , 218 N-(2-butyl-2-ethyl-5-methyl-3,4hexadienyl)-lY3-propanediamine, 77 butylpyrimidine nucleosides, 134 p-t-butylvinylsulfone, 123
cadmium chloride, 89 cambendazole, 120, 124 camphidines, 187 candicidin, 107 cannabichromene, 27 cannabicyclol, 27 cannabidiol (CBD), 27, 254 cannabigerol, 27 cannabinol (CBN) , 254 0-carbamyl-D-serine, 240 4-carbethoxy-5-(3,3-dimethyl-l-triazino)-2-phenylimidazole, 147 carbidopa (MK 486), 20 carbocyclic purine derivatives, 142 carboxamide ribonucleosides, 134 carboxanilides, 198 carbuterol, 86 carmantadine (Sch 15427), 21 CBD (cannabidiol), 27, 254 CBN (cannabinol), 254 CCNU, 146 cefamandole, 98 cefazolin, 98
COMPOUND NAME AND CODE NUMBER INDEX cefoxitin, 96, 98 celesticetins, 101 centpyraquin(69-183), 3 centrophenoxine (meclofenoxate), 216 chenodeoxycholic acid, 179 chloramphenicol, 100 a-chlordane, 204 chlorgyline, 235 2-chloroadenosine, 76 0-chloroalanine, 237 chlorocyanoethyl colchicine, 230 2-chloromethyl-l,4-naphthoquinone, 146 7a-(chloropropyl)-testosterone, 227 14-chloro-prostaglandin Ez, 164 14-chloro-prostaglandin F 2 a , 164 chloroquine, 115 5-chlorosalicylic acid, 199 chlorpromazine, 2 chlorthalidone (Hygrotonm) , 71 cholestyramine, 175 chromomycin A3, 110 CI-440 (flufenamic acid), 81, 87 CI-473 (mefenamic acid), 81, 87, 168, 208 CI-628, 226 CI-680, 226 CI-683 (pyrazapon) , 5 CI-686, 3 CI-747, 13 CI-750, 14 cinmetacin, 198 C1 110,393, 32 clindamycin, 119 clindamycin 2-palmitate, 100 clindamycin 2-phosphate, 96, 100 clobazam (H-4723), 6 clofibrate, 77, 81, 175, 176, 177 clonazepam (Ro5-4023), 4 clonidine (Catapresm, ST-155), 57 clonixin, 197 clotrimazole, (Bay b 5097), 108, 109 clozapine (HF-1854), 1 cocaine abuse, 38 coenzyme Q, 116 colestipol, 177 Combipresm, 57 cortisone, 225 coumarin, 78 CP-24O-S, 62
311
CP-15,973 (sudoxicam), 76 CP-19106 (trimazosin) , 58 CP-12299-1 (prazosin), 58 CRD-401, 70 cromolyn sodium, 85, 91 croton oil, 155 cryptomycin, 109 CS-359, 68 cyclazocine, 32, 40 cyclic AMP, 87, 142, 143, 228 cyclic GMP, 88 cycloalkyl lactamirnides (RMI 11,894), 197 cyclocytidine, 133 Nb-cyclohexyladenosine, 77 2-cyclohexylthioadenosine, 77 cyclophosphamide, 196, 219 10,11(B)-cyclopropyl-prostaglandin
A2 , 165 cyclorphan, 41, 42 D-cycloserine, 240 cyproheptadine, 78 cytarabine (ara-C, cytosine arabinoside), 132, 139, 141, 158 cytosine arabinoside (ara-C, cytarabine), 132, 139, 141, 158
D-32, 68 D-40TA, 5 D 145 (DMAA), 21 daunomycin, 139, 144 DCAA, 69 DDS (diaminodipttenylsulfone) , 116 3-deaza-5-fluorouridine, 141 1-deazapurines, 142 3-deazauridine, 141 debrisoquin (Declinafl) , 57 (+)-decanoylcarnitine, 187 decoquinate, 118 AS-decynoyl thioester, 234 4-deformy1-4-viny1pyridoxa1, 141 ent-13,14-dehydro-prostaglandin E,, 163 13,14-dehydro-prostaglandin E,, 163, 164 13,14-dehydro-prostaglandin Fza, 163, 164 4'-demethylepipodophyllotoxin 9(4,6-0-2-thenylidene-B-g-gluco-
pyranoside) , 144
312
COMPOUND NAME AND CODE NUMBER INDEX
N-demethyl-4'-pentyl clindamycin, 119 deoxy-fluoro-Q-glucopyranoses, 143 (+)-ent-ll-deoxy-15-methyl prostaglandin El, 165 3- ( 2-deoxy-8-2-erythro-pentofuranosyl)-2,3-dihydro-l, 3-6H-oxazine2,6-dione, 141 9-deoxy-prostaglandin El, 165 11-deoxy-prostaglandin El, 165 11-deoxy-prostaglandin Ez, 165 11-deoxy-prostaglandin F2a, 165 4-deoxypyridoxine, 141 2'-deoxyuridine (UDR), 132 DES (diethylstilbestrol), 226 detrenyl, 235 dextran, 78 DH-581 (probucol, BiphenabidB), 177 2,4-diamino-6,7-bis(aralkyl)pteridine
derivatives, 140 2,2'-diaminodiethyldisulfide, 218 4,6-diamino-1,2-dihydro-2,2-dimethyl-
1-phenyl-s-triazine derivatives, 140 diaminodiphenylsulfone (DDS), 116 diamphenethide, 122 diazoacetylcholine, 229 2-diazoestrone sulfate, 226 5-diazo-4-oxo-L-norvaline, 241 6-diazo-5-oxo-L-norleucine, 241 diazoxide (Hyperstat@ I . V . ) , 57 N6,02'-dibutyryl cyclic AMP 143 dibutryl cyclic GMP, 88 dichlorvos, 120 diethanolamine, 82 N-[ 2- (diethylamino)ethyl]-(bbiphenyloxy)acetamide, 77 diethylcarbamazine, 91, 121 4'-diethyleneoxythiosemicarbazone derivatives, 145 diethylstilbestrol (DES) , 226 13-dihydrodaunomycin HC1, 144 1,2-dihydro-l-(2-deoxy-~-~-ribofurano-
syl)-2-oxopyrazine-4-oxide, 142 3,4-dihydroxybenzoic acid, 229 2,7-dihydroxynaphthalene, 168 6,6'-dihydroxythiobinupharidine, 110 diisopropylammonium dichloroacetate, 187 dimethisterone, 89
dimethylaminoethanol, 218 15,16-dimethyl-prostaglandin Ez,
164 5- (3,3-dimethyl-l-triazeno) imidazole-4-carboxamide, 147 N,N-dimethyltryptamine (DMT) , 31, 32 2,4-dinitrochlorobenzene (DNCB), 155 diphenidol, 69 diphenyleneiodonium, 187 5,5-diphenylhydantoin, 216 diphetarsone, 121 diphtheria toxin, 144 N,N-dipropyltryptamine, 32 dipyridamole, 76 disophenol, 120 disopyramide phosphate (Norpace@) , 69 ditazol ( S 222), 76, 199 Ditran@, 32 diviminol (viminol, Z 424), 14, 15 DMAA (D 1451, 21 DMHP, 30, 257 DMT (N,N-dimethyltryptamine) , 31, 32 DNCB, 155 DOB, 31, 32 DOET, 31, 32 DOM, 29, 31, 32 DON , 141 L-dopa(1evodopa) , 19, 141 dopamantine, 22 dopamine, 19 doxycycline, 102 edeine D, 101 elantrine (Ex 10-029), 23 EN-1661 (bisobrin), 81 enduracidin, 102 epinephrine, 90 epithioandrostanol, 143 ergot alkaloids, 145 eritadenine (Lentinacin), 177 erythromycins, 101 erythromycylamines, 101 estradiol, azide and diazoketone, 226 estradiol mustard, 146 estrogen, 176
COMPOUND NAME AND CODE NUMBER INDEX estrone, azides and diazoketone, 226 ET 495 (trivastal) , 22 ethacrynic acid, 204 ethoxyquin, 218 1,l'-ethylenebis(1-nitrosourea), 146 ethylestranol, 82 3-ethyl-8-methyl-l,3,8- triazabicyclo[ 4,4,O]decan-2-one, 121 etoxadrol, 15 Ex10-029 (elantrine), 23 EX 11-5828, 2
313
gentamicin, 98, 99 Gerobreka, 82 glibornuride (Ro 6-4563), 186 gliclazide (S 1702), 82, 186 glipizide (K 4024), 186 glucagon, 185 glyburide (HB-419, U-26,452), 186 glycyrrhetinic acid, 207 gold thiomalate, 200 GP 45840 (naltaren), 198 growth hormone, 185 GTP (guanosine triphosphate), 226 guanabenz (WY-8678, BR 750, FLA 137), 58 guanadrel (U-28,288D), 58 guancydine, 5% guanosine triphosphate (GTP), 226
F151, 121 FD-008, 61 fenoprofen, 197, 199 filipin, 110, 111 FL 1060, 96 FLA - 137 (guanabenz, BR 750, WY-86781, 58 H-4723(clobazam), 6 fletazepam (Sch 15,698), 4 H87/07, 68 flufenamic acid (CI-440), 81, 87 H93/26, 68 fluorene (bisalkamine esters), 132 halazepam (Sch 12,041), 3 fluorenol (bisallcamine esters), 132 halofenate (MK-185), 176 fluorenone (bisalkamine esters) , 132 haloprogin (Halotex@), 108 2-fluoroalkanoic acids, 110 hamycin, 110 5-fluorocytosine (Ancobon@), 107, 108, HB-419 (glyburide, U-26,452), 186 110, 111 HBB (hydroxybenzylbenzimidazole), 5-fluoro-2'-deoxyuridine (FUDR) , 133 134 9a-fluoro-11$-hydroxybenzo[d, e,] testos- hellibrigenin iodoacetate, 229 heparin, 78 terone 17-acetate, 143 hep tachlor, 204 5-fluor0-3-pyridinemethano1, 178 heroin abuse, 38 6-fluoropyridoxal, 140, 141 heterocyclic alkanoic acid, 198 6-fluoropyridoxamine, 140, 141 heterocyclic carboxamide, 198 6-fluoropyridoxol, 140, 141 hexestrol, azides and diazoketones, 5-fluorouracil, 110, 111, 139, 141 226 fluperamide, 14 hexoprenaline, 86 flurbiprofen (U-27,182) , 76 HF-1854 (clozapine), 1 folinic acid, 140 histamine, 88 6-fomylpurine thiosemicarbazone deHOE33258, 121 rivatives, 145 homoazasteroid mustards, 146 FPL55712, 91, 92 hycanthone, 123 FUDR(5-fluoro-2'-deoxyuridine), 133 hydrocortisone, 90 furosemide, 208 hydroxybenzylbenzimidazole (HBB) , fusaric acid, 60 134 fusidic acid 111 4-hydroxy-clonidine, 57 6~-hydroxy-levallorphan, 12 G-33,040 (opipramol), 6 6B-hydroxy-naltrexone, 12 gastrin, 228 p-hydroxynorephedrine, 61 GCNU, 146 2-hydroxystilbamidine, 107 geldanomycin, 131
314
COMPOUND NAME AND CODE NUMBER INDEX
7-hydroxy-A‘-tetrahydrocannabinol, 28, 30 hypoxanthine arabinoside, 132 (RO 20-1724), 88 kzprofen, 168, 197 ICI 50172 (practolol),
59, 67, 68,
70
ICI 58,301, 88 ICI 63,197, 88 ICI 66,082, 59 ID-540, 4 idoxuridine(IUDR), 132 indandione, 78 indomethacin, 75, 87, 168, 197, 198 indoramine, 58 inosine, 89 insulin, 182 interferon, 131 interferon inducers, 131 iodoacetamide, 229 iodoacetic acid, 229 16a-iodo-3-acetoxyestrone, 225 21-iodoacetylcortisone, 225 N-iodoacetyl-3,5-dimethoxy-4-hydroxyphenylethylamine, 229 3-0-iodoacetyl estradiol, 225 3-0-iodoacetyl estrone, 225 5-iodo-2’-deoxyuridine, 230 ipronidazole, 119 iproveratril (verapamil), 70 isoetharine, 86 8-iso-~-homoestradio1, 143 8-iso-g-homoestrone, 143 4-isopropyl-a-methyl-1-cyclohexene1-acetic acid, 81 isoprinosine, 135 isopropyl atropine (Sch lOOO), 89 isoproterenol, 86 isoquinoline, 135 isosetoclavine, 28 IUDR (idoxuridine) , 132
K 4024 (glipizide), 186 K 4277, 198 K-4423, 68 K8 592, 68 ketoprofen (OrudisB, RP-19583), Koe 1366 (bunitrolol), 68
197
L-6150, 62 L-8040, 71 lankacidins, 101 lasalocid (X537A), 118 LB-46 (pindolol), 59, 68 lecithin, 179 lenperone (AHR-2277), 3 L-leucine dehydrogenase, 144 leucovorin, 140 levallorphan, 32, 42 levamisole, 120 levarin complex, component A , , levodopa (L-dopa) , 19, 141 lincomycin, 100 lincomycin esters, 101 LL-1530, 70 lomofungin, 111 loperamide, 14 LSD, 28, 30, 31, 32 LSD abuse, 38 LU 6-062, 14 lymphocyte chalone, 200
109
maridomycins, 101 mannan, 143 McN-2981 (tolmetin), 197 mebendazole, 120 meclofenamic acid, 168 meclofenoxate (centrophenoxine), 216 mefenamic acid (CI-473), 81, 87, 168, 208 megesterol acetate, 89 5-mercapto-2-deoxyuridine
(Mum),
155 2-mercaptoethylamine, 218 6-mercaptopurineY 139, 141, 196 6-mercaptopurine ribonucleoside triphosphate, 228 4-mercuriestradiol, 226 mescaline, 29, 30, 31, 32 methadone abuse, 42, 43 methadone maintenance, 39, 41, 42 methadone:naloxone combination, 11 methanesulfonic acid esters of amino glycols, 146 L-methioninase, 144 methotrexate, 139, 140 methotrexate esters, 140
COMPOUND NAME AND CODE NUMBER INDEX
methyl adamantanespiro-3'-pyrrolidine, 134 methylapogalanthamine, 60 a-methyldopa, 61 3-methylisoxazole carboxylic acid, 187 methyl lidocaine, 69 7-O-methyllincomycin, 100 ll-methyl-prostaglandin A , , 165 8-methyl-prostaglandin C 2 , 163 16(R)-methyl-prostaglandin E2, 164 16(S)-methyl-prostaglandin E l , 164 2-methylthioadenosine-5'-monophos-
phate, 76 17~-methylthiol-4-androsten-3-one, 227 6-methylthiopurine derivatives, 142 meticlorpindol, 118 metrifonate, 123 metronidazole, 96 miconazole (R14889), 108 milipertine (Win 18,935), 3 mimosine, 141 minocycline, 102 minoxidil (U-10,858), 58 MJ 1999 (sotalol), 204 MJ-9465-2, 62 MK-185 (halofenate) , 176 MK-270, 187 MK 486 (carbidopa), 20 MK-950 (timolol), 59, 68 MMDA, 29, 32 monensin, 118 monoguanidines, 187 morantel, 120 MURD, 155 myristicin, 29 nalbuphine, 43 nalidixic acid, 111 nalorphine, 41, 42 naloxone, 40, 42 naloxone combinations, 43 naltaren (GP 45840), 198 naltrexone, 40, 42 naproxen, 168, 197 napthaleneacetic acid, 198 N B T I , 23 neohomofolic acid, 140 nephrotensin, 52
315
nicergoline, 31 nicotinic acid, 82, 175 niflumic acid, 81, 168 nifurtimox, 119 nitroglycerin, 71, 72 nordopan glucosides, 142 norepinephrine, 90 norpropoxyphene, 16 19-nortestosterone, 224 19-nortestosterone acetate, 224 novastat, 118 nutmeg abuse, 1 nystatin, 107, 109, 110, 111 octoclothepin, 2 octometothepin, 2 opipramol (G-33,040), 6 ORF-8063 (triflubazepam), 5 oxamicetin, 102 oxamniquine, 123 oxazole, 199 oxibendazole, 121 16a, 17a- oxidoestradiol, 226 168, 178-oxidoestradiol, 226 oxilorphan [ (-) -BC-26051, 11, 41, 42 oxisuran, 196 6-oxoestradiol, 226 oxprenolol (Trasicofl), 60, 71 p-oxprenolol, 68 oxyclozanide, 124 oxycodone-naloxone, 43
papain, 89 papaverine, 207 paregoric-naloxone, 43 pargyline, 235 paromomycin, 124 penicillamine, 199 penicillin G, 77 pentazocine, 42 perathiepin, 1 perhexilene (PexidB), 71 phenformin, 82, 187 phenobarbital, 179 phenylalanine ammonia lyase, 143 phenylbutazone, 168, 197, 198, 199, 208, 281, 286 phenylisopropylamines, 29, 31 phosgene, 89 ,
316
COMPOUND NAME AND CODE NUMBER INDEX
phytohemagglutinin, 89 pindolol (LB-46, ViskenB), 59, 68 2-piperazino-4(3H)quinazolinone monoacetate, 187 pivampicillin, 95 platinum (11) complexes, 145 polifungin, 109 polydeoxy-4-thiothymidylic acid, 230 poly I:C, 131 polymyxin, 102 polymyxin B, 110 polyphloretin phosphate (PPP), 87 poly-4-vinylpyridine, 146 potassium canrenoate, 69 PPP (polyphloretin phosphate), 87 practolol (EraldinB, ICI 50172), 59, 67, 68, 70 prazosin (CP-12299-1), 58 PR-G-138, 62 PR-H-286 BS, 196 primaquine, 115 probucol (DH-581, BiphenabidB), 177 procainamide, 216 progesterone, 89, 225, 227 prolactin, 141 propargylamine, 236 propranolol ( InderalB) , 57, 59, 68, 70, 71, 72, 204 prostaglandin, 162 prostaglandin A l , 166 53, 60, 162, 163, prostaglandin A,, 165, 166 prostaglandin CZ, 163 prostaglandin Dz, 164, 166, 167 prostaglandin El, 75, 87, 166 prostaglandin Ez, 53, 82, 87, 164, 167 8-epi-prostaglandin F l u , 165 prostaglandin F 2 a , 53, 70, 87, 163, 164, 166, 167 prostaglandin FzS, 87 psilocin, 29 psilocybin, 31 pyrazapon (CI-683), 5 pyrazomycin, 142 pyrbuterol, 86 3-p yridinemethan01 , 178 pyrimethamine, 115 pyrimidine nucleosides, 132
pyrrolidone carboxylic acid, 141 quinacrine, 116 quinazolines (SL-512),
198
R 11,333 (bromoperidol), 2 R 14889 (miconazole), 108 rafoxanide, 122 rhizobitoxine, 238 rifampicin (rifamycin) , 100 rifampin, 110, 111, 129 rifamycin (rifampicin) , 100 rifamycin B derivatives, 131 rifamycin SV derivatives, 129 rimiterol (WG253), 86 RMI9918, 91, 92 RMI 11,894 (cycloalkyl lactamimides), 187 Ro 4-4602 (benserazide), 20 Ro 5-4023 (clonazepam), 4 Ro 6-4563 (glibornuride), 186 RO 7 - 1 ~ 1 , 119 RO 8-4192, 4 RO 20-1724 (I 1 , 88 robenedine, ?y8 RP-19583 (OrudisB, ketoprofen) , 197 RX 67668, 32 S-222 (ditazol), 76, 199 S-1702 (gliclazide), 82, 186 S-2395, 62, 68 S-8527, 177 salbutamol (albuterol), 80, 86 salicylate, 197, 198 salmefamol, 86 Sandoz 44-549, 63 sarcolysine, 146 Sch 1000 (isopropyl atropine), 89 Sch 12,041 (halazepam), 3 Sch 15,280 (azanator maleate), 91, 92 Sch 15427 (carmantadine), 21 Sch 15,698 (fletazepam), 4 scopolamine, 32 seclazone (W-2354), 199 silicon compounds, 145 SKF 525-A, 110 SKF 3301-A, 110 SKF 16467-A, 110
COMPOUND NAME AND CODE NUMBER INDEX SKF-D 39162, 91, 92 SL-512 (quinazolines), 198 sodium salicylate, 199 sodium selenite, 145 somantin, 185 somatostatin, 185 sorbitol, 186 sotalol (MJ 1999), 204 soterenol, 86 SP54@, 82 SP106 (Abbott 40656), 258 spectinomycin, 98 SQ 18,506, 119 SRS-A, 91 St-155 (clonidine, Catapresm), 57 stanozolol, 82 streptokinase, 79 streptovaricins A, C, D, 131 streptozotocin, 146 strophanthidine, bromoacetate, 229 sudoxicam (CP-15,973), 76 sulfinpyrazone, 78 sulfamethoxydiazine, 115 psulfamoylcarbanilic acid, pentachloro-, tribromo- and triiodophenyl esters of, 109 p-sulfamoylcarbanilic acid, polyhalophenyl esters of, 109 sulfaorthodimethoxine, 286 TEIB, 155 terbutaline, 86 testosterone, 224, 227 3-(tetraacetyl glucopyranos-2-yl)-l(2-chloroethy1)-l-nitrosourea
(GCNU), 146 tetracycline, 102 A'-tetrahydro cannabinol, 60 tetrahydrocannabinols, 27, 28, 30, 31 Th1165a, 86 A"-THC, 256 A'-THC, 253, 254, 255, 256, 257 theophylline, 88, 207 thermolysin, 89 thiabendazole, 120 17B-thio-4-androsten-3-one, 226 thiophanate, 121 thio-TEPA analogs, 145 4-thiouridine triphosphate, 230
317
tibric acid, 176 ticarcillin (BRL 2288), 97 tilorone, 154, 194 tho101(MK-950) , 59, 68 tobramycin, 98, 99 tolamolol(UK 6558), 60, 67 tolbutamide, 82, 186, 204, 208 tolmetin (McN-2981), 197 tolnaftate (Tinactin@), 108 p-toluoyl chloride phenylhydrazone, 121, 124 topolymycin, 131 triarylethylene antiestrogens, 226 triazolam (U-33,030), 5 triflubazepam (ORF-8063), 5 trifluorothymidine (F3T), 132 trimazosin (CP-19106), 58 2,3,4-trimethoxyphenylethylamine, 31
trimetoquinol,
86
2,3,5-trisethyleneiminobenzo-
quinone (TEIB), 155 N-trisubstituted methylimidazoles, 109 Triton WR1339 (formaldehyde polymer of polyoxyethylene ether of octylphenol) , 154 trivastal (ET 495), 22 tubercidin, 123 U-5092, 6 U-10,858 (minoxidil), 58 U-11,lOOA, 226 U-13,625, 6 U-24,568, 199 U-26,452 (glyburide, HB-419), 186 U-27,182, (flurbiprofen), 76 U-28,288D (guanadrel), 58 U-31,889 (alprazolam), 5 U-31,920 (uldazepam), 5 U-31,957, 5 U-33,030 (triazolam), 5 U-35,005, 5 U-42,126, 109 UDR (2' -deoxyuridine) , 132 UK 6558 (tolamolol), 60 uldazepam (U-31,920), 5 UM-272, 68, 71 urokinase, 80
318
COMPOUND NAME AND CODE NUMBER INDEX
6-ureidopurines,
142
valinomycin, 204 v e r a p a m i l ( i p r o v e r a t r i l ) , 70 v i m i n o l ( d i v i m i n o l , Z 424), 1 4 , 15 v i n c r i s t i n e , 139, 144 VirazoleB, 134 v i t a m i n A , 200 v i t a m i n C ( a s c o r b i c a c i d ) , 83, 128 v i t a m i n E, 200 W-1984, 63 w-2354 ( s e c l a z o n e ) , 199 W-2587, 63 W36095, 70 WG253 ( r i m i t e r o l ) , 86 Win 1 8 , 9 3 5 ( m i l i p e r t i n e ) , 3 WR30090, 115 WR33063, 115 WY-8678 (guanabenz, BR 750, FLA 1 3 7 ) ~ 58 77 Wy-23,049, X537A ( l a s a l o c i d ) ,
YB-2, 68 yttrium-90,
118
199
Z 424 (viminol, d i v i m i n o l ) ,
zymosan,
14, 1 5
143
61137, 1 9 7 9411x27, 226 124735L, 6 1 69-183 ( c e n t p y r a q u i n ) , 3
A
4
B 5 C G D
I
L a F ‘I G O ti 1 1 2
J 3
